Sample records for abundant non-protein amino

  1. Detection of non-protein amino acids in the presence of protein amino acids. II.

    NASA Technical Reports Server (NTRS)

    Shapshak, P.; Okaji, M.

    1972-01-01

    Studies conducted with the JEOL 5AH amino acid analyzer are described. This instrument makes possible the programming of the chromatographic process. Data are presented showing the separations of seventeen non-protein amino acids in the presence of eighteen protein amino acids. It is pointed out that distinct separations could be obtained in the case of a number of chemically similar compounds, such as ornithine and lysine, N-amidino alanine and arginine, and iminodiacetic acid and S-carboxymethyl cysteine and aspartic acid.

  2. An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins.

    PubMed

    Singh-Blom, Amrita; Hughes, Randall A; Ellington, Andrew D

    2014-05-20

    Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Effects of non-protein amino acids on survival and locomotion of Osmia bicornis.

    PubMed

    Felicioli, Antonio; Sagona, Simona; Galloni, Marta; Bortolotti, Laura; Bogo, Gherardo; Guarnieri, Massimo; Nepi, Massimo

    2018-04-17

    To investigate the effects of two non-protein amino acids, β-alanine and γ-amino butyric acid (GABA), on Osmia bicornis survival and locomotion, two groups of caged bees were fed with sugar syrup enriched with β-alanine and GABA, respectively. A further control group was fed with sugar syrup. Five behavioural categories were chosen according to the principle of parsimony and intrinsic unitary consistency from start to end, and recorded by scan sampling: two states (remaining under paper or in tubes) and three events (walking on net, feeding from flower and flying). We also analysed the amino acid content of haemolymph sampled from an additional 45 bees fed the same diets (15 per diet type). Bees fed with ß-alanine had a significantly shorter survival than those fed with control and GABA diets. The GABA diet induced higher locomotion than β-alanine. The former non-protein amino acid was only detected in the haemolymph of bees fed GABA. The results suggest that insects consuming non-protein amino-acid-rich diets absorb and transfer these substances to the haemolymph and that non-protein amino acids affect survival and locomotion. Ecological consequences are discussed in the framework of plant reproductive biology. This article is protected by copyright. All rights reserved. © 2018 The Royal Entomological Society.

  4. Computational and Statistical Analyses of Amino Acid Usage and Physico-Chemical Properties of the Twelve Late Embryogenesis Abundant Protein Classes

    PubMed Central

    Jaspard, Emmanuel; Macherel, David; Hunault, Gilles

    2012-01-01

    Late Embryogenesis Abundant Proteins (LEAPs) are ubiquitous proteins expected to play major roles in desiccation tolerance. Little is known about their structure - function relationships because of the scarcity of 3-D structures for LEAPs. The previous building of LEAPdb, a database dedicated to LEAPs from plants and other organisms, led to the classification of 710 LEAPs into 12 non-overlapping classes with distinct properties. Using this resource, numerous physico-chemical properties of LEAPs and amino acid usage by LEAPs have been computed and statistically analyzed, revealing distinctive features for each class. This unprecedented analysis allowed a rigorous characterization of the 12 LEAP classes, which differed also in multiple structural and physico-chemical features. Although most LEAPs can be predicted as intrinsically disordered proteins, the analysis indicates that LEAP class 7 (PF03168) and probably LEAP class 11 (PF04927) are natively folded proteins. This study thus provides a detailed description of the structural properties of this protein family opening the path toward further LEAP structure - function analysis. Finally, since each LEAP class can be clearly characterized by a unique set of physico-chemical properties, this will allow development of software to predict proteins as LEAPs. PMID:22615859

  5. Amino Acid Flux from Metabolic Network Benefits Protein Translation: the Role of Resource Availability.

    PubMed

    Hu, Xiao-Pan; Yang, Yi; Ma, Bin-Guang

    2015-06-09

    Protein translation is a central step in gene expression and affected by many factors such as codon usage bias, mRNA folding energy and tRNA abundance. Despite intensive previous studies, how metabolic amino acid supply correlates with protein translation efficiency remains unknown. In this work, we estimated the amino acid flux from metabolic network for each protein in Escherichia coli and Saccharomyces cerevisiae by using Flux Balance Analysis. Integrated with the mRNA expression level, protein abundance and ribosome profiling data, we provided a detailed description of the role of amino acid supply in protein translation. Our results showed that amino acid supply positively correlates with translation efficiency and ribosome density. Moreover, with the rank-based regression model, we found that metabolic amino acid supply facilitates ribosome utilization. Based on the fact that the ribosome density change of well-amino-acid-supplied genes is smaller than poorly-amino-acid-supply genes under amino acid starvation, we reached the conclusion that amino acid supply may buffer ribosome density change against amino acid starvation and benefit maintaining a relatively stable translation environment. Our work provided new insights into the connection between metabolic amino acid supply and protein translation process by revealing a new regulation strategy that is dependent on resource availability.

  6. Enantiomeric separation of non-protein amino acids by electrokinetic chromatography.

    PubMed

    Pérez-Míguez, Raquel; Marina, María Luisa; Castro-Puyana, María

    2016-10-07

    New analytical methodologies enabling the enantiomeric separation of a group of non-protein amino acids of interest in the pharmaceutical and food analysis fields were developed in this work using Electrokinetic Chromatography. The use of FMOC as derivatization reagent and the subsequent separation using acidic conditions (formate buffer at pH 2.0) and anionic cyclodextrins as chiral selectors allowed the chiral separation of eight from the ten non-protein amino acids studied. Pyroglutamic acid, norvaline, norleucine, 3,4-dihydroxyphenilalanine, 2-aminoadipic acid, and selenomethionine were enantiomericaly separated using sulfated-α-CD while sulfated-γ-CD enabled the enantiomeric separation of norvaline, 3,4-dihydroxyphenilalanine, 2-aminoadipic acid, selenomethionie, citrulline, and pipecolic acid. Moreover, the potential of the developed methodologies was demonstrated in the analysis of citrulline and its enantiomeric impurity in food supplements. For that purpose, experimental and instrumental variables were optimized and the analytical characteristics of the proposed method were evaluated. LODs of 2.1×10 -7 and 1.8×10 -7 M for d- and l-citrulline, respectively, were obtained. d-Cit was not detectable in any of the six food supplement samples analyzed showing that the effect of storage time on the racemization of citrulline was negligible. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Predicting Thermodynamic Behaviors of Non-Protein Amino Acids as a Function of Temperature and pH

    NASA Astrophysics Data System (ADS)

    Kitadai, Norio

    2016-03-01

    Why does life use α-amino acids exclusively as building blocks of proteins? To address that fundamental question from an energetic perspective, this study estimated the standard molal thermodynamic data for three non-α-amino acids (β-alanine, γ-aminobutyric acid, and ɛ-aminocaproic acid) and α-amino- n-butyric acid in their zwitterionic, negative, and positive ionization states based on the corresponding experimental measurements reported in the literature. Temperature dependences of their heat capacities were described based on the revised Helgeson-Kirkham-Flowers (HKF) equations of state. The obtained dataset was then used to calculate the standard molal Gibbs energies ( ΔG o) of the non-α-amino acids as a function of temperature and pH. Comparison of their ΔG o values with those of α-amino acids having the same molecular formula showed that the non-α-amino acids have similar ΔG o values to the corresponding α-amino acids in physiologically relevant conditions (neutral pH, <100 °C). In acidic and alkaline pH, the non-α-amino acids are thermodynamically more stable than the corresponding α-ones over a broad temperature range. These results suggest that the energetic cost of synthesis is not an important selection pressure to incorporate α-amino acids into biological systems.

  8. Predicting Thermodynamic Behaviors of Non-Protein Amino Acids as a Function of Temperature and pH.

    PubMed

    Kitadai, Norio

    2016-03-01

    Why does life use α-amino acids exclusively as building blocks of proteins? To address that fundamental question from an energetic perspective, this study estimated the standard molal thermodynamic data for three non-α-amino acids (β-alanine, γ-aminobutyric acid, and ε-aminocaproic acid) and α-amino-n-butyric acid in their zwitterionic, negative, and positive ionization states based on the corresponding experimental measurements reported in the literature. Temperature dependences of their heat capacities were described based on the revised Helgeson-Kirkham-Flowers (HKF) equations of state. The obtained dataset was then used to calculate the standard molal Gibbs energies (∆G (o)) of the non-α-amino acids as a function of temperature and pH. Comparison of their ∆G (o) values with those of α-amino acids having the same molecular formula showed that the non-α-amino acids have similar ∆G (o) values to the corresponding α-amino acids in physiologically relevant conditions (neutral pH, <100 °C). In acidic and alkaline pH, the non-α-amino acids are thermodynamically more stable than the corresponding α-ones over a broad temperature range. These results suggest that the energetic cost of synthesis is not an important selection pressure to incorporate α-amino acids into biological systems.

  9. Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis

    NASA Astrophysics Data System (ADS)

    Hong, Seok Hoon; Kwon, Yong-Chan; Jewett, Michael

    2014-06-01

    Incorporating non-standard amino acids (NSAAs) into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS) systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (>1 g/L) and long-lasting (>10 h in batch operation) CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications.

  10. New Insights into Amino Acid Preservation in the Early Oceans using Modern Analytical Techniques

    NASA Astrophysics Data System (ADS)

    Parker, E. T.; Brinton, K. L.; Burton, A. S.; Glavin, D. P.; Dworkin, J. P.; Bada, J.

    2015-12-01

    Protein- and non-protein-amino acids likely occupied the oceans at the time of the origin and evolution of life. Primordial soup-, hydrothermal vent-, and meteoritic-processes likely contributed to this early chemical inventory. Prebiotic synthesis and carbonaceous meteorite studies suggest that non-protein amino acids were likely more abundant than their protein-counterparts. Amino acid preservation before abiotic and biotic destruction is key to biomarker availability in paleoenvironments and remains an important uncertainty. To constrain primitive amino acid lifetimes, a 1992 archived seawater/beach sand mixture was spiked with D,L-alanine, D,L-valine (Val), α-aminoisobutyric acid (α-AIB), D,L-isovaline (Iva), and glycine (Gly). Analysis by high performance liquid chromatography with fluorescence detection (HPLC-FD) showed that only D-Val and non-protein amino acids were abundant after 2250 days. The mixture was re-analyzed in 2012 using HPLC-FD and a triple quadrupole mass spectrometer (QqQ-MS). The analytical results 20 years after the inception of the experiment were strikingly similar to those after 2250 days. To confirm that viable microorganisms were still present, the mixture was re-spiked with Gly in 2012. Aliquots were collected immediately after spiking, and at 5- and 9-month intervals thereafter. Final HPLC-FD/QqQ-MS analyses were performed in 2014. The 2014 analyses revealed that only α-AIB, D,L-Iva, and D-Val remained abundant. The disappearance of Gly indicated that microorganisms still lived in the mixture and were capable of consuming protein amino acids. These findings demonstrate that non-protein amino acids are minimally impacted by biological degradation and thus have very long lifetimes under these conditions. Primitive non-protein amino acids from terrestrial synthesis, or meteorite in-fall, likely experienced greater preservation than protein amino acids in paleo-oceanic environments. Such robust molecules may have reached a steady

  11. New Insights into Amino Acid Preservation in the Early Oceans Using Modern Analytical Techniques

    NASA Technical Reports Server (NTRS)

    Parker, Eric T.; Brinton, Karen L.; Burton, Aaron S.; Glavin, Daniel P.; Dworkin, Jason P.; Bada, Jeffrey L.

    2015-01-01

    Protein- and non-protein-amino acids likely occupied the oceans at the time of the origin and evolution of life. Primordial soup-, hydrothermal vent-, and meteoritic-processes likely contributed to this early chemical inventory. Prebiotic synthesis and carbonaceous meteorite studies suggest that non-protein amino acids were likely more abundant than their protein-counterparts. Amino acid preservation before abiotic and biotic destruction is key to biomarker availability in paleoenvironments and remains an important uncertainty. To constrain primitive amino acid lifetimes, a 1992 archived seawater/beach sand mixture was spiked with D,L-alanine, D,L-valine (Val), alpha-aminoisobutyric acid (alpha-AIB), D,L-isovaline (Iva), and glycine (Gly). Analysis by high performance liquid chromatography with fluorescence detection (HPLC-FD) showed that only D-Val and non-protein amino acids were abundant after 2250 days. The mixture was re-analyzed in 2012 using HPLC-FD and a triple quadrupole mass spectrometer (QqQ-MS). The analytical results 20 years after the inception of the experiment were strikingly similar to those after 2250 days. To confirm that viable microorganisms were still present, the mixture was re-spiked with Gly in 2012. Aliquots were collected immediately after spiking, and at 5- and 9-month intervals thereafter. Final HPLC-FD/QqQ-MS analyses were performed in 2014. The 2014 analyses revealed that only alpha-AIB, D,L-Iva, and D-Val remained abundant. The disappearance of Gly indicated that microorganisms still lived in the mixture and were capable of consuming protein amino acids. These findings demonstrate that non-protein amino acids are minimally impacted by biological degradation and thus have very long lifetimes under these conditions. Primitive non-protein amino acids from terrestrial synthesis, or meteorite in-fall, likely experienced great-er preservation than protein amino acids in paleo-oceanic environments. Such robust molecules may have reached a

  12. Amino acid repletion does not decrease muscle protein catabolism during hemodialysis.

    PubMed

    Raj, Dominic S C; Adeniyi, Oladipo; Dominic, Elizabeth A; Boivin, Michel A; McClelland, Sandra; Tzamaloukas, Antonios H; Morgan, Nancy; Gonzales, Lawrence; Wolfe, Robert; Ferrando, Arny

    2007-06-01

    Intradialytic protein catabolism is attributed to loss of amino acids in the dialysate. We investigated the effect of amino acid infusion during hemodialysis (HD) on muscle protein turnover and amino acid transport kinetics by using stable isotopes of phenylalanine, leucine, and lysine in eight patients with end-stage renal disease (ESRD). Subjects were studied at baseline (pre-HD), 2 h of HD without amino acid infusion (HD-O), and 2 h of HD with amino acid infusion (HD+AA). Amino acid depletion during HD-O augmented the outward transport of amino acids from muscle into the vein. Increased delivery of amino acids to the leg during HD+AA facilitated the transport of amino acids from the artery into the intracellular compartment. Increase in muscle protein breakdown was more than the increase in synthesis during HD-O (46.7 vs. 22.3%, P < 0.001). Net balance (nmol.min(-1).100 ml (-1)) was more negative during HD-O compared with pre-HD (-33.7 +/- 1.5 vs. -6.0 +/- 2.3, P < 0.001). Despite an abundant supply of amino acids, the net balance (-16.9 +/- 1.8) did not switch from net release to net uptake. HD+AA induced a proportional increase in muscle protein synthesis and catabolism. Branched chain amino acid catabolism increased significantly from baseline during HD-O and did not decrease during HD+AA. Protein synthesis efficiency, the fraction of amino acid in the intracellular pool that is utilized for muscle protein synthesis decreased from 42.1% pre-HD to 33.7 and 32.6% during HD-O and HD+AA, respectively (P < 0.01). Thus amino acid repletion during HD increased muscle protein synthesis but did not decrease muscle protein breakdown.

  13. Comparing Amino Acid Abundances and Distributions Across Carbonaceous Chondrite Groups

    NASA Technical Reports Server (NTRS)

    Burton, Aaron S.; Callahan, Michael P.; Glavin, Daniel P.; Elsila, Jamie E.; Dworkin, Jason P.

    2012-01-01

    Meteorites are grouped according to bulk properties such as chemical composition and mineralogy. These parameters can vary significantly among the different carbonaceous chondrite groups (CI, CM, CO, CR, CH, CB, CV and CK). We have determined the amino acid abundances of more than 30 primary amino acids in meteorites from each of the eight groups, revealing several interesting trends. There are noticeable differences in the structural diversity and overall abundances of amino acids between meteorites from the different chondrite groups. Because meteorites may have been an important source of amino acids to the prebiotic Earth and these organic compounds are essential for life as we know it, the observed variations of these molecules may have been important for the origins of life.

  14. DNA–protein π-interactions in nature: abundance, structure, composition and strength of contacts between aromatic amino acids and DNA nucleobases or deoxyribose sugar

    PubMed Central

    Wilson, Katie A.; Kellie, Jennifer L.; Wetmore, Stacey D.

    2014-01-01

    Four hundred twenty-eight high-resolution DNA–protein complexes were chosen for a bioinformatics study. Although 164 crystal structures (38% of those searched) contained no interactions, 574 discrete π–contacts between the aromatic amino acids and the DNA nucleobases or deoxyribose were identified using strict criteria, including visual inspection. The abundance and structure of the interactions were determined by unequivocally classifying the contacts as either π–π stacking, π–π T-shaped or sugar–π contacts. Three hundred forty-four nucleobase–amino acid π–π contacts (60% of all interactions identified) were identified in 175 of the crystal structures searched. Unprecedented in the literature, 230 DNA–protein sugar–π contacts (40% of all interactions identified) were identified in 137 crystal structures, which involve C–H···π and/or lone–pair···π interactions, contain any amino acid and can be classified according to sugar atoms involved. Both π–π and sugar–π interactions display a range of relative monomer orientations and therefore interaction energies (up to –50 (–70) kJ mol−1 for neutral (charged) interactions as determined using quantum chemical calculations). In general, DNA–protein π-interactions are more prevalent than perhaps currently accepted and the role of such interactions in many biological processes may yet to be uncovered. PMID:24744240

  15. Effects of Long-Term Protein Restriction on Meat Quality, Muscle Amino Acids, and Amino Acid Transporters in Pigs.

    PubMed

    Yin, Jie; Li, Yuying; Zhu, Xiaotong; Han, Hui; Ren, Wenkai; Chen, Shuai; Bin, Peng; Liu, Gang; Huang, Xingguo; Fang, Rejun; Wang, Bin; Wang, Kai; Sun, Liping; Li, Tiejun; Yin, Yulong

    2017-10-25

    This study aimed to investigate the long-term effects of protein restriction from piglets to finishing pigs for 16 weeks on meat quality, muscle amino acids, and amino acid transporters. Thirty-nine piglets were randomly divided into three groups: a control (20-18-16% crude protein, CP) and two protein restricted groups (17-15-13% CP and 14-12-10% CP). The results showed that severe protein restriction (14-12-10% CP) inhibited feed intake and body weight, while moderate protein restriction (17-15-13% CP) had little effect on growth performance in pigs. Meat quality (i.e., pH, color traits, marbling, water-holding capacity, and shearing force) were tested, and the results exhibited that 14-12-10% CP treatment markedly improved muscle marbling score and increased yellowness (b*). pH value (45 min) was significantly higher in 17-15-13% CP group than that in other groups. In addition, protein restriction reduced muscle histone, arginine, valine, and isoleucine abundances and enhanced glycine and lysine concentrations compared with the control group, while the RT-PCR results showed that protein restriction downregulated amino acids transporters. Mechanistic target of rapamycin (mTOR) signaling pathway was inactivated in the moderate protein restricted group (17-15-13% CP), while severe protein restriction with dietary 14-12-10% CP markedly enhanced mTOR phosphorylation. In conclusion, long-term protein restriction affected meat quality and muscle amino acid metabolism in pigs, which might be associated with mTOR signaling pathway.

  16. Enhanced Bio-hydrogen Production from Protein Wastewater by Altering Protein Structure and Amino Acids Acidification Type

    PubMed Central

    Xiao, Naidong; Chen, Yinguang; Chen, Aihui; Feng, Leiyu

    2014-01-01

    Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control. PMID:24495932

  17. Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type.

    PubMed

    Xiao, Naidong; Chen, Yinguang; Chen, Aihui; Feng, Leiyu

    2014-02-05

    Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control.

  18. Enhanced Bio-hydrogen Production from Protein Wastewater by Altering Protein Structure and Amino Acids Acidification Type

    NASA Astrophysics Data System (ADS)

    Xiao, Naidong; Chen, Yinguang; Chen, Aihui; Feng, Leiyu

    2014-02-01

    Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control.

  19. Human Protein and Amino Acid Requirements.

    PubMed

    Hoffer, L John

    2016-05-01

    Human protein and amino acid nutrition encompasses a wide, complex, frequently misunderstood, and often contentious area of clinical research and practice. This tutorial explains the basic biochemical and physiologic principles that underlie our current understanding of protein and amino acid nutrition. The following topics are discussed: (1) the identity, measurement, and essentiality of nutritional proteins; (2) the definition and determination of minimum requirements; (3) nutrition adaptation; (4) obligatory nitrogen excretion and the minimum protein requirement; (5) minimum versus optimum protein intakes; (6) metabolic responses to surfeit and deficient protein intakes; (7) body composition and protein requirements; (8) labile protein; (9) N balance; (10) the principles of protein and amino acid turnover, including an analysis of the controversial indicator amino acid oxidation technique; (11) general guidelines for evaluating protein turnover articles; (12) amino acid turnover versus clearance; (13) the protein content of hydrated amino acid solutions; (14) protein requirements in special situations, including protein-catabolic critical illness; (15) amino acid supplements and additives, including monosodium glutamate and glutamine; and (16) a perspective on the future of protein and amino acid nutrition research. In addition to providing practical information, this tutorial aims to demonstrate the importance of rigorous physiologic reasoning, stimulate intellectual curiosity, and encourage fresh ideas in this dynamic area of human nutrition. In general, references are provided only for topics that are not well covered in modern textbooks. © 2016 American Society for Parenteral and Enteral Nutrition.

  20. Nature's starships. I. Observed abundances and relative frequencies of amino acids in meteorites

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cobb, Alyssa K.; Pudritz, Ralph E., E-mail: cobbak@mcmaster.ca, E-mail: pudritz@physics.mcmaster.ca

    The class of meteorites called carbonaceous chondrites are examples of material from the solar system which have been relatively unchanged from the time of their initial formation. These meteorites have been classified according to the temperatures and physical conditions of their parent planetesimals. We collate available data on amino acid abundance in these meteorites and plot the concentrations of different amino acids for each meteorite within various meteorite subclasses. We plot average concentrations for various amino acids across meteorites separated by subclass and petrologic type. We see a predominance in the abundance and variety of amino acids in CM2 andmore » CR2 meteorites. The range in temperature corresponding to these subclasses indicates high degrees of aqueous alteration, suggesting aqueous synthesis of amino acids. Within the CM2 and CR2 subclasses, we identify trends in relative frequencies of amino acids to investigate how common amino acids are as a function of their chemical complexity. These two trends (total abundance and relative frequencies) can be used to constrain formation parameters of amino acids within planetesimals. Our organization of the data supports an onion shell model for the temperature structure of planetesimals. The least altered meteorites (type 3) and their amino acids originated near cooler surface regions. The most active amino acid synthesis likely took place at intermediate depths (type 2). The most altered materials (type 1) originated furthest toward parent body cores. This region is likely too hot to either favor amino acid synthesis or for amino acids to be retained after synthesis.« less

  1. Translation system engineering in Escherichia coli enhances non-canonical amino acid incorporation into proteins.

    PubMed

    Gan, Rui; Perez, Jessica G; Carlson, Erik D; Ntai, Ioanna; Isaacs, Farren J; Kelleher, Neil L; Jewett, Michael C

    2017-05-01

    The ability to site-specifically incorporate non-canonical amino acids (ncAAs) into proteins has made possible the study of protein structure and function in fundamentally new ways, as well as the bio synthesis of unnatural polymers. However, the task of site-specifically incorporating multiple ncAAs into proteins with high purity and yield continues to present a challenge. At the heart of this challenge lies the lower efficiency of engineered orthogonal translation system components compared to their natural counterparts (e.g., translation elements that specifically use a ncAA and do not interact with the cell's natural translation apparatus). Here, we show that evolving and tuning expression levels of multiple components of an engineered translation system together as a whole enhances ncAA incorporation efficiency. Specifically, we increase protein yield when incorporating multiple p-azido-phenylalanine(pAzF) residues into proteins by (i) evolving the Methanocaldococcus jannaschii p-azido-phenylalanyl-tRNA synthetase anti-codon binding domain, (ii) evolving the elongation factor Tu amino acid-binding pocket, and (iii) tuning the expression of evolved translation machinery components in a single vector. Use of the evolved translation machinery in a genomically recoded organism lacking release factor one enabled enhanced multi-site ncAA incorporation into proteins. We anticipate that our approach to orthogonal translation system development will accelerate and expand our ability to site-specifically incorporate multiple ncAAs into proteins and biopolymers, advancing new horizons for synthetic and chemical biotechnology. Biotechnol. Bioeng. 2017;114: 1074-1086. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  2. Conformational Entropy of Intrinsically Disordered Proteins from Amino Acid Triads

    PubMed Central

    Baruah, Anupaul; Rani, Pooja; Biswas, Parbati

    2015-01-01

    This work quantitatively characterizes intrinsic disorder in proteins in terms of sequence composition and backbone conformational entropy. Analysis of the normalized relative composition of the amino acid triads highlights a distinct boundary between globular and disordered proteins. The conformational entropy is calculated from the dihedral angles of the middle amino acid in the amino acid triad for the conformational ensemble of the globular, partially and completely disordered proteins relative to the non-redundant database. Both Monte Carlo (MC) and Molecular Dynamics (MD) simulations are used to characterize the conformational ensemble of the representative proteins of each group. The results show that the globular proteins span approximately half of the allowed conformational states in the Ramachandran space, while the amino acid triads in disordered proteins sample the entire range of the allowed dihedral angle space following Flory’s isolated-pair hypothesis. Therefore, only the sequence information in terms of the relative amino acid triad composition may be sufficient to predict protein disorder and the backbone conformational entropy, even in the absence of well-defined structure. The predicted entropies are found to agree with those calculated using mutual information expansion and the histogram method. PMID:26138206

  3. Utilizing knowledge base of amino acids structural neighborhoods to predict protein-protein interaction sites.

    PubMed

    Jelínek, Jan; Škoda, Petr; Hoksza, David

    2017-12-06

    Protein-protein interactions (PPI) play a key role in an investigation of various biochemical processes, and their identification is thus of great importance. Although computational prediction of which amino acids take part in a PPI has been an active field of research for some time, the quality of in-silico methods is still far from perfect. We have developed a novel prediction method called INSPiRE which benefits from a knowledge base built from data available in Protein Data Bank. All proteins involved in PPIs were converted into labeled graphs with nodes corresponding to amino acids and edges to pairs of neighboring amino acids. A structural neighborhood of each node was then encoded into a bit string and stored in the knowledge base. When predicting PPIs, INSPiRE labels amino acids of unknown proteins as interface or non-interface based on how often their structural neighborhood appears as interface or non-interface in the knowledge base. We evaluated INSPiRE's behavior with respect to different types and sizes of the structural neighborhood. Furthermore, we examined the suitability of several different features for labeling the nodes. Our evaluations showed that INSPiRE clearly outperforms existing methods with respect to Matthews correlation coefficient. In this paper we introduce a new knowledge-based method for identification of protein-protein interaction sites called INSPiRE. Its knowledge base utilizes structural patterns of known interaction sites in the Protein Data Bank which are then used for PPI prediction. Extensive experiments on several well-established datasets show that INSPiRE significantly surpasses existing PPI approaches.

  4. Differential abundance analysis of mesocarp protein from high- and low-yielding oil palms associates non-oil biosynthetic enzymes to lipid biosynthesis.

    PubMed

    Ooi, Tony Eng Keong; Yeap, Wan Chin; Daim, Leona Daniela Jeffery; Ng, Boon Zean; Lee, Fong Chin; Othman, Ainul Masni; Appleton, David Ross; Chew, Fook Tim; Kulaveerasingam, Harikrishna

    2015-01-01

    The oil palm Elaeis guineensis Jacq. which produces the highest yield per unit land area of the oil crops is the most important commercial oil crop in South East Asia. The fleshy mesocarp of oil palm fruit, where oil is mostly derived from, contains up to 90 % dry weight of oil (one of the most concentrated in plant tissues). Hence, there is attention given to gain insights into the processes of oil deposition in this oil rich tissue. For that purpose, two-dimensional differential gel electrophoresis (DIGE) coupled with western assays, were used here to analyze differential protein levels in genetically-related high-and low-yielding oil palm mesocarps. From the DIGE comparative analysis in combination with western analysis, 41 unique differentially accumulated proteins were discovered. Functional categorization of these proteins placed them in the metabolisms of lipid, carbohydrate, amino acids, energy, structural proteins, as well as in other functions. In particular, higher abundance of fructose-1,6-biphosphate aldolase combined with reduced level of triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase may be indicative of important flux balance changes in glycolysis, while amino acid metabolism also appeared to be closely linked with oil yield. Forty-one proteins in several important biological pathways were identified as exhibiting differential in abundance at critical oil production stages. These confirm that oil yield is a complex trait involving the regulation of genes in multiple biological pathways. The results also provide insights into key control points of lipid biosynthesis in oil palm and can assist in the development of genetic markers for use in oil palm breeding programmes.

  5. Bacillus anthracis Overcomes an Amino Acid Auxotrophy by Cleaving Host Serum Proteins

    PubMed Central

    Terwilliger, Austen; Swick, Michelle C.; Pflughoeft, Kathryn J.; Pomerantsev, Andrei; Lyons, C. Rick; Koehler, Theresa M.

    2015-01-01

    ABSTRACT Bacteria sustain an infection by acquiring nutrients from the host to support replication. The host sequesters these nutrients as a growth-restricting strategy, a concept termed “nutritional immunity.” Historically, the study of nutritional immunity has centered on iron uptake because many bacteria target hemoglobin, an abundant circulating protein, as an iron source. Left unresolved are the mechanisms that bacteria use to attain other nutrients from host sources, including amino acids. We employed a novel medium designed to mimic the chemical composition of human serum, and we show here that Bacillus anthracis, the causative agent of anthrax disease, proteolyzes human hemoglobin to liberate essential amino acids which enhance its growth. This property can be traced to the actions of InhA1, a secreted metalloprotease, and extends to at least three other serum proteins, including serum albumin. The results suggest that we must also consider proteolysis of key host proteins to be a way for bacterial pathogens to attain essential nutrients, and we provide an experimental framework to determine the host and bacterial factors involved in this process. IMPORTANCE The mechanisms by which bacterial pathogens acquire nutrients during infection are poorly understood. Here we used a novel defined medium that approximates the chemical composition of human blood serum, blood serum mimic (BSM), to better model the nutritional environment that pathogens encounter during bacteremia. Removing essential amino acids from BSM revealed that two of the most abundant proteins in blood—hemoglobin and serum albumin—can satiate the amino acid requirement for Bacillus anthracis, the causative agent of anthrax. We further demonstrate that hemoglobin is proteolyzed by the secreted protease InhA1. These studies highlight that common blood proteins can be a nutrient source for bacteria. They also challenge the historical view that hemoglobin is solely an iron source for

  6. Amino Acid Stability in the Early Oceans

    NASA Technical Reports Server (NTRS)

    Parker, E. T.; Brinton, K. L.; Burton, A. S.; Glavin, D. P.; Dworkin, J. P.; Bada, J. L.

    2015-01-01

    It is likely that a variety of amino acids existed in the early oceans of the Earth at the time of the origin and early evolution of life. "Primordial soup", hydrothermal vent, and meteorite based processes could have contributed to such an inventory. Several "protein" amino acids were likely present, however, based on prebiotic synthesis experiments and carbonaceous meteorite studies, non-protein amino acids, which are rare on Earth today, were likely the most abundant. An important uncertainty is the length of time these amino acids could have persisted before their destruction by abiotic and biotic processes. Prior to life, amino acid concentrations in the oceans were likely regulated by circulation through hydro-thermal vents. Today, the entire ocean circulates through vent systems every 10(exp 7) years. On the early Earth, this value was likely smaller due to higher heat flow and thus marine amino acid life-time would have been shorter. After life, amino acids in the oceans could have been assimilated by primitive organisms.

  7. Comparative characterization of random-sequence proteins consisting of 5, 12, and 20 kinds of amino acids

    PubMed Central

    Tanaka, Junko; Doi, Nobuhide; Takashima, Hideaki; Yanagawa, Hiroshi

    2010-01-01

    Screening of functional proteins from a random-sequence library has been used to evolve novel proteins in the field of evolutionary protein engineering. However, random-sequence proteins consisting of the 20 natural amino acids tend to aggregate, and the occurrence rate of functional proteins in a random-sequence library is low. From the viewpoint of the origin of life, it has been proposed that primordial proteins consisted of a limited set of amino acids that could have been abundantly formed early during chemical evolution. We have previously found that members of a random-sequence protein library constructed with five primitive amino acids show high solubility (Doi et al., Protein Eng Des Sel 2005;18:279–284). Although such a library is expected to be appropriate for finding functional proteins, the functionality may be limited, because they have no positively charged amino acid. Here, we constructed three libraries of 120-amino acid, random-sequence proteins using alphabets of 5, 12, and 20 amino acids by preselection using mRNA display (to eliminate sequences containing stop codons and frameshifts) and characterized and compared the structural properties of random-sequence proteins arbitrarily chosen from these libraries. We found that random-sequence proteins constructed with the 12-member alphabet (including five primitive amino acids and positively charged amino acids) have higher solubility than those constructed with the 20-member alphabet, though other biophysical properties are very similar in the two libraries. Thus, a library of moderate complexity constructed from 12 amino acids may be a more appropriate resource for functional screening than one constructed from 20 amino acids. PMID:20162614

  8. Protein and amino acid nutrition

    USDA-ARS?s Scientific Manuscript database

    Dairy cow protein and amino acid nutrition have a significant role in sustainable dairying. Protein, amino acids, and nitrogen are inextricably linked through effects in the rumen, metabolism of the cow, and environmental nutrient management. Feeding systems have been making progress toward emphasiz...

  9. Inferring Selection on Amino Acid Preference in Protein Domains

    PubMed Central

    Durbin, Richard

    2009-01-01

    Models that explicitly account for the effect of selection on new mutations have been proposed to account for “codon bias” or the excess of “preferred” codons that results from selection for translational efficiency and/or accuracy. In principle, such models can be applied to any mutation that results in a preferred allele, but in most cases, the fitness effect of a specific mutation cannot be predicted. Here we show that it is possible to assign preferred and unpreferred states to amino acid changing mutations that occur in protein domains. We propose that mutations that lead to more common amino acids (at a given position in a domain) can be considered “preferred alleles” just as are synonymous mutations leading to codons for more abundant tRNAs. We use genome-scale polymorphism data to show that alleles for preferred amino acids in protein domains occur at higher frequencies in the population, as has been shown for preferred codons. We show that this effect is quantitative, such that there is a correlation between the shift in frequency of preferred alleles and the predicted fitness effect. As expected, we also observe a reduction in the numbers of polymorphisms and substitutions at more important positions in domains, consistent with stronger selection at those positions. We examine the derived allele frequency distribution and polymorphism to divergence ratios of preferred and unpreferred differences and find evidence for both negative and positive selections acting to maintain protein domains in the human population. Finally, we analyze a model for selection on amino acid preferences in protein domains and find that it is consistent with the quantitative effects that we observe. PMID:19095755

  10. Extraterrestrial Amino Acids in the Almahata Sitta Meteorite

    NASA Technical Reports Server (NTRS)

    Glavin, Daniel P.; Aubrey, Andrew D.; Callahan, Michael P.; Dworkin, Jason P.; Elsila, Jamie E.; Parker, Eric T.; Bada, Jeffrey L.

    2010-01-01

    Amino acid analysis of a meteorite fragment of asteroid 2008 TC3 called Almahata Sitta was carried out using reverse-phase liquid chromatography coupled with UV fluorescence detection and time-of-flight mass spectrometry (LC-FD/ToF-MS) as part of a sample analysis consortium. LC-FD/ToF-MS analyses of hot-water extracts from the meteorite revealed a complex distribution of two- to seven-carbon aliphatic amino acids and one- to three-carbon amines with abundances ranging from 0.5 to 149 parts-per-billion (ppb). The enantiomeric ratios of the amino acids alanine, R-amino-n-butyric acid (beta-ABA), 2-amino-2-methylbutanoic acid (isovaline), and 2-aminopentanoic acid (norvaline) in the meteorite were racemic (D/L approximately 1), indicating that these amino acids are indigenous to the meteorite and not terrestrial contaminants. Several other non-protein amino acids were also identified in the meteorite above background levels including alpha-aminoisobutyric acid (alpha-AIB), 4-amino-2- methylbutanoic acid, 4-amino-3-methylbutanoic acid, and 3-, 4-, and 5-aminopentanoic acid. The total abundances of isovaline and alpha-AIB in Almahata Sitta are 1000 times lower than the abundances of these amino acids found in the CM carbonaceous chondrite Murchison. The extremely low abundances and unusual distribution of five carbon amino acids in Almahata Sitta compared to Cl, CM, and CR carbonaceous chondrites may reflect extensive thermal alteration of amino acids on the parent asteroid by partial melting during formation or subsequent impact shock heating. It is also possible that amino acids were synthesized by catalytic reactions on the parent body after asteroid 2008 TC3 cooled to lower temperatures.

  11. Flavor Compounds in Pixian Broad-Bean Paste: Non-Volatile Organic Acids and Amino Acids.

    PubMed

    Lin, Hongbin; Yu, Xiaoyu; Fang, Jiaxing; Lu, Yunhao; Liu, Ping; Xing, Yage; Wang, Qin; Che, Zhenming; He, Qiang

    2018-05-29

    Non-volatile organic acids and amino acids are important flavor compounds in Pixian broad-bean paste, which is a traditional Chinese seasoning product. In this study, non-volatile organic acids, formed in the broad-bean paste due to the metabolism of large molecular compounds, are qualitatively and quantitatively determined by high-performance liquid chromatography (HPLC). Amino acids, mainly produced by hydrolysis of soybean proteins, were determined by the amino acid automatic analyzer. Results indicated that seven common organic acids and eighteen common amino acids were found in six Pixian broad-bean paste samples. The content of citric acid was found to be the highest in each sample, between 4.1 mg/g to 6.3 mg/g, and malic acid were between 2.1 mg/g to 3.6 mg/g ranked as the second. Moreover, fumaric acid was first detected in fermented bean pastes albeit with a low content. For amino acids, savory with lower sour taste including glutamine (Gln), glutamic acid (Glu), aspartic acid (Asp) and asparagines (Asn) were the most abundant, noted to be 6.5 mg/g, 4.0 mg/g, 6.4 mg/g, 4.9 mg/g, 6.2 mg/g and 10.2 mg/g, and bitter taste amino acids followed. More importantly, as important flavor materials in Pixian broad-bean paste, these two groups of substances are expected to be used to evaluate and represent the flavor quality of Pixian broad-bean paste. Moreover, the results revealed that citric acid, glutamic acid, methionine and proline were the most important flavor compounds. These findings are agreat contribution for evaluating the quality and further assessment of Pixian broad-bean paste.

  12. PLASMA PROTEIN AND HEMOGLOBIN PRODUCTION : DELETION OF INDIVIDUAL AMINO ACIDS FROM GROWTH MIXTURE OF TEN ESSENTIAL AMINO ACIDS. SIGNIFICANT CHANGES IN URINARY NITROGEN.

    PubMed

    Robscheit-Robbins, F S; Miller, L L; Whipple, G H

    1947-02-28

    Given healthy dogs fed abundant iron and protein-free or low protein diets with sustained anemia and hypoproteinemia, we can study the capacity of these animals to produce simultaneously new hemoglobin and plasma protein. Reserve stores of blood protein-building materials are measurably depleted and levels of 6 to 8 gm. per cent for hemoglobin and 4 to 5 gm. per cent for plasma protein can be maintained for weeks or months depending upon the intake of food proteins or amino acid mixtures. These dogs are very susceptible to infection and various poisons. Dogs tire of these diets and loss of appetite terminates many experiments. Under these conditions (double depletion) standard growth mixtures of essential amino acids are tested to show the response in blood protein output and urinary nitrogen balance. As a part of each tabulated experiment one of the essential amino acids is deleted from the complete growth mixture to compare such response with that of the whole mixture. Methionine, threonine, phenylalanine, and tryptophane when singly eliminated from the complete amino acid mixture do effect a sharp rise in urinary nitrogen. This loss of urinary nitrogen is corrected when the individual amino acid is replaced in the mixture. Histidine, lysine, and valine have a moderate influence upon urinary nitrogen balance toward nitrogen conservation. Leucine, isoleucine, and arginine have minimal or no effect upon urinary nitrogen balance when these individual amino acids are deleted from the complete growth mixture of amino acids during 3 to 4 week periods. Tryptophane and to a less extent phenylalanine and threonine when returned to the amino acid mixture are associated with a conspicuous preponderance of plasma protein output over the hemoglobin output (Table 4). Arginine, lysine, and histidine when returned to the amino acid mixture are associated with a large preponderance of hemoglobin output. Various amino acid mixtures under these conditions may give a positive

  13. Predicting the Dynamics of Protein Abundance

    PubMed Central

    Mehdi, Ahmed M.; Patrick, Ralph; Bailey, Timothy L.; Bodén, Mikael

    2014-01-01

    Protein synthesis is finely regulated across all organisms, from bacteria to humans, and its integrity underpins many important processes. Emerging evidence suggests that the dynamic range of protein abundance is greater than that observed at the transcript level. Technological breakthroughs now mean that sequencing-based measurement of mRNA levels is routine, but protocols for measuring protein abundance remain both complex and expensive. This paper introduces a Bayesian network that integrates transcriptomic and proteomic data to predict protein abundance and to model the effects of its determinants. We aim to use this model to follow a molecular response over time, from condition-specific data, in order to understand adaptation during processes such as the cell cycle. With microarray data now available for many conditions, the general utility of a protein abundance predictor is broad. Whereas most quantitative proteomics studies have focused on higher organisms, we developed a predictive model of protein abundance for both Saccharomyces cerevisiae and Schizosaccharomyces pombe to explore the latitude at the protein level. Our predictor primarily relies on mRNA level, mRNA–protein interaction, mRNA folding energy and half-life, and tRNA adaptation. The combination of key features, allowing for the low certainty and uneven coverage of experimental observations, gives comparatively minor but robust prediction accuracy. The model substantially improved the analysis of protein regulation during the cell cycle: predicted protein abundance identified twice as many cell-cycle-associated proteins as experimental mRNA levels. Predicted protein abundance was more dynamic than observed mRNA expression, agreeing with experimental protein abundance from a human cell line. We illustrate how the same model can be used to predict the folding energy of mRNA when protein abundance is available, lending credence to the emerging view that mRNA folding affects translation

  14. Predicting the dynamics of protein abundance.

    PubMed

    Mehdi, Ahmed M; Patrick, Ralph; Bailey, Timothy L; Bodén, Mikael

    2014-05-01

    Protein synthesis is finely regulated across all organisms, from bacteria to humans, and its integrity underpins many important processes. Emerging evidence suggests that the dynamic range of protein abundance is greater than that observed at the transcript level. Technological breakthroughs now mean that sequencing-based measurement of mRNA levels is routine, but protocols for measuring protein abundance remain both complex and expensive. This paper introduces a Bayesian network that integrates transcriptomic and proteomic data to predict protein abundance and to model the effects of its determinants. We aim to use this model to follow a molecular response over time, from condition-specific data, in order to understand adaptation during processes such as the cell cycle. With microarray data now available for many conditions, the general utility of a protein abundance predictor is broad. Whereas most quantitative proteomics studies have focused on higher organisms, we developed a predictive model of protein abundance for both Saccharomyces cerevisiae and Schizosaccharomyces pombe to explore the latitude at the protein level. Our predictor primarily relies on mRNA level, mRNA-protein interaction, mRNA folding energy and half-life, and tRNA adaptation. The combination of key features, allowing for the low certainty and uneven coverage of experimental observations, gives comparatively minor but robust prediction accuracy. The model substantially improved the analysis of protein regulation during the cell cycle: predicted protein abundance identified twice as many cell-cycle-associated proteins as experimental mRNA levels. Predicted protein abundance was more dynamic than observed mRNA expression, agreeing with experimental protein abundance from a human cell line. We illustrate how the same model can be used to predict the folding energy of mRNA when protein abundance is available, lending credence to the emerging view that mRNA folding affects translation efficiency

  15. Amino-Acid Network Clique Analysis of Protein Mutation Non-Additive Effects: A Case Study of Lysozme.

    PubMed

    Ming, Dengming; Chen, Rui; Huang, He

    2018-05-10

    Optimizing amino-acid mutations in enzyme design has been a very challenging task in modern bio-industrial applications. It is well known that many successful designs often hinge on extensive correlations among mutations at different sites within the enzyme, however, the underpinning mechanism for these correlations is far from clear. Here, we present a topology-based model to quantitively characterize non-additive effects between mutations. The method is based on the molecular dynamic simulations and the amino-acid network clique analysis. It examines if the two mutation sites of a double-site mutation fall into to a 3-clique structure, and associates such topological property of mutational site spatial distribution with mutation additivity features. We analyzed 13 dual mutations of T4 phage lysozyme and found that the clique-based model successfully distinguishes highly correlated or non-additive double-site mutations from those additive ones whose component mutations have less correlation. We also applied the model to protein Eglin c whose structural topology is significantly different from that of T4 phage lysozyme, and found that the model can, to some extension, still identify non-additive mutations from additive ones. Our calculations showed that mutation non-additive effects may heavily depend on a structural topology relationship between mutation sites, which can be quantitatively determined using amino-acid network k -cliques. We also showed that double-site mutation correlations can be significantly altered by exerting a third mutation, indicating that more detailed physicochemical interactions should be considered along with the network clique-based model for better understanding of this elusive mutation-correlation principle.

  16. Perturbations of Amino Acid Metabolism Associated with Glyphosate-Dependent Inhibition of Shikimic Acid Metabolism Affect Cellular Redox Homeostasis and Alter the Abundance of Proteins Involved in Photosynthesis and Photorespiration1[W][OA

    PubMed Central

    Vivancos, Pedro Diaz; Driscoll, Simon P.; Bulman, Christopher A.; Ying, Liu; Emami, Kaveh; Treumann, Achim; Mauve, Caroline; Noctor, Graham; Foyer, Christine H.

    2011-01-01

    The herbicide glyphosate inhibits the shikimate pathway of the synthesis of amino acids such as phenylalanine, tyrosine, and tryptophan. However, much uncertainty remains concerning precisely how glyphosate kills plants or affects cellular redox homeostasis and related processes in glyphosate-sensitive and glyphosate-resistant crop plants. To address this issue, we performed an integrated study of photosynthesis, leaf proteomes, amino acid profiles, and redox profiles in the glyphosate-sensitive soybean (Glycine max) genotype PAN809 and glyphosate-resistant Roundup Ready Soybean (RRS). RRS leaves accumulated much more glyphosate than the sensitive line but showed relatively few changes in amino acid metabolism. Photosynthesis was unaffected by glyphosate in RRS leaves, but decreased abundance of photosynthesis/photorespiratory pathway proteins was observed together with oxidation of major redox pools. While treatment of a sensitive genotype with glyphosate rapidly inhibited photosynthesis and triggered the appearance of a nitrogen-rich amino acid profile, there was no evidence of oxidation of the redox pools. There was, however, an increase in starvation-associated and defense proteins. We conclude that glyphosate-dependent inhibition of soybean leaf metabolism leads to the induction of defense proteins without sustained oxidation. Conversely, the accumulation of high levels of glyphosate in RRS enhances cellular oxidation, possibly through mechanisms involving stimulation of the photorespiratory pathway. PMID:21757634

  17. Amino acids in the Yamato carbonaceous chrondrite from Antarctica

    NASA Technical Reports Server (NTRS)

    Shimoyama, A.; Ponnamperuma, C.; Yanai, K.

    1979-01-01

    Evidence for the presence of amino acids of extraterrestrial origin in the Antarctic Yamato carbonaceous chrondrite is presented. Hydrolyzed and nonhydrolyzed water-extracted amino acid samples from exterior, middle and interior portions of the meteorite were analyzed by an amino acid analyzer and by gas chromatography of N-TFA-isopropyl amino acid derivatives. Nine protein and six nonprotein amino acids were detected in the meteorite at abundances between 34 and less than one nmole/g, with equal amounts in interior and exterior portions. Nearly equal abundances of the D and L enantiomers of alanine, aspartic acid and glutamic acid were found, indicating the abiotic, therefore extraterrestrial, origin of the amino acids. The Antarctic environment and the uniformity of protein amino acid abundances are discussed as evidence against the racemization of terrestrially acquired amino acids, and similarities between Yamato amino acid compositions and the amino acid compositions of the Murchison and Murray type II carbonaceous chrondrites are indicated.

  18. Extraterrestrial Amino Acids in the Almahata Sitta Meteorite

    NASA Technical Reports Server (NTRS)

    Glavin, Daniel P.; Aubrey, Andrew D.; Callahan, Michael P.; Dworkin, Jason P.; Elsila, Jamie E.; Parker, Eric T.; Bada, Jeffrey L.

    2009-01-01

    Amino acid analysis of a meteorite fragment of asteroid 2008 TC(sub 3) called Almahata Sitta was carried out using reverse-phase high-perfo rmance liquid chromatography coupled with UV fluorescence detection a nd time-of-flight mass spectrometry (HPLC-FD/ToF-MS) as part of a sam ple analysis consortium. HPLC analyses of hot-water extracts from the meteorite revealed a complex distribution of two- to six-carbon aliph atic amino acids and one- to three carbon amines with abundances rang ing from 0.5 to 149 parts-per-billion (ppb). The enantiomeric ratios of the amino acids alanine, Beta-amino-n-butyric acid (Beta-ABA), 2-amino-2- methylbutanoic acid (isovaline), and 2-aminopentanoic acid (no rvaline) in the meteorite were racemic (D/L approximately 1), indicat ing that these amino acids are indigenous to the meteorite and not te rrestrial contaminants. Several other non-protein amino acids were also identified in the meteorite above background levels including alpha -aminoisobutyric acid (alpha-AIB), 4-amino-2- methybutanoic acid, 4-a mino-3-methylbutanoic acid, and 3-, 4-, and 5-aminopentanoic acid. Th e total abundances of isovaline and AlB in Almahata Sitta are approximately 1000 times lower than the abundances of these amino acids found in the CM carbonaceous meteorite Murchison. The extremely love abund ances and unusual distribution of five carbon amino acids in Almahata Sitta compared to Cl, CM, and CR carbonaceous meteorites and may be due to extensive thermal alteration of amino acids on the parent aster oid by partial melting during formation or impact shock heating.

  19. The Amino Acid Composition of the Sutter's Mill Carbonaceous Chondrite

    NASA Technical Reports Server (NTRS)

    Glavin, D. P.; Burton, A. S.; Elsila, J. E.; Dworkin, J. P.; Yin, Q. Z.; Cooper, G.; Jenniskens, P.

    2012-01-01

    In contrast to the Murchison meteorite which had a complex distribution of amino acids with a total C2 to Cs amino acid abundance of approx.14,000 parts-per-billion (ppb) [2], the Sutters Mill meteorite was found to be highly depleted in amino acids. Much lower abundances (approx.30 to 180 ppb) of glycine, beta-alanine, L-alanine and L-serine were detected in SM2 above procedural blank levels indicating that this meteorite sample experienced only minimal terrestrial amino acid contamination after its fall to Earth. Carbon isotope measurements will be necessary to establish the origin of glycine and beta-alanine in SM2. Other non-protein amino acids that are rare on Earth, yet commonly found in other CM meteorites such as aaminoisobutyric acid (alpha-AIB) and isovaline, were not identified in SM2. However, traces of beta-AIB (approx.1 ppb) were detected in SM2 and could be" extraterrestrial in origin. The low abundances of amino acids in the Sutter's Mill meteorite is consistent with mineralogical evidence that at least some parts of the Sutter's Mill meteorite parent body experienced extensive aqueous and/or thermal alteration.

  20. NCAD, a database integrating the intrinsic conformational preferences of non-coded amino acids

    PubMed Central

    Revilla-López, Guillem; Torras, Juan; Curcó, David; Casanovas, Jordi; Calaza, M. Isabel; Zanuy, David; Jiménez, Ana I.; Cativiela, Carlos; Nussinov, Ruth; Grodzinski, Piotr; Alemán, Carlos

    2010-01-01

    Peptides and proteins find an ever-increasing number of applications in the biomedical and materials engineering fields. The use of non-proteinogenic amino acids endowed with diverse physicochemical and structural features opens the possibility to design proteins and peptides with novel properties and functions. Moreover, non-proteinogenic residues are particularly useful to control the three-dimensional arrangement of peptidic chains, which is a crucial issue for most applications. However, information regarding such amino acids –also called non-coded, non-canonical or non-standard– is usually scattered among publications specialized in quite diverse fields as well as in patents. Making all these data useful to the scientific community requires new tools and a framework for their assembly and coherent organization. We have successfully compiled, organized and built a database (NCAD, Non-Coded Amino acids Database) containing information about the intrinsic conformational preferences of non-proteinogenic residues determined by quantum mechanical calculations, as well as bibliographic information about their synthesis, physical and spectroscopic characterization, conformational propensities established experimentally, and applications. The architecture of the database is presented in this work together with the first family of non-coded residues included, namely, α-tetrasubstituted α-amino acids. Furthermore, the NCAD usefulness is demonstrated through a test-case application example. PMID:20455555

  1. Protein Design Using Unnatural Amino Acids

    NASA Astrophysics Data System (ADS)

    Bilgiçer, Basar; Kumar, Krishna

    2003-11-01

    With the increasing availability of whole organism genome sequences, understanding protein structure and function is of capital importance. Recent developments in the methodology of incorporation of unnatural amino acids into proteins allow the exploration of proteins at a very detailed level. Furthermore, de novo design of novel protein structures and function is feasible with unprecedented sophistication. Using examples from the literature, this article describes the available methods for unnatural amino acid incorporation and highlights some recent applications including the design of hyperstable protein folds.

  2. Human baby hair amino acid natural abundance 15N-isotope values are not related to the 15N-isotope values of amino acids in mother's breast milk protein.

    PubMed

    Romek, Katarzyna M; Julien, Maxime; Frasquet-Darrieux, Marine; Tea, Illa; Antheaume, Ingrid; Hankard, Régis; Robins, Richard J

    2013-12-01

    Since exclusively breast-suckled infants obtain their nutrient only from their mother's milk, it might be anticipated that a correlation will exist between the (15)N/(14)N isotope ratios of amino acids of protein of young infants and those supplied by their mother. The work presented here aimed to determine whether amino nitrogen transfer from human milk to infant hair protein synthesized within the first month of life conserves the maternal isotopic signature or whether post-ingestion fractionation dominates the nitrogen isotope spectrum. The study was conducted at 1 month post-birth on 100 mother-infant pairs. Isotope ratios (15)N/(14)N and (13)C/(12)C were measured using isotope ratio measurement by Mass Spectrometry (irm-MS) for whole maternal milk, and infant hair and (15)N/(14)N ratios were also measured by GC-irm-MS for the N-pivaloyl-O-isopropyl esters of amino acids obtained from the hydrolysis of milk and hair proteins. The δ(15)N and δ(13)C (‰) were found to be significantly higher in infant hair than in breast milk (δ(15)N, P < 0.001; δ(13)C, P < 0.001). Furthermore, the δ(15)N (‰) of individual amino acids in infant hair was also significantly higher than that in maternal milk (P < 0.001). By calculation, the observed shift in isotope ratio was shown not to be accounted for by the amino acid composition of hair and milk proteins, indicating that it is not simply due to differences in the composition in the proteins present. Rather, it would appear that each pool-mother and infant-turns over independently, and that fractionation in infant N-metabolism even in the first month of life dominates over the nutrient N-content.

  3. Ontogenetic change in the abundance of mycosporine-like amino acids in non-zooxanthellate coral larvae

    NASA Astrophysics Data System (ADS)

    Yakovleva, Irina M.; Baird, Andrew H.

    2005-11-01

    Although mycosporine-like amino acids (MAAs) have been extensively investigated in reef-building corals, the sources of these MAAs and the process of their interconversion remain a topic of interest. Here we examined ontogenetic change in the abundance of MAAs in planula larvae of the spawning scleractinian coral Goniastrea retiformis in the absence of zooxanthellae and other dietary input. In order to examine the potential contribution of prokaryotes in the synthesis of MAAs in animal tissue, one group of larvae were treated with the antibiotic rifampicin. High concentrations of MAAs (mycosporine-glycine, shinorine, palythinol, asterina-330), were present in the asymbiotic eggs and adults; however, no MAAs were present in the endosymbiotic zooxanthellae. We documented a steady decline in the total MAA concentrations through time in larvae treated with rifampicin; however, in the absence of antibiotic there was a significant increase in the concentration of MAAs, driven by a sharp increase in the abundance of shinorine and palythinol between day 3 and 7. Our results suggest that MAA synthesis and conversion in G. retiformis larvae occurred in the absence of symbiotic zooxanthellae, and indicate a possible contribution of prokaryotes associated with the animal tissue to these processes.

  4. Amino acid composition and biological effects of supplementing broad bean and corn proteins with Nigella sativa (black cumin) cake protein.

    PubMed

    al-Gaby, A M

    1998-10-01

    The biological effects of supplementing broad bean (Vicia faba) or corn (Zea maize) meal protein with black cumin (Nigella sativa) cake protein as well as their amino acid composition were investigated. The percentage of total protein content of Nigella cake was 22.7%. Lysine is existent in abundant amounts in faba meal protein, while leucine is the most abundant in corn meal protein (chemical score = 156) and valine is higher in Nagella cake protein. compared with rats fed sole corn or faba meal protein, substitution of 25% of corn or faba meal protein with Nigella cake protein in the diet remarkably raised the growth rate of rats and resulted in significant higher levels of rat total serum lipids and triglycerides. Also, the supplemented diet caused significant increases in serum total protein and its two fractions albumin and globulin and insignificantly increase the activity of serum phosphatases and transaminases within normal ranges. The supplementation did not have any adverse nutritional effects in the levels of lipid fractions in the serum.

  5. The tip and hidden part of the iceberg: Proteinogenic and non-proteinogenic aliphatic amino acids.

    PubMed

    Fichtner, Maximilian; Voigt, Kerstin; Schuster, Stefan

    2017-01-01

    Amino acids are the essential building blocks of proteins and, therefore, living organisms. While the focus often lies on the canonical or proteinogenic amino acids, there is also a large number of non-canonical amino acids to explore. Some of them are part of toxins or antibiotics in fungi, bacteria or animals (e.g. sponges). Some others operate at the translational level like an "undercover agent". Here we give an overview of natural aliphatic amino acids, up to a side chain length of five carbons, without rings and with an unmodified backbone, and have a closer look on each of them. Some of them are dehydro amino acids with double or even triple bonds. Moreover, we outline mathematical methods for enumerating the complete list of all potential aliphatic amino acids of a given chain length. This should be of interest for synthetic biology. Most non-proteinogenic amino acids are found within fungi, with particularly many produced by Amanita species as defence chemicals. Several are incorporated into peptide antibiotics. Some of the amino acids occur due to broad substrate specificity of the branched-chain amino acid synthesis pathways. A large variety of amino acids were also found in the Murchison meteorite. Non-proteinogenic amino acids are of interest for numerous medical applications: discovery of new antibiotics, support in designing synthetic antibiotics, improvement of protein and peptide pharmaceuticals by avoiding incorporation of non-canonical amino acids, study of toxic cyanobacteria and other applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. The Abundant Class III Chitinase Homolog in Young Developing Banana Fruits Behaves as a Transient Vegetative Storage Protein and Most Probably Serves as an Important Supply of Amino Acids for the Synthesis of Ripening-Associated Proteins1

    PubMed Central

    Peumans, Willy J.; Proost, Paul; Swennen, Rony L.; Van Damme, Els J.M.

    2002-01-01

    Analyses of the protein content and composition revealed dramatic changes in gene expression during in situ banana (Musa spp.) fruit formation/ripening. The total banana protein content rapidly increases during the first 60 to 70 d, but remains constant for the rest of fruit formation/ripening. During the phase of rapid protein accumulation, an inactive homolog of class III chitinases accounts for up to 40% (w/v) of the total protein. Concomitant with the arrest of net protein accumulation, the chitinase-related protein (CRP) progressively decreases and several novel proteins appear in the electropherograms. Hence, CRP behaves as a fruit-specific vegetative storage protein that accumulates during early fruit formation and serves as a source of amino acids for the synthesis of ripening-associated proteins. Analyses of individual proteins revealed that a thaumatin-like protein, a β-1,3-glucanase, a class I chitinase, and a mannose-binding lectin are the most abundant ripening-associated proteins. Because during the ripening of prematurely harvested bananas, similar changes take place as in the in situ ripening bananas, CRP present in immature fruits is a sufficient source of amino acids for a quasi-normal synthesis of ripening-associated proteins. However, it is evident that the conversion of CRP in ripening-associated proteins takes place at an accelerated rate, especially when climacteric ripening is induced by ethylene. The present report also includes a discussion of the accumulation of the major banana allergens and the identification of suitable promoters for the production of vaccines in transgenic bananas. PMID:12376669

  7. Regulation of intestinal protein metabolism by amino acids.

    PubMed

    Bertrand, Julien; Goichon, Alexis; Déchelotte, Pierre; Coëffier, Moïse

    2013-09-01

    Gut homeostasis plays a major role in health and may be regulated by quantitative and qualitative food intake. In the intestinal mucosa, an intense renewal of proteins occurs, at approximately 50% per day in humans. In some pathophysiological conditions, protein turnover is altered and may contribute to intestinal or systemic diseases. Amino acids are key effectors of gut protein turnover, both as constituents of proteins and as regulatory molecules limiting intestinal injury and maintaining intestinal functions. Many studies have focused on two amino acids: glutamine, known as the preferential substrate of rapidly dividing cells, and arginine, another conditionally essential amino acid. The effects of glutamine and arginine on protein synthesis appear to be model and condition dependent, as are the involved signaling pathways. The regulation of gut protein degradation by amino acids has been minimally documented until now. This review will examine recent data, helping to better understand how amino acids regulate intestinal protein metabolism, and will explore perspectives for future studies.

  8. The Effect of Column and Eluent Fluorination on the Retention and Separation of non-Fluorinated Amino Acids and Proteins by HPLC

    PubMed Central

    Joyner, Katherine; Wang, Weizhen; Yu, Yihua Bruce

    2011-01-01

    The effect of column and eluent fluorination on the retention and separation of non-fluorinated amino acids and proteins in HPLC is investigated. A side-by-side comparison of fluorocarbon column and eluents (F-column and F-eluents) with their hydrocarbon counterparts (H-column and H-eluents) in the separation of a group of 33 analytes, including 30 amino acids and 3 proteins, is conducted. The H-column and the F-column contain the n-C8H17 group and n-C8F17 group, respectively, in their stationary phases. The H-eluents include ethanol (EtOH) and isopropanol (ISP) while the F-eluents include trifluoroethanol (TFE) and hexafluorosopropanol (HFIP). The 2 columns and 4 eluents generated 8 (column, eluent) pairs that produce 264 retention time data points for the 33 analytes. A statistical analysis of the retention time data reveals that although the H-column is better than the F-column in analyte separation and H-eluents are better than F-eluents in analyte retention, the more critical factor is the proper pairing of column with eluent. Among the conditions explored in this project, optimal retention and separation is achieved when the fluorocarbon column is paired with ethanol, even though TFE is the most polar one among the 4 eluents. This result shows fluorocarbon columns have much potential in chromatographic analysis and separation of non-fluorinated amino acids and proteins. PMID:21318121

  9. Sensitivity of whole body protein synthesis to amino acid administration during short-term bed rest.

    PubMed

    Biolo, Gianni; Ciocchi, Beniamino; Lebenstedt, Marion; Heer, Martina; Guarnieri, Gianfranco

    2002-07-01

    We tested the hypothesis that a reduced stimulation of whole-body protein synthesis by amino acid administration represents a major mechanism for the bed rest-induced loss of lean body mass. Healthy young subjects and matched controls were studied on the last day of a 14-day bed rest or ambulatory period, as part of the overall protocol "Short-term Bed Rest - Integrated Physiology" set up by the German Aerospace Centre (DLR) in co-operation with the European Space Agency. A balanced mixture of essential and non-essential amino acids was intravenously infused in the postabsorptive state for 3 hours at the rate of 0.1 g/kg/hour. The oxidative and non-oxidative (i.e., to protein synthesis) disposal of the infused leucine was determined by stable isotope and mass spectrometry techniques. The clearance of total infused amino acids tended to be greater (P=0.07) in the ambulatory group than in the bed rest group. When leucine clearance was partitioned between its oxidative and non-oxidative (i.e., to protein synthesis) components, the results indicated that the oxidative disposal was not statistically different in the bed rest and in the ambulatory groups. In contrast, the non-oxidative leucine disposal (i.e., to protein synthesis) was about 20% greater (P<0.01) in the ambulatory group than in the bed rest group. In conclusion, these preliminary data suggest that 14-day bed rest impairs the ability to utilise exogenous amino acids for protein synthesis.

  10. Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence.

    PubMed

    Baumann, Tobias; Schmitt, Franz-Josef; Pelzer, Almut; Spiering, Vivian Jeanette; Freiherr von Sass, Georg Johannes; Friedrich, Thomas; Budisa, Nediljko

    2018-04-27

    Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore's conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced "cyan" variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence

  11. Late Embryogenesis Abundant (LEA) proteins in legumes

    PubMed Central

    Battaglia, Marina; Covarrubias, Alejandra A.

    2013-01-01

    Plants are exposed to different external conditions that affect growth, development, and productivity. Water deficit is one of these adverse conditions caused by drought, salinity, and extreme temperatures. Plants have developed different responses to prevent, ameliorate or repair the damage inflicted by these stressful environments. One of these responses is the activation of a set of genes encoding a group of hydrophilic proteins that typically accumulate to high levels during seed dehydration, at the last stage of embryogenesis, hence named Late Embryogenesis Abundant (LEA) proteins. LEA proteins also accumulate in response to water limitation in vegetative tissues, and have been classified in seven groups based on their amino acid sequence similarity and on the presence of distinctive conserved motifs. These proteins are widely distributed in the plant kingdom, from ferns to angiosperms, suggesting a relevant role in the plant response to this unfavorable environmental condition. In this review, we analyzed the LEA proteins from those legumes whose complete genomes have been sequenced such as Phaseolus vulgaris, Glycine max, Medicago truncatula, Lotus japonicus, Cajanus cajan, and Cicer arietinum. Considering their distinctive motifs, LEA proteins from the different groups were identified, and their sequence analysis allowed the recognition of novel legume specific motifs. Moreover, we compile their transcript accumulation patterns based on publicly available data. In spite of the limited information on these proteins in legumes, the analysis and data compiled here confirm the high correlation between their accumulation and water deficit, reinforcing their functional relevance under this detrimental conditions. PMID:23805145

  12. Early changes in tissue amino acid metabolism and nutrient routing in rats fed a high-fat diet: evidence from natural isotope abundances of nitrogen and carbon in tissue proteins.

    PubMed

    Mantha, Olivier L; Polakof, Sergio; Huneau, Jean-François; Mariotti, François; Poupin, Nathalie; Zalko, Daniel; Fouillet, Helene

    2018-05-01

    Little is known about how diet-induced obesity and insulin resistance affect protein and amino acid (AA) metabolism in tissues. The natural relative abundances of the heavy stable isotopes of C (δ 13C) and N (δ 15N) in tissue proteins offer novel and promising biomarkers of AA metabolism. They, respectively, reflect the use of dietary macronutrients for tissue AA synthesis and the relative metabolic use of tissue AA for oxidation v. protein synthesis. In this study, δ 13C and δ 15N were measured in the proteins of various tissues in young adult rats exposed perinatally and/or fed after weaning with a normal- or a high-fat (HF) diet, the aim being to characterise HF-induced tissue-specific changes in AA metabolism. HF feeding was shown to increase the routing of dietary fat to all tissue proteins via non-indispensable AA synthesis, but did not affect AA allocation between catabolic and anabolic processes in most tissues. However, the proportion of AA directed towards oxidation rather than protein synthesis was increased in the small intestine and decreased in the tibialis anterior muscle and adipose tissue. In adipose tissue, the AA reallocation was observed in the case of perinatal or post-weaning exposure to HF, whereas in the small intestine and tibialis anterior muscle the AA reallocation was only observed after HF exposure that covered both the perinatal and post-weaning periods. In conclusion, HF exposure induced an early reorganisation of AA metabolism involving tissue-specific effects, and in particular a decrease in the relative allocation of AA to oxidation in several peripheral tissues.

  13. Conservation of Shannon's redundancy for proteins. [information theory applied to amino acid sequences

    NASA Technical Reports Server (NTRS)

    Gatlin, L. L.

    1974-01-01

    Concepts of information theory are applied to examine various proteins in terms of their redundancy in natural originators such as animals and plants. The Monte Carlo method is used to derive information parameters for random protein sequences. Real protein sequence parameters are compared with the standard parameters of protein sequences having a specific length. The tendency of a chain to contain some amino acids more frequently than others and the tendency of a chain to contain certain amino acid pairs more frequently than other pairs are used as randomness measures of individual protein sequences. Non-periodic proteins are generally found to have random Shannon redundancies except in cases of constraints due to short chain length and genetic codes. Redundant characteristics of highly periodic proteins are discussed. A degree of periodicity parameter is derived.

  14. The use of amino acid indices for assessing organic matter quality and microbial abundance in deep-sea Antarctic sediments of IODP Expedition 318

    USGS Publications Warehouse

    Carr, Stephanie A; Mills, Christopher T.; Mandernack, Kevin W

    2016-01-01

    The Adélie Basin, located offshore of the Wilkes Land margin, experiences unusually high sedimentation rates (~ 2 cm yr− 1) for the Antarctic coast. This study sought to compare depthwise changes in organic matter (OM) quantity and quality with changes in microbial biomass with depth at this high-deposition site and an offshore continental margin site. Sediments from both sites were collected during the International Ocean Drilling (IODP) Program Expedition 318. Viable microbial biomass was estimated from concentrations of bacterial-derived phospholipid fatty acids, while OM quality was assessed using four different amino acid degradation proxies. Concentrations of total hydrolysable amino acids (THAA) measured from the continental margin suggest an oligotrophic environment, with THAA concentrations representing only 2% of total organic carbon with relative proportions of non-protein amino acids β-alanine and γ-aminobutyric acid as high as 40%. In contrast, THAA concentrations from the near-shore Adélie Basin represent 40%–60% of total organic carbon. Concentrations of β-alanine and γ-aminobutyric acid were often below the detection limit and suggest that the OM of the basin as labile. DI values in surface sediments at the Adélie and margin sites were measured to be + 0.78 and − 0.76, reflecting labile and more recalcitrant OM, respectively. Greater DI values in deeper and more anoxic portions of both cores correlated positively with increased relative concentrations of phenylalanine plus tyrosine and may represent a change of redox conditions, rather than OM quality. This suggests that DI values calculated along chemical profiles should be interpreted with caution. THAA concentrations, the percentage of organic carbon (CAA%) and total nitrogen (NAA%) represented by amino acids at both sites demonstrated a significant positive correlation with bacterial abundance estimates. These data suggest that the selective degradation of amino acids, as

  15. Protein and Essential Amino Acids to Protect Musculoskeletal Health during Spaceflight: Evidence of a Paradox?

    PubMed Central

    Hackney, Kyle J.; English, Kirk L.

    2014-01-01

    Long-duration spaceflight results in muscle atrophy and a loss of bone mineral density. In skeletal muscle tissue, acute exercise and protein (e.g., essential amino acids) stimulate anabolic pathways (e.g., muscle protein synthesis) both independently and synergistically to maintain neutral or positive net muscle protein balance. Protein intake in space is recommended to be 12%–15% of total energy intake (≤1.4 g∙kg−1∙day−1) and spaceflight is associated with reduced energy intake (~20%), which enhances muscle catabolism. Increasing protein intake to 1.5–2.0 g∙kg−1∙day−1 may be beneficial for skeletal muscle tissue and could be accomplished with essential amino acid supplementation. However, increased consumption of sulfur-containing amino acids is associated with increased bone resorption, which creates a dilemma for musculoskeletal countermeasures, whereby optimizing skeletal muscle parameters via essential amino acid supplementation may worsen bone outcomes. To protect both muscle and bone health, future unloading studies should evaluate increased protein intake via non-sulfur containing essential amino acids or leucine in combination with exercise countermeasures and the concomitant influence of reduced energy intake. PMID:25370374

  16. Mammalian target of rapamycin signalling modulates amino acid uptake by regulating transporter cell surface abundance in primary human trophoblast cells.

    PubMed

    Rosario, Fredrick J; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

    2013-02-01

    Abnormal fetal growth increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Emerging evidence suggests that changes in placental amino acid transport directly contribute to altered fetal growth. However, the molecular mechanisms regulating placental amino acid transport are largely unknown. Here we combined small interfering (si) RNA-mediated silencing approaches with protein expression/localization and functional studies in cultured primary human trophoblast cells to test the hypothesis that mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) regulate amino acid transporters by post-translational mechanisms. Silencing raptor (inhibits mTORC1) or rictor (inhibits mTORC2) markedly decreased basal System A and System L amino acid transport activity but had no effect on growth factor-stimulated amino acid uptake. Simultaneous inhibition of mTORC1 and 2 completely inhibited both basal and growth factor-stimulated amino acid transport activity. In contrast, mTOR inhibition had no effect on serotonin transport. mTORC1 or mTORC2 silencing markedly decreased the plasma membrane expression of specific System A (SNAT2, SLC38A2) and System L (LAT1, SLC7A5) transporter isoforms without affecting global protein expression. In conclusion, mTORC1 and mTORC2 regulate human trophoblast amino acid transporters by modulating the cell surface abundance of specific transporter isoforms. This is the first report showing regulation of amino acid transport by mTORC2. Because placental mTOR activity and amino acid transport are decreased in human intrauterine growth restriction our data are consistent with the possibility that dysregulation of placental mTOR plays an important role in the development of abnormal fetal growth.

  17. Conformational landscape of isolated capped amino acids: on the nature of non-covalent interactions*

    NASA Astrophysics Data System (ADS)

    González, Jorge; Martínez, Rodrigo; Fernández, José A.; Millan, Judith

    2017-08-01

    The intramolecular interactions for isolated capped amino acids were investigated computationally by characterizing the conformers for selected amino acids with charged (arginine), polar (asparagine and glutamine), non-polar (alanine, valine and isoleucine), and aromatic (phenylalanine, tryptophan and tyrosine) side chains. The computational method applied combined a molecular mechanics conformational search (with an MMFFs forced field) followed by structural and vibrational density-functional calculations (M06-2X with a triple- ζ Pople's basis set). The intramolecular forces in each amino acid were analyzed with the Non-Covalent Interactions (NCI) analysis. The results for the 15 most stable conformers studied showed that the structure of isolated capped amino acids resembles those found in proteins. In particular, the two most stable conformers of the nine amino acids investigated exhibit γ L and β L conformations with 7- and 5-membered rings, respectively, as a result of the balance between non-covalent interactions (hydrogen bonds and van der Waals).

  18. Hunting for low abundant redox proteins in plant plasma membranes.

    PubMed

    Lüthje, Sabine; Hopff, David; Schmitt, Anna; Meisrimler, Claudia-Nicole; Menckhoff, Ljiljana

    2009-04-13

    Nowadays electron transport (redox) systems in plasma membranes appear well established. Members of the flavocytochrome b family have been identified by their nucleotide acid sequences and characterized on the transcriptional level. For their gene products functions have been demonstrated in iron uptake and oxidative stress including biotic interactions, abiotic stress factors and plant development. In addition, NAD(P)H-dependent oxidoreductases and b-type cytochromes have been purified and characterized from plasma membranes. Several of these proteins seem to belong to the group of hypothetical or unknown proteins. Low abundance and the lack of amino acid sequence data for these proteins still hamper their functional analysis. Consequently, little is known about the physiological function and regulation of these enzymes. In recent years evidence has been presented for the existence of microdomains (so-called lipid rafts) in plasma membranes and their interaction with specific membrane proteins. The identification of redox systems in detergent insoluble membranes supports the idea that redox systems may have important functions in signal transduction, stress responses, cell wall metabolism, and transport processes. This review summarizes our present knowledge on plasma membrane redox proteins and discusses alternative strategies to investigate the function and regulation of these enzymes.

  19. Enigmatic Isovaline: Investigating the Stability, Racemization, and Formation of a Non-biological Meteoritic Amino Acid

    NASA Technical Reports Server (NTRS)

    Hudson, Reggie; Moore, Marla; Lewis, Ariel; Dworkin, Jason

    2008-01-01

    Among the Murchison meteoritic amino acids, isovaline stands out as being both nonbiological (non-protein) and having a relatively high abundance. While approximately equal amounts of D- and L-isovaline have been reported in Murchison and other CM meteorites, the molecule's structure appears to prohibit its racemization in aqueous solutions. We recently have investigated the low-temperature solid-phase chemistry of both isovaline and valine with an eye toward each molecule's formation, stability, and possible interconversions of D and L enantiomers. Ion-irradiated isovaline- and valinecontaining ices were examined by IR spectroscopy and highly-sensitive liquid chromatography/time-of-flight mass spectral methods to assess both amino acid destruction and racemization. Samples were studied in the presence and in the absence of water-ice, and the destruction of both isovaline and valine was measured as a function of radiation dose. In addition, we have undertaken experiments to synthesize isovaline, valine, and their amino acid isomers by solid-phase radiation-chemical pathways other than the oft-invoked Strecker process. This presentation will review and summarize some of our recent findings. -- Our work has been supported by a grant to the Goddard Center for Astrobiology through the NASA Astrobiology Institute. Experiments were performed in the Cosmic Ice Laboratory (RLH, MHM, AL) and the Astrobiology Analytical Laboratory (JPD, DPG) at the NASA Goddard Space Flight Center.

  20. Evidence from Meteorites for Multiple Possible Amino Acid Alphabets for the Origins of Life

    NASA Technical Reports Server (NTRS)

    Burton, A. S.; Elsila, J. E.; Callahan, M. P.; Glavin, D. P.; Dworkin, J. P.

    2015-01-01

    A key question for the origins of life is understanding which amino acids made up the first proteins synthesized during the origins of life. The canonical set of 20 - 22 amino acids used in proteins are all alpha-amino, alpha-hydrogen isomers that, nevertheless, show considerable variability in properties including size, hydrophobicity, and ionizability. Abiotic amino acid synthesis experiments such as Miller-Urey spark discharge reactions produce a set of up to 23 amino acids, depending on starting materials and reaction conditions, with significant abundances of both alpha- and non-alpha-amino acid isomers. These two sets of amino acids do not completely overlap; of the 23 spark discharge amino acids, only 11 are used in modern proteins. Furthermore, because our understanding of conditions on the early Earth are limited, it is unclear which set(s) of conditions employed in spark discharge or hydrothermal reactions are correct, leaving us with significant uncertainty about the amino acid alphabet available for the origins of life on Earth. Meteorites, the surviving remnants of asteroids and comets that fall to the Earth, offer the potential to study authentic samples of naturally-occurring abiotic chemistry, and thus can provide an alternative approach to constraining the amino acid library during the origins of life.

  1. Suppression of muscle protein turnover and amino acid degradation by dietary protein deficiency

    NASA Technical Reports Server (NTRS)

    Tawa, N. E. Jr; Goldberg, A. L.

    1992-01-01

    To define the adaptations that conserve amino acids and muscle protein when dietary protein intake is inadequate, rats (60-70 g final wt) were fed a normal or protein-deficient (PD) diet (18 or 1% lactalbumin), and their muscles were studied in vitro. After 7 days on the PD diet, both protein degradation and synthesis fell 30-40% in skeletal muscles and atria. This fall in proteolysis did not result from reduced amino acid supply to the muscle and preceded any clear decrease in plasma amino acids. Oxidation of branched-chain amino acids, glutamine and alanine synthesis, and uptake of alpha-aminoisobutyrate also fell by 30-50% in muscles and adipose tissue of PD rats. After 1 day on the PD diet, muscle protein synthesis and amino acid uptake decreased by 25-40%, and after 3 days proteolysis and leucine oxidation fell 30-45%. Upon refeeding with the normal diet, protein synthesis also rose more rapidly (+30% by 1 day) than proteolysis, which increased significantly after 3 days (+60%). These different time courses suggest distinct endocrine signals for these responses. The high rate of protein synthesis and low rate of proteolysis during the first 3 days of refeeding a normal diet to PD rats contributes to the rapid weight gain ("catch-up growth") of such animals.

  2. Topology of RNA–protein nucleobase–amino acid π–π interactions and comparison to analogous DNA–protein π–π contacts

    PubMed Central

    Wilson, Katie A.; Holland, Devany J.; Wetmore, Stacey D.

    2016-01-01

    The present work analyzed 120 high-resolution X-ray crystal structures and identified 335 RNA–protein π-interactions (154 nonredundant) between a nucleobase and aromatic (W, H, F, or Y) or acyclic (R, E, or D) π-containing amino acid. Each contact was critically analyzed (including using a visual inspection protocol) to determine the most prevalent composition, structure, and strength of π-interactions at RNA–protein interfaces. These contacts most commonly involve F and U, with U:F interactions comprising one-fifth of the total number of contacts found. Furthermore, the RNA and protein π-systems adopt many different relative orientations, although there is a preference for more parallel (stacked) arrangements. Due to the variation in structure, the strength of the intermolecular forces between the RNA and protein components (as determined from accurate quantum chemical calculations) exhibits a significant range, with most of the contacts providing significant stability to the associated RNA–protein complex (up to −65 kJ mol−1). Comparison to the analogous DNA–protein π-interactions emphasizes differences in RNA– and DNA–protein π-interactions at the molecular level, including the greater abundance of RNA contacts and the involvement of different nucleobase/amino acid residues. Overall, our results provide a clearer picture of the molecular basis of nucleic acid–protein binding and underscore the important role of these contacts in biology, including the significant contribution of π–π interactions to the stability of nucleic acid–protein complexes. Nevertheless, more work is still needed in this area in order to further appreciate the properties and roles of RNA nucleobase–amino acid π-interactions in nature. PMID:26979279

  3. Rewiring protein synthesis: From natural to synthetic amino acids.

    PubMed

    Fan, Yongqiang; Evans, Christopher R; Ling, Jiqiang

    2017-11-01

    The protein synthesis machinery uses 22 natural amino acids as building blocks that faithfully decode the genetic information. Such fidelity is controlled at multiple steps and can be compromised in nature and in the laboratory to rewire protein synthesis with natural and synthetic amino acids. This review summarizes the major quality control mechanisms during protein synthesis, including aminoacyl-tRNA synthetases, elongation factors, and the ribosome. We will discuss evolution and engineering of such components that allow incorporation of natural and synthetic amino acids at positions that deviate from the standard genetic code. The protein synthesis machinery is highly selective, yet not fixed, for the correct amino acids that match the mRNA codons. Ambiguous translation of a codon with multiple amino acids or complete reassignment of a codon with a synthetic amino acid diversifies the proteome. Expanding the genetic code with synthetic amino acids through rewiring protein synthesis has broad applications in synthetic biology and chemical biology. Biochemical, structural, and genetic studies of the translational quality control mechanisms are not only crucial to understand the physiological role of translational fidelity and evolution of the genetic code, but also enable us to better design biological parts to expand the proteomes of synthetic organisms. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Regulation of protein synthesis by amino acids in muscle of neonates

    PubMed Central

    Suryawan, Agus; Davis, Teresa A.

    2011-01-01

    The marked increase in skeletal muscle mass during the neonatal period is largely due to a high rate of postprandial protein synthesis that is modulated by an enhanced sensitivity to insulin and amino acids. The amino acid signaling pathway leading to the stimulation of protein synthesis has not been fully elucidated. Among the amino acids, leucine is considered to be a principal anabolic agent that regulates protein synthesis. mTORC1, which controls protein synthesis, has been implicated as a target for leucine. Until recently, there have been few studies exploring the role of amino acids in enhancing muscle protein synthesis in vivo. In this review, we discuss amino acid-induced protein synthesis in muscle in the neonate, focusing on current knowledge of the role of amino acids in the activation of mTORC1 leading to mRNA translation. The role of the amino acid transporters, SNAT2, LAT1, and PAT, in the modulation of mTORC1 activation and the role of amino acids in the activation of putative regulators of mTORC1, i.e., raptor, Rheb, MAP4K3, Vps34, and Rag GTPases, are discussed. PMID:21196241

  5. A method for analysing small samples of floral pollen for free and protein-bound amino acids.

    PubMed

    Stabler, Daniel; Power, Eileen F; Borland, Anne M; Barnes, Jeremy D; Wright, Geraldine A

    2018-02-01

    Pollen provides floral visitors with essential nutrients including proteins, lipids, vitamins and minerals. As an important nutrient resource for pollinators, including honeybees and bumblebees, pollen quality is of growing interest in assessing available nutrition to foraging bees. To date, quantifying the protein-bound amino acids in pollen has been difficult and methods rely on large amounts of pollen, typically more than 1 g. More usual is to estimate a crude protein value based on the nitrogen content of pollen, however, such methods provide no information on the distribution of essential and non-essential amino acids constituting the proteins.Here, we describe a method of microwave-assisted acid hydrolysis using low amounts of pollen that allows exploration of amino acid composition, quantified using ultra high performance liquid chromatography (UHPLC), and a back calculation to estimate the crude protein content of pollen.Reliable analysis of protein-bound and free amino acids as well as an estimation of crude protein concentration was obtained from pollen samples as low as 1 mg. Greater variation in both protein-bound and free amino acids was found in pollen sample sizes <1 mg. Due to the variability in recovery of amino acids in smaller sample sizes, we suggest a correction factor to apply to specific sample sizes of pollen in order to estimate total crude protein content.The method described in this paper will allow researchers to explore the composition of amino acids in pollen and will aid research assessing the available nutrition to pollinating animals. This method will be particularly useful in assaying the pollen of wild plants, from which it is difficult to obtain large sample weights.

  6. Monitoring Replication Protein A (RPA) dynamics in homologous recombination through site-specific incorporation of non-canonical amino acids.

    PubMed

    Pokhrel, Nilisha; Origanti, Sofia; Davenport, Eric Parker; Gandhi, Disha; Kaniecki, Kyle; Mehl, Ryan A; Greene, Eric C; Dockendorff, Chris; Antony, Edwin

    2017-09-19

    An essential coordinator of all DNA metabolic processes is Replication Protein A (RPA). RPA orchestrates these processes by binding to single-stranded DNA (ssDNA) and interacting with several other DNA binding proteins. Determining the real-time kinetics of single players such as RPA in the presence of multiple DNA processors to better understand the associated mechanistic events is technically challenging. To overcome this hurdle, we utilized non-canonical amino acids and bio-orthogonal chemistry to site-specifically incorporate a chemical fluorophore onto a single subunit of heterotrimeric RPA. Upon binding to ssDNA, this fluorescent RPA (RPAf) generates a quantifiable change in fluorescence, thus serving as a reporter of its dynamics on DNA in the presence of multiple other DNA binding proteins. Using RPAf, we describe the kinetics of facilitated self-exchange and exchange by Rad51 and mediator proteins during various stages in homologous recombination. RPAf is widely applicable to investigate its mechanism of action in processes such as DNA replication, repair and telomere maintenance. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Monitoring Replication Protein A (RPA) dynamics in homologous recombination through site-specific incorporation of non-canonical amino acids

    PubMed Central

    Pokhrel, Nilisha; Origanti, Sofia; Davenport, Eric Parker; Gandhi, Disha; Kaniecki, Kyle; Mehl, Ryan A.; Greene, Eric C.; Dockendorff, Chris

    2017-01-01

    Abstract An essential coordinator of all DNA metabolic processes is Replication Protein A (RPA). RPA orchestrates these processes by binding to single-stranded DNA (ssDNA) and interacting with several other DNA binding proteins. Determining the real-time kinetics of single players such as RPA in the presence of multiple DNA processors to better understand the associated mechanistic events is technically challenging. To overcome this hurdle, we utilized non-canonical amino acids and bio-orthogonal chemistry to site-specifically incorporate a chemical fluorophore onto a single subunit of heterotrimeric RPA. Upon binding to ssDNA, this fluorescent RPA (RPAf) generates a quantifiable change in fluorescence, thus serving as a reporter of its dynamics on DNA in the presence of multiple other DNA binding proteins. Using RPAf, we describe the kinetics of facilitated self-exchange and exchange by Rad51 and mediator proteins during various stages in homologous recombination. RPAf is widely applicable to investigate its mechanism of action in processes such as DNA replication, repair and telomere maintenance. PMID:28934470

  8. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

    2011-03-22

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  9. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

    2008-10-07

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  10. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

    2011-12-06

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  11. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

    2012-02-14

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  12. Covariation of Peptide Abundances Accurately Reflects Protein Concentration Differences*

    PubMed Central

    Pirmoradian, Mohammad

    2017-01-01

    Most implementations of mass spectrometry-based proteomics involve enzymatic digestion of proteins, expanding the analysis to multiple proteolytic peptides for each protein. Currently, there is no consensus of how to summarize peptides' abundances to protein concentrations, and such efforts are complicated by the fact that error control normally is applied to the identification process, and do not directly control errors linking peptide abundance measures to protein concentration. Peptides resulting from suboptimal digestion or being partially modified are not representative of the protein concentration. Without a mechanism to remove such unrepresentative peptides, their abundance adversely impacts the estimation of their protein's concentration. Here, we present a relative quantification approach, Diffacto, that applies factor analysis to extract the covariation of peptides' abundances. The method enables a weighted geometrical average summarization and automatic elimination of incoherent peptides. We demonstrate, based on a set of controlled label-free experiments using standard mixtures of proteins, that the covariation structure extracted by the factor analysis accurately reflects protein concentrations. In the 1% peptide-spectrum match-level FDR data set, as many as 11% of the peptides have abundance differences incoherent with the other peptides attributed to the same protein. If not controlled, such contradicting peptide abundance have a severe impact on protein quantifications. When adding the quantities of each protein's three most abundant peptides, we note as many as 14% of the proteins being estimated as having a negative correlation with their actual concentration differences between samples. Diffacto reduced the amount of such obviously incorrectly quantified proteins to 1.6%. Furthermore, by analyzing clinical data sets from two breast cancer studies, our method revealed the persistent proteomic signatures linked to three subtypes of breast cancer

  13. A Dominant Conformational Role for Amino Acid Diversity in Minimalist Protein-Protein Interfaces

    PubMed Central

    Gilbreth, Ryan N.; Esaki, Kaori; Koide, Akiko; Sidhu, Sachdev S.; Koide, Shohei

    2008-01-01

    Recent studies have shown that highly simplified interaction surfaces consisting of combinations of just two amino acids, Tyr and Ser, exhibit high affinity and specificity. The high functional levels of such minimalist interfaces might thus indicate small contributions of greater amino acid diversity seen in natural interfaces. Toward addressing this issue, we have produced a pair of binding proteins built on the fibronectin type III scaffold, termed “monobodies”. One monobody contains the Tyr/Ser binary-code interface (termed YS) and the other contains an expanded amino acid diversity interface (YSX), but both bind to an identical target, maltose binding protein (MBP). The YSX monobody bound with higher affinity, a slower off rate and a more favorable enthalpic contribution than the YS monobody. High-resolution x-ray crystal structures revealed that both proteins bound to an essentially identical epitope, providing a unique opportunity to directly investigate the role of amino acid diversity in a protein interaction interface. Surprisingly, Tyr still dominates the YSX paratope and the additional amino acid types are primarily used to conformationally optimize contacts made by tyrosines. Scanning mutagenesis showed that while all contacting Tyr side-chains are essential in the YS monobody, the YSX interface was more tolerant to mutations. These results suggest that the conformational, not chemical, diversity of additional types of amino acids provided higher functionality and evolutionary robustness, supporting the dominant role of Tyr and the importance of conformational diversity in forming protein interaction interfaces. PMID:18602117

  14. Snake venoms are integrated systems, but abundant venom proteins evolve more rapidly.

    PubMed

    Aird, Steven D; Aggarwal, Shikha; Villar-Briones, Alejandro; Tin, Mandy Man-Ying; Terada, Kouki; Mikheyev, Alexander S

    2015-08-28

    While many studies have shown that extracellular proteins evolve rapidly, how selection acts on them remains poorly understood. We used snake venoms to understand the interaction between ecology, expression level, and evolutionary rate in secreted protein systems. Venomous snakes employ well-integrated systems of proteins and organic constituents to immobilize prey. Venoms are generally optimized to subdue preferred prey more effectively than non-prey, and many venom protein families manifest positive selection and rapid gene family diversification. Although previous studies have illuminated how individual venom protein families evolve, how selection acts on venoms as integrated systems, is unknown. Using next-generation transcriptome sequencing and mass spectrometry, we examined microevolution in two pitvipers, allopatrically separated for at least 1.6 million years, and their hybrids. Transcriptomes of parental species had generally similar compositions in regard to protein families, but for a given protein family, the homologs present and concentrations thereof sometimes differed dramatically. For instance, a phospholipase A2 transcript comprising 73.4 % of the Protobothrops elegans transcriptome, was barely present in the P. flavoviridis transcriptome (<0.05 %). Hybrids produced most proteins found in both parental venoms. Protein evolutionary rates were positively correlated with transcriptomic and proteomic abundances, and the most abundant proteins showed positive selection. This pattern holds with the addition of four other published crotaline transcriptomes, from two more genera, and also for the recently published king cobra genome, suggesting that rapid evolution of abundant proteins may be generally true for snake venoms. Looking more broadly at Protobothrops, we show that rapid evolution of the most abundant components is due to positive selection, suggesting an interplay between abundance and adaptation. Given log-scale differences in toxin

  15. Use of conserved key amino acid positions to morph protein folds.

    PubMed

    Reddy, Boojala V B; Li, Wilfred W; Bourne, Philip E

    2002-07-15

    By using three-dimensional (3D) structure alignments and a previously published method to determine Conserved Key Amino Acid Positions (CKAAPs) we propose a theoretical method to design mutations that can be used to morph the protein folds. The original Paracelsus challenge, met by several groups, called for the engineering of a stable but different structure by modifying less than 50% of the amino acid residues. We have used the sequences from the Protein Data Bank (PDB) identifiers 1ROP, and 2CRO, which were previously used in the Paracelsus challenge by those groups, and suggest mutation to CKAAPs to morph the protein fold. The total number of mutations suggested is less than 40% of the starting sequence theoretically improving the challenge results. From secondary structure prediction experiments of the proposed mutant sequence structures, we observe that each of the suggested mutant protein sequences likely folds to a different, non-native potentially stable target structure. These results are an early indicator that analyses using structure alignments leading to CKAAPs of a given structure are of value in protein engineering experiments. Copyright 2002 Wiley Periodicals, Inc.

  16. Comparative analysis of barophily-related amino acid content in protein domains of Pyrococcus abyssi and Pyrococcus furiosus.

    PubMed

    Yafremava, Liudmila S; Di Giulio, Massimo; Caetano-Anollés, Gustavo

    2013-01-01

    Amino acid substitution patterns between the nonbarophilic Pyrococcus furiosus and its barophilic relative P. abyssi confirm that hydrostatic pressure asymmetry indices reflect the extent to which amino acids are preferred by barophilic archaeal organisms. Substitution patterns in entire protein sequences, shared protein domains defined at fold superfamily level, domains in homologous sequence pairs, and domains of very ancient and very recent origin now provide further clues about the environment that led to the genetic code and diversified life. The pyrococcal proteomes are very similar and share a very early ancestor. Relative amino acid abundance analyses showed that biases in the use of amino acids are due to their shared fold superfamilies. Within these repertoires, only two of the five amino acids that are preferentially barophilic, aspartic acid and arginine, displayed this preference significantly and consistently across structure and in domains appearing in the ancestor. The more primordial asparagine, lysine and threonine displayed a consistent preference for nonbarophily across structure and in the ancestor. Since barophilic preferences are already evident in ancient domains that are at least ~3 billion year old, we conclude that barophily is a very ancient trait that unfolded concurrently with genetic idiosyncrasies in convergence towards a universal code.

  17. Parsing the life-shortening effects of dietary protein: effects of individual amino acids

    PubMed Central

    Bouchebti, Sofia; Bazazi, Sepideh; Le Hesran, Sophie; Puga, Camille; Latil, Gérard; Simpson, Stephen J.

    2017-01-01

    High-protein diets shorten lifespan in many organisms. Is it because protein digestion is energetically costly or because the final products (the amino acids) are harmful? To answer this question while circumventing the life-history trade-off between reproduction and longevity, we fed sterile ant workers on diets based on whole proteins or free amino acids. We found that (i) free amino acids shortened lifespan even more than proteins; (ii) the higher the amino acid-to-carbohydrate ratio, the shorter ants lived and the lower their lipid reserves; (iii) for the same amino acid-to-carbohydrate ratio, ants eating free amino acids had more lipid reserves than those eating whole proteins; and (iv) on whole protein diets, ants seem to regulate food intake by prioritizing sugar, while on free amino acid diets, they seem to prioritize amino acids. To test the effect of the amino acid profile, we tested diets containing proportions of each amino acid that matched the ant's exome; surprisingly, longevity was unaffected by this change. We further tested diets with all amino acids under-represented except one, finding that methionine, serine, threonine and phenylalanine are especially harmful. All together, our results show certain amino acids are key elements behind the high-protein diet reduction in lifespan. PMID:28053059

  18. Parsing the life-shortening effects of dietary protein: effects of individual amino acids.

    PubMed

    Arganda, Sara; Bouchebti, Sofia; Bazazi, Sepideh; Le Hesran, Sophie; Puga, Camille; Latil, Gérard; Simpson, Stephen J; Dussutour, Audrey

    2017-01-11

    High-protein diets shorten lifespan in many organisms. Is it because protein digestion is energetically costly or because the final products (the amino acids) are harmful? To answer this question while circumventing the life-history trade-off between reproduction and longevity, we fed sterile ant workers on diets based on whole proteins or free amino acids. We found that (i) free amino acids shortened lifespan even more than proteins; (ii) the higher the amino acid-to-carbohydrate ratio, the shorter ants lived and the lower their lipid reserves; (iii) for the same amino acid-to-carbohydrate ratio, ants eating free amino acids had more lipid reserves than those eating whole proteins; and (iv) on whole protein diets, ants seem to regulate food intake by prioritizing sugar, while on free amino acid diets, they seem to prioritize amino acids. To test the effect of the amino acid profile, we tested diets containing proportions of each amino acid that matched the ant's exome; surprisingly, longevity was unaffected by this change. We further tested diets with all amino acids under-represented except one, finding that methionine, serine, threonine and phenylalanine are especially harmful. All together, our results show certain amino acids are key elements behind the high-protein diet reduction in lifespan. © 2017 The Author(s).

  19. Amino acid homeostasis and signalling in mammalian cells and organisms

    PubMed Central

    Bröer, Angelika

    2017-01-01

    Cells have a constant turnover of proteins that recycle most amino acids over time. Net loss is mainly due to amino acid oxidation. Homeostasis is achieved through exchange of essential amino acids with non-essential amino acids and the transfer of amino groups from oxidised amino acids to amino acid biosynthesis. This homeostatic condition is maintained through an active mTORC1 complex. Under amino acid depletion, mTORC1 is inactivated. This increases the breakdown of cellular proteins through autophagy and reduces protein biosynthesis. The general control non-derepressable 2/ATF4 pathway may be activated in addition, resulting in transcription of genes involved in amino acid transport and biosynthesis of non-essential amino acids. Metabolism is autoregulated to minimise oxidation of amino acids. Systemic amino acid levels are also tightly regulated. Food intake briefly increases plasma amino acid levels, which stimulates insulin release and mTOR-dependent protein synthesis in muscle. Excess amino acids are oxidised, resulting in increased urea production. Short-term fasting does not result in depletion of plasma amino acids due to reduced protein synthesis and the onset of autophagy. Owing to the fact that half of all amino acids are essential, reduction in protein synthesis and amino acid oxidation are the only two measures to reduce amino acid demand. Long-term malnutrition causes depletion of plasma amino acids. The CNS appears to generate a protein-specific response upon amino acid depletion, resulting in avoidance of an inadequate diet. High protein levels, in contrast, contribute together with other nutrients to a reduction in food intake. PMID:28546457

  20. Modulating protein adsorption onto hydroxyapatite particles using different amino acid treatments

    PubMed Central

    Lee, Wing-Hin; Loo, Ching-Yee; Van, Kim Linh; Zavgorodniy, Alexander V.; Rohanizadeh, Ramin

    2012-01-01

    Hydroxyapatite (HA) is a material of choice for bone grafts owing to its chemical and structural similarities to the mineral phase of hard tissues. The combination of osteogenic proteins with HA materials that carry and deliver the proteins to the bone-defective areas will accelerate bone regeneration. The study investigated the treatment of HA particles with different amino acids such as serine (Ser), asparagine (Asn), aspartic acid (Asp) and arginine (Arg) to enhance the adsorption ability of HA carrier for delivering therapeutic proteins to the body. The crystallinity of HA reduced when amino acids were added during HA preparation. Depending on the types of amino acid, the specific surface area of the amino acid-functionalized HA particles varied from 105 to 149 m2 g–1. Bovine serum albumin (BSA) and lysozyme were used as model proteins for adsorption study. The protein adsorption onto the surface of amino acid-functionalized HA depended on the polarities of HA particles, whereby, compared with lysozyme, BSA demonstrated higher affinity towards positively charged Arg-HA. Alternatively, the binding affinity of lysozyme onto the negatively charged Asp-HA was higher when compared with BSA. The BSA and lysozyme adsorptions onto the amino acid-functionalized HA fitted better into the Freundlich than Langmuir model. The amino acid-functionalized HA particles that had higher protein adsorption demonstrated a lower protein-release rate. PMID:21957116

  1. Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance.

    PubMed

    Morimoto, Shimpei; Yahara, Koji

    2018-03-01

    Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes ( ADC17 and KIN1 ) that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning) that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after accounting for potential

  2. A reduced amino acid alphabet for understanding and designing protein adaptation to mutation.

    PubMed

    Etchebest, C; Benros, C; Bornot, A; Camproux, A-C; de Brevern, A G

    2007-11-01

    Protein sequence world is considerably larger than structure world. In consequence, numerous non-related sequences may adopt similar 3D folds and different kinds of amino acids may thus be found in similar 3D structures. By grouping together the 20 amino acids into a smaller number of representative residues with similar features, sequence world simplification may be achieved. This clustering hence defines a reduced amino acid alphabet (reduced AAA). Numerous works have shown that protein 3D structures are composed of a limited number of building blocks, defining a structural alphabet. We previously identified such an alphabet composed of 16 representative structural motifs (5-residues length) called Protein Blocks (PBs). This alphabet permits to translate the structure (3D) in sequence of PBs (1D). Based on these two concepts, reduced AAA and PBs, we analyzed the distributions of the different kinds of amino acids and their equivalences in the structural context. Different reduced sets were considered. Recurrent amino acid associations were found in all the local structures while other were specific of some local structures (PBs) (e.g Cysteine, Histidine, Threonine and Serine for the alpha-helix Ncap). Some similar associations are found in other reduced AAAs, e.g Ile with Val, or hydrophobic aromatic residues Trp with Phe and Tyr. We put into evidence interesting alternative associations. This highlights the dependence on the information considered (sequence or structure). This approach, equivalent to a substitution matrix, could be useful for designing protein sequence with different features (for instance adaptation to environment) while preserving mainly the 3D fold.

  3. Carbonyl-based blue autofluorescence of proteins and amino acids

    PubMed Central

    Niyangoda, Chamani; Miti, Tatiana; Breydo, Leonid; Uversky, Vladimir

    2017-01-01

    Intrinsic protein fluorescence is inextricably linked to the near-UV autofluorescence of aromatic amino acids. Here we show that a novel deep-blue autofluorescence (dbAF), previously thought to emerge as a result of protein aggregation, is present at the level of monomeric proteins and even poly- and single amino acids. Just as its aggregation-related counterpart, this autofluorescence does not depend on aromatic residues, can be excited at the long wavelength edge of the UV and emits in the deep blue. Differences in dbAF excitation and emission peaks and intensities from proteins and single amino acids upon changes in solution conditions suggest dbAF’s sensitivity to both the chemical identity and solution environment of amino acids. Autofluorescence comparable to dbAF is emitted by carbonyl-containing organic solvents, but not those lacking the carbonyl group. This implicates the carbonyl double bonds as the likely source for the autofluorescence in all these compounds. Using beta-lactoglobulin and proline, we have measured the molar extinction coefficients and quantum yields for dbAF in the monomeric state. To establish its potential utility in monitoring protein biophysics, we show that dbAF emission undergoes a red-shift comparable in magnitude to tryptophan upon thermal denaturation of lysozyme, and that it is sensitive to quenching by acrylamide. Carbonyl dbAF therefore provides a previously neglected intrinsic optical probe for investigating the structure and dynamics of amino acids, proteins and, by extension, DNA and RNA. PMID:28542206

  4. Elevated tropospheric ozone increased grain protein and amino acid content of a hybrid rice without manipulation by planting density.

    PubMed

    Zhou, Xiaodong; Zhou, Juan; Wang, Yunxia; Peng, Bin; Zhu, Jianguo; Yang, Lianxin; Wang, Yulong

    2015-01-01

    Rising tropospheric ozone affects crop yield and quality. Rice protein concentration, which is closely associated with eating/cooking quality, is of critical importance to nutritional quality. The ozone effect on amino acids of rice grains was little known, especially grown under different cultivation conditions. A hybrid rice cultivar Shanyou 63 was grown in 2010 and 2011 to investigate the interactive effect of ozone exposure and planting density on rice protein quality in a free-air ozone enrichment system. The content of protein, total amino acids (TAA), total essential (TEAA) and non-essential amino acids (TNEAA) in rice grain was increased by 12-14% with elevated ozone. A similar significant response to ozone was observed for concentrations of the seven essential and eight non-essential amino acids. In contrast, elevated ozone caused a small but significant decrease in percentage of TEAA to TAA. The year effect was significant for all measured traits; however, interactions of ozone with year or planting density were not detected. The study suggested that season-long elevation of ozone concentration to projected 2050 levels will increase protein and amino acids of Shanyou 63, and crop management such as changing planting density might not alter the impact. © 2014 Society of Chemical Industry.

  5. DIRProt: a computational approach for discriminating insecticide resistant proteins from non-resistant proteins.

    PubMed

    Meher, Prabina Kumar; Sahu, Tanmaya Kumar; Banchariya, Anjali; Rao, Atmakuri Ramakrishna

    2017-03-24

    Insecticide resistance is a major challenge for the control program of insect pests in the fields of crop protection, human and animal health etc. Resistance to different insecticides is conferred by the proteins encoded from certain class of genes of the insects. To distinguish the insecticide resistant proteins from non-resistant proteins, no computational tool is available till date. Thus, development of such a computational tool will be helpful in predicting the insecticide resistant proteins, which can be targeted for developing appropriate insecticides. Five different sets of feature viz., amino acid composition (AAC), di-peptide composition (DPC), pseudo amino acid composition (PAAC), composition-transition-distribution (CTD) and auto-correlation function (ACF) were used to map the protein sequences into numeric feature vectors. The encoded numeric vectors were then used as input in support vector machine (SVM) for classification of insecticide resistant and non-resistant proteins. Higher accuracies were obtained under RBF kernel than that of other kernels. Further, accuracies were observed to be higher for DPC feature set as compared to others. The proposed approach achieved an overall accuracy of >90% in discriminating resistant from non-resistant proteins. Further, the two classes of resistant proteins i.e., detoxification-based and target-based were discriminated from non-resistant proteins with >95% accuracy. Besides, >95% accuracy was also observed for discrimination of proteins involved in detoxification- and target-based resistance mechanisms. The proposed approach not only outperformed Blastp, PSI-Blast and Delta-Blast algorithms, but also achieved >92% accuracy while assessed using an independent dataset of 75 insecticide resistant proteins. This paper presents the first computational approach for discriminating the insecticide resistant proteins from non-resistant proteins. Based on the proposed approach, an online prediction server DIRProt has

  6. Amino acids of Diclidophora merlangi (Monogenea).

    PubMed

    Arme, C; Whyte, A

    1975-02-01

    The level of free amino acids in Diclidophora merlangi is high, comprising over 500 mu moles/g ethanol extracted dry weight. A single amino acid, proline, constitutes some 70% of the total pool. Analysis of parasite protein and host blood and mucus revealed low proline levels, suggesting that the high free pool content was not related to a requirement for protein systhesis or to its abundance in the diet of the worm. Experiments revealed that proline was not involved specifically in osmoregulation, and the reasons for the large amounts present in Diclidophora remain unknown.

  7. Exploiting Amino Acid Composition for Predicting Protein-Protein Interactions

    PubMed Central

    Roy, Sushmita; Martinez, Diego; Platero, Harriett; Lane, Terran; Werner-Washburne, Margaret

    2009-01-01

    Background Computational prediction of protein interactions typically use protein domains as classifier features because they capture conserved information of interaction surfaces. However, approaches relying on domains as features cannot be applied to proteins without any domain information. In this paper, we explore the contribution of pure amino acid composition (AAC) for protein interaction prediction. This simple feature, which is based on normalized counts of single or pairs of amino acids, is applicable to proteins from any sequenced organism and can be used to compensate for the lack of domain information. Results AAC performed at par with protein interaction prediction based on domains on three yeast protein interaction datasets. Similar behavior was obtained using different classifiers, indicating that our results are a function of features and not of classifiers. In addition to yeast datasets, AAC performed comparably on worm and fly datasets. Prediction of interactions for the entire yeast proteome identified a large number of novel interactions, the majority of which co-localized or participated in the same processes. Our high confidence interaction network included both well-studied and uncharacterized proteins. Proteins with known function were involved in actin assembly and cell budding. Uncharacterized proteins interacted with proteins involved in reproduction and cell budding, thus providing putative biological roles for the uncharacterized proteins. Conclusion AAC is a simple, yet powerful feature for predicting protein interactions, and can be used alone or in conjunction with protein domains to predict new and validate existing interactions. More importantly, AAC alone performs at par with existing, but more complex, features indicating the presence of sequence-level information that is predictive of interaction, but which is not necessarily restricted to domains. PMID:19936254

  8. Dynamics of amino acid utilization in Phaeobacter inhibens DSM 17395.

    PubMed

    Zech, Hajo; Hensler, Michael; Koßmehl, Sebastian; Drüppel, Katharina; Wöhlbrand, Lars; Trautwein, Kathleen; Colby, Thomas; Schmidt, Jürgen; Reinhardt, Richard; Schmidt-Hohagen, Kerstin; Schomburg, Dietmar; Rabus, Ralf

    2013-10-01

    Time-resolved utilization of multiple amino acids by Phaeobacter inhibens DSM 17395 was studied during growth with casamino acids. The 15 detected amino acids could be grouped according to depletion rate into four different categories, i.e. from rapid (category I) to nondepletion (category IV). Upon entry into stationary growth phase, amino acids of category I (e.g. glutamate) were (almost) completely depleted, while those of categories II (e.g. leucine) and III (e.g. serine) were further consumed at varying rates and to different extents. Thus, cultures entered stationary growth phase despite the ample presence of organic nutrients, i.e. under nonlimiting conditions. Integrated proteomic and metabolomic analysis identified 1747 proteins and 94 intracellular metabolites. Of these, 180 proteins and 86 metabolites displayed altered abundance levels during growth. Most strikingly, abundance and activity profiles of alanine dehydrogenase concomitantly increased with the onset of enhanced alanine utilization during transition into stationary growth phase. Most enzymes of amino acid and central metabolism, however, displayed unaltered abundances across exponential and stationary growth phases. In contrast, metabolites of the Entner-Doudoroff pathway and gluconeogenesis as well as cellular fatty acids increased markedly in abundance in early stationary growth phase. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Distribution of stable free radicals among amino acids of isolated soy proteins.

    PubMed

    Lei, Qingxin; Liebold, Christopher M; Boatright, William L; Shah Jahan, M

    2010-09-01

    Application of deuterium sulfide to powdered isolated soy proteins (ISP) was used to quench stable free radicals and produce a single deuterium label on amino acids where free radicals reside. The deuterium labels rendered increases of isotope ratio for the specific ions of radical-bearing amino acids. Isotope ratio measurements were achieved by gas chromatography/mass spectrometry (GC/MS) analyses after the amino acids were released by acidic hydrolysis and converted to volatile derivatives with propyl chloroformate. The isotope enrichment data showed the stable free radicals were located on Ala, Gly, Leu, Ile, Asx (Asp+Asn), Glx (Glu+Gln), and Trp but not on Val, Pro, Met, Phe, Lys, and His. Due to the low abundance of Ser, Thr, and Cys derivatives and the impossibility to accurately measure their isotope ratios, the radical bearing status for these amino acids remained undetermined even though their derivatives were positively identified from ISP hydrolysates. The relative isotope enrichment for radical-bearing amino acids Ala, Gly, Leu, Ile, Asx (Asp+Asn), Glx (Glu+Gln), and Trp were 8.67%, 2.96%, 2.90%, 3.94%, 6.03%, 3.91%, and 21.48%, respectively. Isotope ratio increase for Tyr was also observed but further investigation revealed such increase was mainly from nonspecific deuterium-hydrogen exchange not free radical quenching. The results obtained from the present study provide important information for a better understanding of the mechanisms of free radical formation and stabilization in "dry" ISP.

  10. Position-specific incorporation of fluorescent non-natural amino acids into maltose-binding protein for detection of ligand binding by FRET and fluorescence quenching.

    PubMed

    Iijima, Issei; Hohsaka, Takahiro

    2009-04-17

    Position-specific incorporation of fluorescent groups is a useful method for analysis of the functions and structures of proteins. We have developed a method for the incorporation of visible-wavelength-fluorescent non-natural amino acids into proteins in a cell-free translation system. Using this technique, we introduced one or two BODIPY-linked amino acids into maltose-binding protein (MBP) to obtain MBP derivatives showing ligand-dependent changes in fluorescence intensity or intensity ratio. BODIPY-FL-aminophenylalanine was incorporated in place of 15 tyrosines, as well as the N-terminal Lys1, and the C-terminal Lys370 of MBP. Fluorescence measurements revealed that MBP containing a BODIPY-FL moiety in place of Tyr210 showed a 13-fold increase in fluorescence upon binding of maltose. Tryptophan-to-phenylalanine substitutions suggest that the increase in fluorescence was the result of a decrease in the quenching of BODIPY-FL by tryptophan located around the binding site. MBP containing a BODIPY-558 moiety also showed a maltose-dependent increase in fluorescence. BODIPY-FL was then additionally incorporated in place of Lys1 of the BODIPY-558-containing MBP as a response to the amber codon. Fluorescence measurements with excitation of BODIPY-FL showed a large change in fluorescence intensity ratio (0.13 to 1.25) upon binding of maltose; this change can be attributed to fluorescence resonance energy transfer (FRET) and maltose-dependent quenching of BODIPY-558. These results demonstrate the usefulness of the position-specific incorporation of fluorescent amino acids in the fluorescence-based detection of protein functions.

  11. Proximate composition, fatty acid analysis and protein digestibility-corrected amino acid score of three Mediterranean cephalopods.

    PubMed

    Zlatanos, Spiros; Laskaridis, Kostas; Feist, Christian; Sagredos, Angelos

    2006-10-01

    Proximate composition, fatty acid analysis and protein digestibility-corrected amino acid score (PDCAAS) in three commercially important cephalopods of the Mediterranean sea (cuttlefish, octopus and squid) were determined. The results of the proximate analysis showed that these species had very high protein:fat ratios similar to lean beef. Docosahexaenoic, palmitic and eicosipentaenoic acid were the most abundant fatty acids among analyzed species. The amount of n-3 fatty acids was higher than that of saturated, monounsaturated and n-6 fatty acids. Despite the fact that cephalopods contain small amounts of fat they were found quite rich in n-3 fatty acids. Finally, PDCAAS indicated that these organisms had a very good protein quality.

  12. Seasonal changes in amino acids, protein and total nitrogen in needles of fertilized Scots pine trees.

    PubMed

    Näsholm, T; Ericsson, A

    1990-09-01

    Seasonal changes in amino acids, protein and total nitrogen in needles of 30-year-old, fertilized Scots pine (Pinus sylvestris L.) trees growing in Northern Sweden were investigated over two years in field experiments. The studied plots had been fertilized annually for 17 years with (i) a high level of N, (ii) a medium level of N, or (iii) a medium level of N, P and K. Trees growing on unfertilized plots served as controls. In control trees, glutamine, glutamic acid, gamma-aminobutyric acid, aspartic acid and proline represented 50-70% of the total free amino acids determined. Arginine was present only in low concentrations in control trees throughout the year, but it was usually the most abundant amino acid in fertilized trees. Glutamine concentrations were high during the spring and summer in both years of study, whereas proline concentrations were high in the spring but otherwise low throughout the year. In the first year of study, glutamic acid concentrations were high during the spring and summer, whereas gamma-aminobutyric acid was present in high concentrations during the winter months. This pattern was less pronounced in the second year of investigation. The concentrations of most amino acids, except glutamic acid, increased in response to fertilization. Nitrogen fertilization increased the foliar concentration of arginine from < 1 micromol g(dw) (-1) in control trees to a maximum of 110 micromol g(dw) (-1). Trees fertilized with nitrogen, phosphorus and potassium had significantly lower arginine concentrations than trees fertilized with the same amount of nitrogen only. Protein concentrations were similar in all fertilized trees but higher than those in control trees. For all treatments, protein concentrations were high in winter and at a minimum in early spring. In summer, the protein concentration remained almost constant except for a temporary decrease which coincided with the expansion of new shoots. Apart from arginine, the amino acid composition of

  13. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [Austin, TX

    2011-08-30

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  14. Site-specific incorporation of redox active amino acids into proteins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  15. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [San Diego, CA

    2012-02-14

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  16. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta; Lital , Schultz; Peter G. , Zhang; Zhiwen

    2010-10-12

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  17. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [San Diego, CA

    2009-02-24

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  18. Protein and Amino Acid Requirements during Pregnancy.

    PubMed

    Elango, Rajavel; Ball, Ronald O

    2016-07-01

    Protein forms an essential component of a healthy diet in humans to support both growth and maintenance. During pregnancy, an exceptional stage of life defined by rapid growth and development, adequate dietary protein is crucial to ensure a healthy outcome. Protein deposition in maternal and fetal tissues increases throughout pregnancy, with most occurring during the third trimester. Dietary protein intake recommendations are based on factorial estimates because the traditional method of determining protein requirements, nitrogen balance, is invasive and undesirable during pregnancy. The current Estimated Average Requirement and RDA recommendations of 0.88 and 1.1 g · kg(-1) · d(-1), respectively, are for all stages of pregnancy. The single recommendation does not take into account the changing needs during different stages of pregnancy. Recently, with the use of the minimally invasive indicator amino acid oxidation method, we defined the requirements to be, on average, 1.2 and 1.52 g · kg(-1) · d(-1) during early (∼16 wk) and late (∼36 wk) stages of pregnancy, respectively. Although the requirements are substantially higher than current recommendations, our values are ∼14-18% of total energy and fit within the Acceptable Macronutrient Distribution Range. Using swine as an animal model we showed that the requirements for several indispensable amino acids increase dramatically during late gestation compared with early gestation. Additional studies should be conducted during pregnancy to confirm the newly determined protein requirements and to determine the indispensable amino acid requirements during pregnancy in humans. © 2016 American Society for Nutrition.

  19. An upper limit to the abundance of lightning-produced amino acids in the Jovian water clouds

    NASA Astrophysics Data System (ADS)

    Bar-Nun, A.; Noy, N.; Podolak, M.

    1984-08-01

    The effect of excess hydrogen on the synthesis of amino acids by high-temperature shock waves in a hydrogen/methane/ammonia/water vapor mixture was studied experimentally. The energy efficiency results, together with the best estimate of the lightning energy dissipation rate on Jupiter from the Voyage data, were used to calculate an upper limit to the rate of amino acid production by lightning in Jovian water clouds. Using reasonable values for the eddy diffusion coefficients within and below the water clouds, the column abundance of lightning-produced amino acids in the clouds was estimated to be 6.2 x 10 to the -6th cm-am. Hence, concentration of amino acids in water droplets would be 8 x 10 to the -8th mole/liter.

  20. Crude protein and essential amino acid requirements in chicks during the first week posthatch.

    PubMed

    Sklan, D; Noy, Y

    2003-05-01

    1. This study examined optimal lysine and sulphur amino acid supply in the first week posthatch in broilers and the relationship between essential amino acids and dietary crude protein during the first week posthatch on performance at 7 d and through marketing. 2. The optimal supply during the 7 d posthatch using a 230 g/kg crude protein diet for sulphur amino acids was 9.1 and for lysine was 10.3-10.8 g/kg with maximal body weight (BW) or feed efficiency as the criteria. 3. Feeding diets with crude protein content ranging from 200 to 260 g/kg with either constant amounts of essential amino acids at different crude protein levels or constant ratios of essential amino acids to crude protein resulted in enhanced performance at 7 but not at 4 d with high protein intake and proportionally increased essential amino acids. 4. Performance on diets with crude protein ranging from 160 to 280 g/kg, with constant ratios of essential amino acid to crude protein, was much enhanced with the high crude protein diets at 7 d. All chicks were transferred to standard diets after 7 d and the BW advantage due to the balanced amino acid-high crude protein diet remained through marketing. 5. Thus increasing essential amino acids in a constant ratio to crude protein enhanced performance during the 7 d posthatch.

  1. Amino acid pair- and triplet-wise groupings in the interior of α-helical segments in proteins.

    PubMed

    de Sousa, Miguel M; Munteanu, Cristian R; Pazos, Alejandro; Fonseca, Nuno A; Camacho, Rui; Magalhães, A L

    2011-02-21

    A statistical approach has been applied to analyse primary structure patterns at inner positions of α-helices in proteins. A systematic survey was carried out in a recent sample of non-redundant proteins selected from the Protein Data Bank, which were used to analyse α-helix structures for amino acid pairing patterns. Only residues more than three positions apart from both termini of the α-helix were considered as inner. Amino acid pairings i, i+k (k=1, 2, 3, 4, 5), were analysed and the corresponding 20×20 matrices of relative global propensities were constructed. An analysis of (i, i+4, i+8) and (i, i+3, i+4) triplet patterns was also performed. These analysis yielded information on a series of amino acid patterns (pairings and triplets) showing either high or low preference for α-helical motifs and suggested a novel approach to protein alphabet reduction. In addition, it has been shown that the individual amino acid propensities are not enough to define the statistical distribution of these patterns. Global pair propensities also depend on the type of pattern, its composition and orientation in the protein sequence. The data presented should prove useful to obtain and refine useful predictive rules which can further the development and fine-tuning of protein structure prediction algorithms and tools. Copyright © 2010 Elsevier Ltd. All rights reserved.

  2. Mechanisms of volatile production from non-sulfur amino acids by irradiation

    NASA Astrophysics Data System (ADS)

    Ahn, Dong Uk; Lee, Eun Joo; Feng, Xi; Zhang, Wangang; Lee, Ji Hwan; Jo, Cheorun; Nam, Kichang

    2016-02-01

    Non-sulfur amino acid monomers were used to study the mechanisms of volatile production in meat by irradiation. Irradiation not only produced many volatiles but also increased the amounts of volatiles from non-sulfur amino acid monomers. The major reaction mechanisms involved in volatile production from each group of the amino acids by irradiation differ significantly. However, we speculate that the radiolysis of amino acid side chains were the major mechanism. In addition, Strecker degradation, especially the production of aldehydes from aliphatic group amino acids, and deamination, isomerization, decarboxylation, cyclic reaction and dehydrogenation of the initial radiolytic products were also contributed to the production of volatile compounds. Each amino acid monomers produced different odor characteristics, but the intensities of odor from all non-sulfur amino acid groups were very weak. This indicated that the contribution of volatiles produced from non-sulfur amino acids was minor. If the volatile compounds from non-sulfur amino acids, especially aldehydes, interact with other volatiles compounds such as sulfur compounds, however, they can contribute to the off-odor of irradiated meat significantly.

  3. NIAS-Server: Neighbors Influence of Amino acids and Secondary Structures in Proteins.

    PubMed

    Borguesan, Bruno; Inostroza-Ponta, Mario; Dorn, Márcio

    2017-03-01

    The exponential growth in the number of experimentally determined three-dimensional protein structures provide a new and relevant knowledge about the conformation of amino acids in proteins. Only a few of probability densities of amino acids are publicly available for use in structure validation and prediction methods. NIAS (Neighbors Influence of Amino acids and Secondary structures) is a web-based tool used to extract information about conformational preferences of amino acid residues and secondary structures in experimental-determined protein templates. This information is useful, for example, to characterize folds and local motifs in proteins, molecular folding, and can help the solution of complex problems such as protein structure prediction, protein design, among others. The NIAS-Server and supplementary data are available at http://sbcb.inf.ufrgs.br/nias .

  4. Prediction of protein S-nitrosylation sites based on adapted normal distribution bi-profile Bayes and Chou's pseudo amino acid composition.

    PubMed

    Jia, Cangzhi; Lin, Xin; Wang, Zhiping

    2014-06-10

    Protein S-nitrosylation is a reversible post-translational modification by covalent modification on the thiol group of cysteine residues by nitric oxide. Growing evidence shows that protein S-nitrosylation plays an important role in normal cellular function as well as in various pathophysiologic conditions. Because of the inherent chemical instability of the S-NO bond and the low abundance of endogenous S-nitrosylated proteins, the unambiguous identification of S-nitrosylation sites by commonly used proteomic approaches remains challenging. Therefore, computational prediction of S-nitrosylation sites has been considered as a powerful auxiliary tool. In this work, we mainly adopted an adapted normal distribution bi-profile Bayes (ANBPB) feature extraction model to characterize the distinction of position-specific amino acids in 784 S-nitrosylated and 1568 non-S-nitrosylated peptide sequences. We developed a support vector machine prediction model, iSNO-ANBPB, by incorporating ANBPB with the Chou's pseudo amino acid composition. In jackknife cross-validation experiments, iSNO-ANBPB yielded an accuracy of 65.39% and a Matthew's correlation coefficient (MCC) of 0.3014. When tested on an independent dataset, iSNO-ANBPB achieved an accuracy of 63.41% and a MCC of 0.2984, which are much higher than the values achieved by the existing predictors SNOSite, iSNO-PseAAC, the Li et al. algorithm, and iSNO-AAPair. On another training dataset, iSNO-ANBPB also outperformed GPS-SNO and iSNO-PseAAC in the 10-fold crossvalidation test.

  5. Specific Labeling of Zinc Finger Proteins using Non-canonical Amino Acids and Copper-free Click Chemistry

    PubMed Central

    Kim, Younghoon; Kim, Sung Hoon; Ferracane, Dean; Katzenellenbogen, John A.

    2012-01-01

    Zinc finger proteins (ZFPs) play a key role in transcriptional regulation and serve as invaluable tools for gene modification and genetic engineering. Development of efficient strategies for labeling metalloproteins such as ZFPs is essential for understanding and controlling biological processes. In this work, we engineered ZFPs containing cysteine-histidine (Cys2-His2) motifs by metabolic incorporation of the unnatural amino acid azidohomoalanine (AHA), followed by specific protein labeling via click chemistry. We show that cyclooctyne promoted [3 + 2] dipolar cycloaddition with azides, known as copper-free click chemistry, provides rapid and specific labeling of ZFPs at high yields as determined by mass spectrometry analysis. We observe that the DNA-binding activity of ZFPs labeled by conventional copper-mediated click chemistry was completely abolished, whereas ZFPs labeled by copper-free click chemistry retain their sequence-specific DNA-binding activity under native conditions, as determined by electrophoretic mobility shift assays, protein microarrays and kinetic binding assays based on Förster resonance energy transfer (FRET). Our work provides a general framework to label metalloproteins such as ZFPs by metabolic incorporation of unnatural amino acids followed by copper-free click chemistry. PMID:22871171

  6. Database of amino acid-nucleotide contacts in contacts in DNA-homeodomain protein

    NASA Astrophysics Data System (ADS)

    Grokhlina, T. I.; Zrelov, P. V.; Ivanov, V. V.; Polozov, R. V.; Chirgadze, Yu. N.; Sivozhelezov, V. S.

    2013-09-01

    The analysis of amino acid-nucleotide contacts in interfaces of the protein-DNA complexes, intended to find consistencies in the protein-DNA recognition, is a complex problem that requires an analysis of the physicochemical characteristics of these contacts and the positions of the participating amino acids and nucleotides in the chains of the protein and the DNA, respectively, as well as conservatism of these contacts. Thus, those heterogeneous data should be systematized. For this purpose we have developed a database of amino acid-nucleotide contacts ANTPC (Amino acid Nucleotide Type Position Conservation) following the archetypal example of the proteins in the homeodomain family. We show that it can be used to compare and classify the interfaces of the protein-DNA complexes.

  7. Determination and stereochemistry of proteinogenic and non-proteinogenic amino acids in Saudi Arabian date fruits.

    PubMed

    Ali, Hatem Salama Mohamed; Alhaj, Omar Amin; Al-Khalifa, Abdulrahman Saleh; Brückner, Hans

    2014-09-01

    Whereas an abundance of literature is available on the occurrence of common proteinogenic amino acids (AAs) in edible fruits of the date palm (Phoenix dactylifera L.), recent reports on non-proteinogenic (non-coded) AAs and amino components are scarce. With emphasis on these components we have analyzed total hydrolysates of twelve cultivars of date fruits using automated ion-exchange chromatography, HPLC employing a fluorescent aminoquinolyl label, and GC-MS of total hydrolysates using the chiral stationary phases Chirasil(®)-L-Val and Lipodex(®) E. Besides common proteinogenic AAs, relatively large amounts of the following non-proteinogenic amino acids were detected: (2S,5R)-5-hydroxypipecolic acid (1.4-4.0 g/kg dry matter, DM), 1-aminocyclopropane-1-carboxylic acid (1.3-2.6 g/kg DM), γ-amino-n-butyric acid (0.5-1.2 g/kg DM), (2S,4R)-4-hydroxyproline (130-230 mg/kg DM), L-pipecolic acid (40-140 mg/kg DM), and 2-aminoethanol (40-160 mg/kg DM) as well as low or trace amounts (<70 mg/kg DM) of L-ornithine, 5-hydroxylysine, β-alanine, and in some samples (<20 mg/kg DM) of (S)-β-aminoisobutyric acid and (<10 mg/kg DM) L-allo-isoleucine. In one date fruit, traces of α-aminoadipic acid could be determined. Enantiomeric analysis of 6 M DCl/D2O hydrolysates of AAs using chiral capillary gas chromatography-mass spectrometry revealed the presence of very low amounts of D-Ala, D-Asp, D-Glu, D-Ser and D-Phe (1.2-0.4%, relative to the corresponding L-enantiomers), besides traces (0.2-1%) of other D-AAs. The possible relevance of non-proteinogenic amino acids in date fruits is briefly addressed.

  8. Identification of Differentially Abundant Proteins of Edwardsiella ictaluri during Iron Restriction

    PubMed Central

    Dumpala, Pradeep R.; Peterson, Brian C.; Lawrence, Mark L.; Karsi, Attila

    2015-01-01

    Edwardsiella ictaluri is a Gram-negative facultative anaerobe intracellular bacterium that causes enteric septicemia in channel catfish. Iron is an essential inorganic nutrient of bacteria and is crucial for bacterial invasion. Reduced availability of iron by the host may cause significant stress for bacterial pathogens and is considered a signal that leads to significant alteration in virulence gene expression. However, the precise effect of iron-restriction on E. ictaluri protein abundance is unknown. The purpose of this study was to identify differentially abundant proteins of E. ictaluri during in vitro iron-restricted conditions. We applied two-dimensional difference in gel electrophoresis (2D-DIGE) for determining differentially abundant proteins and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF MS) for protein identification. Gene ontology and pathway-based functional modeling of differentially abundant proteins was also conducted. A total of 50 unique differentially abundant proteins at a minimum of 2-fold (p ≤ 0.05) difference in abundance due to iron-restriction were detected. The numbers of up- and down-regulated proteins were 37 and 13, respectively. We noted several proteins, including EsrB, LamB, MalM, MalE, FdaA, and TonB-dependent heme/hemoglobin receptor family proteins responded to iron restriction in E. ictaluri. PMID:26168192

  9. Expression-Linked Patterns of Codon Usage, Amino Acid Frequency, and Protein Length in the Basally Branching Arthropod Parasteatoda tepidariorum

    PubMed Central

    Whittle, Carrie A.; Extavour, Cassandra G.

    2016-01-01

    Abstract Spiders belong to the Chelicerata, the most basally branching arthropod subphylum. The common house spider, Parasteatoda tepidariorum, is an emerging model and provides a valuable system to address key questions in molecular evolution in an arthropod system that is distinct from traditionally studied insects. Here, we provide evidence suggesting that codon usage, amino acid frequency, and protein lengths are each influenced by expression-mediated selection in P. tepidariorum. First, highly expressed genes exhibited preferential usage of T3 codons in this spider, suggestive of selection. Second, genes with elevated transcription favored amino acids with low or intermediate size/complexity (S/C) scores (glycine and alanine) and disfavored those with large S/C scores (such as cysteine), consistent with the minimization of biosynthesis costs of abundant proteins. Third, we observed a negative correlation between expression level and coding sequence length. Together, we conclude that protein-coding genes exhibit signals of expression-related selection in this emerging, noninsect, arthropod model. PMID:27017527

  10. Photo-CIDNP NMR spectroscopy of amino acids and proteins.

    PubMed

    Kuhn, Lars T

    2013-01-01

    Photo-chemically induced dynamic nuclear polarization (CIDNP) is a nuclear magnetic resonance (NMR) phenomenon which, among other things, is exploited to extract information on biomolecular structure via probing solvent-accessibilities of tryptophan (Trp), tyrosine (Tyr), and histidine (His) amino acid side chains both in polypeptides and proteins in solution. The effect, normally triggered by a (laser) light-induced photochemical reaction in situ, yields both positive and/or negative signal enhancements in the resulting NMR spectra which reflect the solvent exposure of these residues both in equilibrium and during structural transformations in "real time". As such, the method can offer - qualitatively and, to a certain extent, quantitatively - residue-specific structural and kinetic information on both the native and, in particular, the non-native states of proteins which, often, is not readily available from more routine NMR techniques. In this review, basic experimental procedures of the photo-CIDNP technique as applied to amino acids and proteins are discussed, recent improvements to the method highlighted, and future perspectives presented. First, the basic principles of the phenomenon based on the theory of the radical pair mechanism (RPM) are outlined. Second, a description of standard photo-CIDNP applications is given and it is shown how the effect can be exploited to extract residue-specific structural information on the conformational space sampled by unfolded or partially folded proteins on their "path" to the natively folded form. Last, recent methodological advances in the field are highlighted, modern applications of photo-CIDNP in the context of biological NMR evaluated, and an outlook into future perspectives of the method is given.

  11. Amino Acid Interaction (INTAA) web server.

    PubMed

    Galgonek, Jakub; Vymetal, Jirí; Jakubec, David; Vondrášek, Jirí

    2017-07-03

    Large biomolecules-proteins and nucleic acids-are composed of building blocks which define their identity, properties and binding capabilities. In order to shed light on the energetic side of interactions of amino acids between themselves and with deoxyribonucleotides, we present the Amino Acid Interaction web server (http://bioinfo.uochb.cas.cz/INTAA/). INTAA offers the calculation of the residue Interaction Energy Matrix for any protein structure (deposited in Protein Data Bank or submitted by the user) and a comprehensive analysis of the interfaces in protein-DNA complexes. The Interaction Energy Matrix web application aims to identify key residues within protein structures which contribute significantly to the stability of the protein. The application provides an interactive user interface enhanced by 3D structure viewer for efficient visualization of pairwise and net interaction energies of individual amino acids, side chains and backbones. The protein-DNA interaction analysis part of the web server allows the user to view the relative abundance of various configurations of amino acid-deoxyribonucleotide pairs found at the protein-DNA interface and the interaction energies corresponding to these configurations calculated using a molecular mechanical force field. The effects of the sugar-phosphate moiety and of the dielectric properties of the solvent on the interaction energies can be studied for the various configurations. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Acute supplementation of amino acids increases net protein accretion in IUGR fetal sheep

    PubMed Central

    Rozance, Paul J.; Thorn, Stephanie R.; Friedman, Jacob E.; Hay, William W.

    2012-01-01

    Placental insufficiency decreases fetal amino acid uptake from the placenta, plasma insulin concentrations, and protein accretion, thus compromising normal fetal growth trajectory. We tested whether acute supplementation of amino acids or insulin into the fetus with intrauterine growth restriction (IUGR) would increase net fetal protein accretion rates. Late-gestation IUGR and control (CON) fetal sheep received acute, 3-h infusions of amino acids (with euinsulinemia), insulin (with euglycemia and euaminoacidemia), or saline. Fetal leucine metabolism was measured under steady-state conditions followed by a fetal muscle biopsy to quantify insulin signaling. In CON, increasing amino acid delivery rates to the fetus by 100% increased leucine oxidation rates by 100%. In IUGR, amino acid infusion completely suppressed fetal protein breakdown rates but increased leucine oxidation rate by only 25%, resulting in increased protein accretion rates by 150%. Acute insulin infusion, however, had very little effect on amino acid delivery rates, fetal leucine disposal rates, or fetal protein accretion rates in CON or IUGR fetuses despite robust signaling of the fetal skeletal muscle insulin-signaling cascade. These results indicate that, when amino acids are given directly into the fetal circulation independently of changes in insulin concentrations, IUGR fetal sheep have suppressed protein breakdown rates, thus increasing net fetal protein accretion. PMID:22649066

  13. Deep Coverage Proteomics Identifies More Low-Abundance Missing Proteins in Human Testis Tissue with Q-Exactive HF Mass Spectrometer.

    PubMed

    Wei, Wei; Luo, Weijia; Wu, Feilin; Peng, Xuehui; Zhang, Yao; Zhang, Manli; Zhao, Yan; Su, Na; Qi, YingZi; Chen, Lingsheng; Zhang, Yangjun; Wen, Bo; He, Fuchu; Xu, Ping

    2016-11-04

    Since 2012, missing proteins (MPs) investigation has been one of the critical missions of Chromosome-Centric Human Proteome Project (C-HPP) through various biochemical strategies. On the basis of our previous testis MPs study, faster scanning and higher resolution mass-spectrometry-based proteomics might be conducive to MPs exploration, especially for low-abundance proteins. In this study, Q-Exactive HF (HF) was used to survey proteins from the same testis tissues separated by two separating methods (tricine- and glycine-SDS-PAGE), as previously described. A total of 8526 proteins were identified, of which more low-abundance proteins were uniquely detected in HF data but not in our previous LTQ Orbitrap Velos (Velos) reanalysis data. Further transcriptomics analysis showed that these uniquely identified proteins by HF also had lower expression at the mRNA level. Of the 81 total identified MPs, 74 and 39 proteins were listed as MPs in HF and Velos data sets, respectively. Among the above MPs, 47 proteins (43 neXtProt PE2 and 4 PE3) were ranked as confirmed MPs after verifying with the stringent spectra match and isobaric and single amino acid variants filtering. Functional investigation of these 47 MPs revealed that 11 MPs were testis-specific proteins and 7 MPs were involved in spermatogenesis process. Therefore, we concluded that higher scanning speed and resolution of HF might be factors for improving the low-abundance MP identification in future C-HPP studies. All mass-spectrometry data from this study have been deposited in the ProteomeXchange with identifier PXD004092.

  14. Wet, Carbonaceous Asteroids: Altering Minerals, Changing Amino Acids

    NASA Astrophysics Data System (ADS)

    Taylor, G. J.

    2011-04-01

    Many carbonaceous chondrites contain alteration products from water-rock interactions at low temperature and organic compounds. A fascinating fact known for decades is the presence in some of them of an assortment of organic compounds, including amino acids, sometimes called the building blocks of life. Murchison and other CM carbonaceous chondrites contain hundreds of amino acids. Early measurements indicated that the amino acids in carbonaceous chondrites had equal proportions of L- and D-structures, a situation called racemic. This was in sharp contrast to life on Earth, which heavily favors L- forms. However, beginning in 1997, John Cronin and Sandra Pizzarello (Arizona State University) found L- excesses in isovaline and several other amino acids in the Murchison carbonaceous chondrite. In 2009, Daniel Glavin and Jason Dworkin (Astrobiology Analytical Lab, Goddard Space Flight Center) reported the first independent confirmation of L-isovaline excesses in Murchison using a different analytical technique than employed by Cronin and Pizzarello. Inspired by this work, Daniel Glavin, Michael Callahan, Jason Dworkin, and Jamie Elsila (Astrobiology Analytical Lab, Goddard Space Flight Center), have done an extensive study of the abundance and symmetry of amino acids in carbonaceous chondrites that experienced a range of alteration by water in their parent asteroids. The results show that amino acids are more abundant in the less altered meteorites, implying that aqueous processing changes the mix of amino acids. They also confirmed the enrichment in L-structures of some amino acids, especially isovaline, confirming earlier work. The authors suggest that aqueously-altered planetesimals might have seeded the early Earth with nonracemic amino acids, perhaps explaining why life from microorganisms to people use only L- forms to make proteins. The initial imbalance caused by non-biologic processes in wet asteroids might have been amplified by life on Earth. Alternatively

  15. Comparative proteomic analyses reveal that the regulators of G-protein signaling proteins regulate amino acid metabolism of the rice blast fungus Magnaporthe oryzae.

    PubMed

    Zhang, Haifeng; Ma, Hongyu; Xie, Xin; Ji, Jun; Dong, Yanhan; Du, Yan; Tang, Wei; Zheng, Xiaobo; Wang, Ping; Zhang, Zhengguang

    2014-11-01

    The rice blast fungus Magnaporthe oryzae encodes eight regulators of G-protein (GTP-binding protein) signaling (RGS) proteins MoRgs1-MoRgs8 that orchestrate the growth, asexual/sexual production, appressorium differentiation, and pathogenicity. To address the mechanisms by which MoRgs proteins function, we conducted a 2DE proteome study and identified 82 differentially expressed proteins by comparing five ∆Morgs mutants with wild-type Guy11 strain. We found that the abundances of eight amino acid (AA) biosynthesis or degradation associated proteins were markedly altered in five ∆Morgs mutants, indicating one of the main collective roles for the MoRgs proteins is to influence AA metabolism. We showed that MoRgs proteins have distinct roles in AA metabolism and nutrient responses from growth assays. In addition, we characterized MoLys20 (Lys is lysine), a homocitrate synthase, whose abundance was significantly decreased in the ∆Morgs mutants. The ∆Molys20 mutant is auxotrophic for lys and exogenous lys could partially rescue its auxotrophic defects. Deletion of MoLYS20 resulted in defects in conidiation and infection, as well as pathogenicity on rice. Overall, our results indicate that one of the critical roles for MoRgs proteins is to regulate AA metabolism, and that MoLys20 may be directly or indirectly regulated by MoRgs and participated in lys biosynthesis, thereby affecting fungal development and pathogenicity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Impact of antinutritional factors in food proteins on the digestibility of protein and the bioavailability of amino acids and on protein quality.

    PubMed

    Sarwar Gilani, G; Wu Xiao, Chao; Cockell, Kevin A

    2012-08-01

    Dietary antinutritional factors have been reported to adversely affect the digestibility of protein, bioavailability of amino acids and protein quality of foods. Published data on these negative effects of major dietary antinutritional factors are summarized in this manuscript. Digestibility and the quality of mixed diets in developing countries are considerably lower than of those in developed regions. For example, the digestibility of protein in traditional diets from developing countries such as India, Guatemala and Brazil is considerably lower compared to that of protein in typical North American diets (54-78 versus 88-94 %). Poor digestibility of protein in the diets of developing countries, which are based on less refined cereals and grain legumes as major sources of protein, is due to the presence of less digestible protein fractions, high levels of insoluble fibre, and/or high concentrations of antinutritional factors present endogenously or formed during processing. Examples of naturally occurring antinutritional factors include glucosinolates in mustard and canola protein products, trypsin inhibitors and haemagglutinins in legumes, tannins in legumes and cereals, gossypol in cottonseed protein products, and uricogenic nucleobases in yeast protein products. Heat/alkaline treatments of protein products may yield Maillard reaction compounds, oxidized forms of sulphur amino acids, D-amino acids and lysinoalanine (LAL, an unnatural nephrotoxic amino acid derivative). Among common food and feed protein products, soyabeans are the most concentrated source of trypsin inhibitors. The presence of high levels of dietary trypsin inhibitors from soyabeans, kidney beans or other grain legumes have been reported to cause substantial reductions in protein and amino acid digestibility (up to 50 %) and protein quality (up to 100 %) in rats and/or pigs. Similarly, the presence of high levels of tannins in sorghum and other cereals, fababean and other grain legumes can cause

  17. Effects of antinutritional factors on protein digestibility and amino acid availability in foods.

    PubMed

    Gilani, G Sarwar; Cockell, Kevin A; Sepehr, Estatira

    2005-01-01

    Digestibility of protein in traditional diets from developing countries such as India, Guatemala, and Brazil is considerably lower compared to that of protein in typical North American diets (54-78 versus 88-94%). The presence of less digestible protein fractions, high levels of insoluble fiber, and high concentrations of antinutritional factors in the diets of developing countries, which are based on less refined cereals and grain legumes as major sources of protein, are responsible for poor digestibility of protein. The effects of the presence of some of the important antinutritional factors on protein and amino digestibilities of food and feed products are reviewed in this chapter. Food and feed products may contain a number of antinutritional factors that may adversely affect protein digestibility and amino acid availability. Antinutritional factors may occur naturally, such as glucosinolates in mustard and rapeseed protein products, trypsin inhibitors and hemagglutinins in legumes, tannins in legumes and cereals, phytates in cereals and oilseeds, and gossypol in cottonseed protein products. Antinutritional factors may also be formed during heat/alkaline processing of protein products, yielding Maillard compounds, oxidized forms of sulfur amino acids, D-amino acids, and lysinoalanine (LAL, an unnatural amino acid derivative). The presence of high levels of dietary trypsin inhibitors from soybeans, kidney beans, or other grain legumes can cause substantial reductions in protein and amino acid digestibilities (up to 50%) in rats and pigs. Similarly, the presence of high levels of tannins in cereals, such as sorghum, and grain legumes, such as fababean (Vicia faba L.), can result in significantly reduced protein and amino acid digestibilities (up to 23%) in rats, poultry, and pigs. Studies involving phytase supplementation of production rations for swine or poultry have provided indirect evidence that normally encountered levels of phytates in cereals and legumes

  18. Survival of Amino Acids in Micrometeorites During Atmospheric Entry

    NASA Technical Reports Server (NTRS)

    Glavin, Daniel P.; Bada, Jeffrey L.

    2003-01-01

    The delivery of amino acids by micrometeorites to the early Earth during the period of heavy bombardment could have been a significant source of the Earth's prebiotic amino acid inventory provided that these organic compounds survived atmospheric entry heating. To investigate the sublimation of amino acids from a micrometeorite analog at elevated temperature, grains from the CM-type carbonaceous chondrite Murchison were heated to 550 C inside a glass sublimation apparatus (SA) under reduced pressure. The sublimed residue that had collected on the cold finger of the SA after heating was analyzed for amino acids by HPLC. We found that when the temperature of the meteorite reached approx. 150 C, a large fraction of the amino acid glycine had vaporized from the meteorite, recondensed onto the end of the SA cold finger, and survived as the rest of the grains heated to 550 C. alpha-Aminoisobutryic acid and isovaline, which are two of the most abundant non-protein amino acids in Murchison, did not sublime from the meteorite and were completely destroyed during the heating experiment. Our experimental results suggest that sublimation of glycine present in micrometeorite grains may provide a way for this amino acid to survive atmospheric entry heating at temperatures less than 550 C; all other amino acids apparently are destroyed. Key Words: Amino acids-Exogenous delivery-Micrometeorites-Sublimation.

  19. Separomics applied to the proteomics and peptidomics of low-abundance proteins: Choice of methods and challenges - A review.

    PubMed

    Baracat-Pereira, Maria Cristina; de Oliveira Barbosa, Meire; Magalhães, Marcos Jorge; Carrijo, Lanna Clicia; Games, Patrícia Dias; Almeida, Hebréia Oliveira; Sena Netto, José Fabiano; Pereira, Matheus Rodrigues; de Barros, Everaldo Gonçalves

    2012-06-01

    The enrichment and isolation of proteins are considered limiting steps in proteomic studies. Identification of proteins whose expression is transient, those that are of low-abundance, and of natural peptides not described in databases, is still a great challenge. Plant extracts are in general complex, and contaminants interfere with the identification of proteins involved in important physiological processes, such as plant defense against pathogens. This review discusses the challenges and strategies of separomics applied to the identification of low-abundance proteins and peptides in plants, especially in plants challenged by pathogens. Separomics is described as a group of methodological strategies for the separation of protein molecules for proteomics. Several tools have been used to remove highly abundant proteins from samples and also non-protein contaminants. The use of chromatographic techniques, the partition of the proteome into subproteomes, and an effort to isolate proteins in their native form have allowed the isolation and identification of rare proteins involved in different processes.

  20. Prediction of protein structural classes by Chou's pseudo amino acid composition: approached using continuous wavelet transform and principal component analysis.

    PubMed

    Li, Zhan-Chao; Zhou, Xi-Bin; Dai, Zong; Zou, Xiao-Yong

    2009-07-01

    A prior knowledge of protein structural classes can provide useful information about its overall structure, so it is very important for quick and accurate determination of protein structural class with computation method in protein science. One of the key for computation method is accurate protein sample representation. Here, based on the concept of Chou's pseudo-amino acid composition (AAC, Chou, Proteins: structure, function, and genetics, 43:246-255, 2001), a novel method of feature extraction that combined continuous wavelet transform (CWT) with principal component analysis (PCA) was introduced for the prediction of protein structural classes. Firstly, the digital signal was obtained by mapping each amino acid according to various physicochemical properties. Secondly, CWT was utilized to extract new feature vector based on wavelet power spectrum (WPS), which contains more abundant information of sequence order in frequency domain and time domain, and PCA was then used to reorganize the feature vector to decrease information redundancy and computational complexity. Finally, a pseudo-amino acid composition feature vector was further formed to represent primary sequence by coupling AAC vector with a set of new feature vector of WPS in an orthogonal space by PCA. As a showcase, the rigorous jackknife cross-validation test was performed on the working datasets. The results indicated that prediction quality has been improved, and the current approach of protein representation may serve as a useful complementary vehicle in classifying other attributes of proteins, such as enzyme family class, subcellular localization, membrane protein types and protein secondary structure, etc.

  1. Amino- and carboxyl-terminal amino acid sequences of proteins coded by gag gene of murine leukemia virus

    PubMed Central

    Oroszlan, Stephen; Henderson, Louis E.; Stephenson, John R.; Copeland, Terry D.; Long, Cedric W.; Ihle, James N.; Gilden, Raymond V.

    1978-01-01

    The amino- and carboxyl-terminal amino acid sequences of proteins (p10, p12, p15, and p30) coded by the gag gene of Rauscher and AKR murine leukemia viruses were determined. Among these proteins, p15 from both viruses appears to have a blocked amino end. Proline was found to be the common NH2 terminus of both p30s and both p12s, and alanine of both p10s. The amino-terminal sequences of p30s are identical, as are those of p10s, while the p12 sequences are clearly distinctive but also show substantial homology. The carboxyl-terminal amino acids of both viral p30s and p12s are leucine and phenylalanine, respectively. Rauscher leukemia virus p15 has tyrosine as the carboxyl terminus while AKR virus p15 has phenylalanine in this position. The compositional and sequence data provide definite chemical criteria for the identification of analogous gag gene products and for the comparison of viral proteins isolated in different laboratories. On the basis of amino acid sequences and the previously proposed H-p15-p12-p30-p10-COOH peptide sequence in the precursor polyprotein, a model for cleavage sites involved in the post-translational processing of the precursor coded for by the gag gene is proposed. PMID:206897

  2. PCI-GC-MS-MS approach for identification of non-amino organic acid and amino acid profiles.

    PubMed

    Luan, Hemi; Yang, Lin; Ji, Fenfen; Cai, Zongwei

    2017-03-15

    Alkyl chloroformate have been wildly used for the fast derivatization of metabolites with amino and/or carboxyl groups, coupling of powerful separation and detection systems, such as GC-MS, which allows the comprehensive analysis of non-amino organic acids and amino acids. The reagents involving n-alkyl chloroformate and n-alcohol are generally employed for providing symmetric labeling terminal alkyl chain with the same length. Here, we developed an asymmetric labeling strategy and positive chemical ionization gas chromatography-tandem mass spectrometry (PCI-GC-MS-MS) approach for determination of non-amino organic acids and amino acids, as well as the short chain fatty acids. Carboxylic and amino groups could be selectively labelled by propyl and ethyl groups, respectively. The specific neutral loss of C 3 H 8 O (60Da), C 3 H 5 O 2 (74Da) and C 4 H 8 O 2 (88Da) were useful in the selective identification for qualitative analysis of organic acids and amino acid derivatives. PCI-GC-MS-MS using multiple reaction monitoring (MRM) was applied for semi-quantification of typical non-amino organic acids and amino acids. This method exhibited a wide range of linear range, good regression coefficient (R 2 ) and repeatability. The relative standard deviation (RSD) of targeted metabolites showed excellent intra- and inter-day precision (<5%). Our method provided a qualitative and semi-quantitative PCI-GC-MS-MS, coupled with alkyl chloroformate derivatization. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Reasons for the occurrence of the twenty coded protein amino acids

    NASA Technical Reports Server (NTRS)

    Weber, A. L.; Miller, S. L.

    1981-01-01

    Factors involved in the selection of the 20 protein L-alpha-amino acids during chemical evolution and the early stages of Darwinian evolution are discussed. The selection is considered on the basis of the availability in the primitive ocean, function in proteins, the stability of the amino acid and its peptides, stability to racemization, and stability on the transfer RNA. It is concluded that aspartic acid, glutamic acid, arginine, lysine, serine and possibly threonine are the best choices for acidic, basic and hydroxy amino acids. The hydrophobic amino acids are reasonable choices, except for the puzzling absences of alpha-amino-n-butyric acid, norvaline and norleucine. The choices of the sulfur and aromatic amino acids seem reasonable, but are not compelling. Asparagine and glutamine are apparently not primitive. If life were to arise on another planet, it would be expected that the catalysts would be poly-alpha-amino acids and that about 75% of the amino acids would be the same as on the earth.

  4. Improving protein complex classification accuracy using amino acid composition profile.

    PubMed

    Huang, Chien-Hung; Chou, Szu-Yu; Ng, Ka-Lok

    2013-09-01

    Protein complex prediction approaches are based on the assumptions that complexes have dense protein-protein interactions and high functional similarity between their subunits. We investigated those assumptions by studying the subunits' interaction topology, sequence similarity and molecular function for human and yeast protein complexes. Inclusion of amino acids' physicochemical properties can provide better understanding of protein complex properties. Principal component analysis is carried out to determine the major features. Adopting amino acid composition profile information with the SVM classifier serves as an effective post-processing step for complexes classification. Improvement is based on primary sequence information only, which is easy to obtain. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Non-protein amino acids in Australian acacia seed: implications for food security and recommended processing methods to reduce djenkolic acid.

    PubMed

    Boughton, Berin A; Reddy, Priyanka; Boland, Martin P; Roessner, Ute; Yates, Peter

    2015-07-15

    Seed of Australian acacia species, Acacia colei, Acacia elecantha, Acacia torulosa, Acacia turmida and Acacia saligna, were analysed for the presence of toxic non-protein amino acids and the levels of essential amino acids. Amines were derivatised with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate before analysis using liquid chromatography electrospray ionisation triple quadrupole mass spectrometry (LC-ESI-QQQ-MS). Multiple reaction monitoring (MRM) with optimised transitions and collision energies for each analyte were employed. The known nephrotoxic compound djenkolic acid was found to be present at elevated levels in all species tested. The lowest levels were in A. colei (0.49% w/w) and the highest in A. saligna (1.85% w/w). Observed levels of djenkolic acid are comparable to measured and reported levels found in the djenkol bean. Subsequent testing of seed processing methods showed djenkolic acid levels can be significantly reduced by over 90% by dry roasting at 180 °C rendering the seed safe for human consumption. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Role of Protein and Amino Acids in Infant and Young Child Nutrition: Protein and Amino Acid Needs and Relationship with Child Growth.

    PubMed

    Uauy, Ricardo; Kurpad, Anura; Tano-Debrah, Kwaku; Otoo, Gloria E; Aaron, Grant A; Toride, Yasuhiko; Ghosh, Shibani

    2015-01-01

    Over a third of all deaths of children under the age of five are linked to undernutrition. At a 90% coverage level, a core group of ten interventions inclusive of infant and young child nutrition could save one million lives of children under 5 y of age (15% of all deaths) (Lancet 2013). The infant and young child nutrition package alone could save over 220,000 lives in children under 5 y of age. High quality proteins (e.g. milk) in complementary, supplementary and rehabilitation food products have been found to be effective for good growth. Individual amino acids such as lysine and arginine have been found to be factors linked to growth hormone release in young children via the somatotropic axis and high intakes are inversely associated with fat mass index in pre-pubertal lean girls. Protein intake in early life is positively associated with height and weight at 10 y of age. This paper will focus on examining the role of protein and amino acids in infant and young child nutrition by examining protein and amino acid needs in early life and the subsequent relationship with stunting.

  7. PLASMA PROTEIN PRODUCTION INFLUENCED BY AMINO ACID MIXTURES AND LACK OF ESSENTIAL AMINO ACIDS

    PubMed Central

    Madden, S. C.; Anderson, F. W.; Donovan, J. C.; Whipple, G. H.

    1945-01-01

    When blood plasma proteins are depleted by bleeding with return of red cells suspended in saline (plasmapheresis) it is possible to bring dogs to a steady state of hypoproteinemia and a constant level of plasma protein production if the diet nitrogen intake is controlled and limited. Such dogs are outwardly normal but have a lowered resistance to infection and intoxication and probably to vitamin deficiency. When the diet nitrogen is provided by certain mixtures of the ten growth essential amino acids plus glycine, given intravenously at a rapid rate, plasma protein production is good. The same mixture absorbed subcutaneously at a slower rate may be slightly better utilized. Fed orally the same mixture is better utilized and associated with a lower urinary nitrogen excretion. An ample amino acid mixture for the daily intake of a 10 kilo dog may contain in grams dl-threonine 1.4, dl-valine 3, dl-leucine 3, dl-isoleucine 2, l(+)-lysine·HCl·H2O 2.2, dl-tryptophane 0.3, dl-phenylalanine 2, dl-methionine 1.2, l(+)-histidine·HCl·H2O 1, l(+)-arginine·HCl 1, and glycine 2. Half this quantity is inadequate and not improved by addition of a mixture of alanine, serine, norleucine, proline, hydroxyproline, and tyrosine totalling 1.4 gm. Aspartic acid appears to induce vomiting when added to a mixture of amino acids. The same response has been reported for glutamic acid (8). Omission from the intake of leucine or of leucine and isoleucine results in negative nitrogen balance and rapid weight loss but plasma protein production may be temporarily maintained. It is possible that leucine may be captured from red blood cell destruction. Tryptophane deficiency causes an abrupt decline in plasma protein production. No decline occurred during 2 weeks of histidine deficiency but the urinary nitrogen increased to negative balance. Plasma protein production may be impaired during conditions of dietary deficiency not related to the protein or amino acid intake. Skin lesions and liver

  8. Total amino acid stabilization during cell-free protein synthesis reactions.

    PubMed

    Calhoun, Kara A; Swartz, James R

    2006-05-17

    Limitations in amino acid supply have been recognized as a substantial problem in cell-free protein synthesis reactions. Although enzymatic inhibitors and fed-batch techniques have been beneficial, the most robust way to stabilize amino acids is to remove the responsible enzymatic activities by genetically modifying the source strain used for cell extract preparation. Previous work showed this was possible for arginine, serine, and tryptophan, but cysteine degradation remained a major limitation in obtaining high protein synthesis yields. Through radiolabel techniques, we confirmed that cysteine degradation was caused by the activity of glutamate-cysteine ligase (gene gshA) in the cell extract. Next, we created Escherichia coli strain KC6 that combines a gshA deletion with previously described deletions for arginine, serine, and tryptophan stabilization. Strain KC6 grows well, and active cell extract can be produced from it for cell-free protein synthesis reactions. The extract from strain KC6 maintains stable amino acid concentrations of all 20 amino acids in a 3-h batch reaction. Yields for three different proteins improved 75-250% relative to cell-free expression using the control extract.

  9. Protein and Amino Acid Requirements during Pregnancy123

    PubMed Central

    Elango, Rajavel; Ball, Ronald O

    2016-01-01

    Protein forms an essential component of a healthy diet in humans to support both growth and maintenance. During pregnancy, an exceptional stage of life defined by rapid growth and development, adequate dietary protein is crucial to ensure a healthy outcome. Protein deposition in maternal and fetal tissues increases throughout pregnancy, with most occurring during the third trimester. Dietary protein intake recommendations are based on factorial estimates because the traditional method of determining protein requirements, nitrogen balance, is invasive and undesirable during pregnancy. The current Estimated Average Requirement and RDA recommendations of 0.88 and 1.1 g · kg−1 · d−1, respectively, are for all stages of pregnancy. The single recommendation does not take into account the changing needs during different stages of pregnancy. Recently, with the use of the minimally invasive indicator amino acid oxidation method, we defined the requirements to be, on average, 1.2 and 1.52 g · kg−1 · d−1 during early (∼16 wk) and late (∼36 wk) stages of pregnancy, respectively. Although the requirements are substantially higher than current recommendations, our values are ∼14–18% of total energy and fit within the Acceptable Macronutrient Distribution Range. Using swine as an animal model we showed that the requirements for several indispensable amino acids increase dramatically during late gestation compared with early gestation. Additional studies should be conducted during pregnancy to confirm the newly determined protein requirements and to determine the indispensable amino acid requirements during pregnancy in humans. PMID:27422521

  10. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

    PubMed Central

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

  11. Single Amino Acid Repeats in the Proteome World: Structural, Functional, and Evolutionary Insights

    PubMed Central

    Kumar, Amitha Sampath; Sowpati, Divya Tej; Mishra, Rakesh K.

    2016-01-01

    Microsatellites or simple sequence repeats (SSR) are abundant, highly diverse stretches of short DNA repeats present in all genomes. Tandem mono/tri/hexanucleotide repeats in the coding regions contribute to single amino acids repeats (SAARs) in the proteome. While SSRs in the coding region always result in amino acid repeats, a majority of SAARs arise due to a combination of various codons representing the same amino acid and not as a consequence of SSR events. Certain amino acids are abundant in repeat regions indicating a positive selection pressure behind the accumulation of SAARs. By analysing 22 proteomes including the human proteome, we explored the functional and structural relationship of amino acid repeats in an evolutionary context. Only ~15% of repeats are present in any known functional domain, while ~74% of repeats are present in the disordered regions, suggesting that SAARs add to the functionality of proteins by providing flexibility, stability and act as linker elements between domains. Comparison of SAAR containing proteins across species reveals that while shorter repeats are conserved among orthologs, proteins with longer repeats, >15 amino acids, are unique to the respective organism. Lysine repeats are well conserved among orthologs with respect to their length and number of occurrences in a protein. Other amino acids such as glutamic acid, proline, serine and alanine repeats are generally conserved among the orthologs with varying repeat lengths. These findings suggest that SAARs have accumulated in the proteome under positive selection pressure and that they provide flexibility for optimal folding of functional/structural domains of proteins. The insights gained from our observations can help in effective designing and engineering of proteins with novel features. PMID:27893794

  12. Variability in the carbon isotope composition of individual amino acids in plant proteins from different sources: 1 Leaves.

    PubMed

    Lynch, Anthony H; Kruger, Nicholas J; Hedges, Robert E M; McCullagh, James S O

    2016-05-01

    The natural carbon isotope composition of individual amino acids from plant leaf proteins has been measured to establish potential sources of variability. The plant leaves studied, taken from a range of plant groups (forbs, trees, grasses, and freshwater aquatic plants), showed no significant influence of either season or environment (water and light availability) on their Δδ(13)C values. Plant groups did, however, differ in carbon isotope composition, although no consistent differences were identified at the species level. A discriminant analysis model was constructed which allowed leaves from (1) nettles, (2) Pooideae, (3) other Poales, (4) trees and (5) freshwater higher plants to be distinguished from each other on the basis of their natural abundance (13)C/(12)C ratios of individual amino acids. Differences in carbon isotope composition are known to be retained, to some extent, in the tissues of their consumers, and hence an understanding of compound-specific variation in (13)C/(12)C fractional abundance in plants has the potential to provide dietary insights of value in archaeological and ecological studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Real-time Measurements of Amino Acid and Protein Hydroperoxides Using Coumarin Boronic Acid*

    PubMed Central

    Michalski, Radoslaw; Zielonka, Jacek; Gapys, Ewa; Marcinek, Andrzej; Joseph, Joy; Kalyanaraman, Balaraman

    2014-01-01

    Hydroperoxides of amino acid and amino acid residues (tyrosine, cysteine, tryptophan, and histidine) in proteins are formed during oxidative modification induced by reactive oxygen species. Amino acid hydroperoxides are unstable intermediates that can further propagate oxidative damage in proteins. The existing assays (oxidation of ferrous cation and iodometric assays) cannot be used in real-time measurements. In this study, we show that the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin. 7-Hydroxycoumarin formation was catalase-independent. Based on this observation, we have developed a fluorometric, real-time assay that is adapted to a multiwell plate format. This is the first report showing real-time monitoring of amino acid and protein hydroperoxides using the CBA-based assay. This approach was used to detect protein hydroperoxides in cell lysates obtained from macrophages exposed to visible light and photosensitizer (rose bengal). We also measured the rate constants for the reaction between amino acid hydroperoxides (tyrosyl, tryptophan, and histidine hydroperoxides) and CBA, and these values (7–23 m−1 s−1) were significantly higher than that measured for H2O2 (1.5 m−1 s−1). Using the CBA-based competition kinetics approach, the rate constants for amino acid hydroperoxides with ebselen, a glutathione peroxidase mimic, were also determined, and the values were within the range of 1.1–1.5 × 103 m−1 s−1. Both ebselen and boronates may be used as small molecule scavengers of amino acid and protein hydroperoxides. Here we also show formation of tryptophan hydroperoxide from tryptophan exposed to co-generated fluxes of nitric oxide and superoxide. This observation reveals a new mechanism for amino acid and protein hydroperoxide formation in biological systems. PMID:24928516

  14. Soy-dairy protein blend and whey protein ingestion after resistance exercise increases amino acid transport and transporter expression in human skeletal muscle.

    PubMed

    Reidy, P T; Walker, D K; Dickinson, J M; Gundermann, D M; Drummond, M J; Timmerman, K L; Cope, M B; Mukherjea, R; Jennings, K; Volpi, E; Rasmussen, B B

    2014-06-01

    Increasing amino acid availability (via infusion or ingestion) at rest or postexercise enhances amino acid transport into human skeletal muscle. It is unknown whether alterations in amino acid availability, from ingesting different dietary proteins, can enhance amino acid transport rates and amino acid transporter (AAT) mRNA expression. We hypothesized that the prolonged hyperaminoacidemia from ingesting a blend of proteins with different digestion rates postexercise would enhance amino acid transport into muscle and AAT expression compared with the ingestion of a rapidly digested protein. In a double-blind, randomized clinical trial, we studied 16 young adults at rest and after acute resistance exercise coupled with postexercise (1 h) ingestion of either a (soy-dairy) protein blend or whey protein. Phenylalanine net balance and transport rate into skeletal muscle were measured using stable isotopic methods in combination with femoral arteriovenous blood sampling and muscle biopsies obtained at rest and 3 and 5 h postexercise. Phenylalanine transport into muscle and mRNA expression of select AATs [system L amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, system A amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, cationic amino acid transporter 1/SLC7A1] increased to a similar extent in both groups (P < 0.05). However, the ingestion of the protein blend resulted in a prolonged and positive net phenylalanine balance during postexercise recovery compared with whey protein (P < 0.05). Postexercise myofibrillar protein synthesis increased similarly between groups. We conclude that, while both protein sources enhanced postexercise AAT expression, transport into muscle, and myofibrillar protein synthesis, postexercise ingestion of a protein blend results in a slightly prolonged net amino acid balance across the leg compared with whey protein. Copyright © 2014 the American Physiological Society.

  15. Soy-dairy protein blend and whey protein ingestion after resistance exercise increases amino acid transport and transporter expression in human skeletal muscle

    PubMed Central

    Reidy, P. T.; Walker, D. K.; Dickinson, J. M.; Gundermann, D. M.; Drummond, M. J.; Timmerman, K. L.; Cope, M. B.; Mukherjea, R.; Jennings, K.; Volpi, E.

    2014-01-01

    Increasing amino acid availability (via infusion or ingestion) at rest or postexercise enhances amino acid transport into human skeletal muscle. It is unknown whether alterations in amino acid availability, from ingesting different dietary proteins, can enhance amino acid transport rates and amino acid transporter (AAT) mRNA expression. We hypothesized that the prolonged hyperaminoacidemia from ingesting a blend of proteins with different digestion rates postexercise would enhance amino acid transport into muscle and AAT expression compared with the ingestion of a rapidly digested protein. In a double-blind, randomized clinical trial, we studied 16 young adults at rest and after acute resistance exercise coupled with postexercise (1 h) ingestion of either a (soy-dairy) protein blend or whey protein. Phenylalanine net balance and transport rate into skeletal muscle were measured using stable isotopic methods in combination with femoral arteriovenous blood sampling and muscle biopsies obtained at rest and 3 and 5 h postexercise. Phenylalanine transport into muscle and mRNA expression of select AATs [system L amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, system A amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, cationic amino acid transporter 1/SLC7A1] increased to a similar extent in both groups (P < 0.05). However, the ingestion of the protein blend resulted in a prolonged and positive net phenylalanine balance during postexercise recovery compared with whey protein (P < 0.05). Postexercise myofibrillar protein synthesis increased similarly between groups. We conclude that, while both protein sources enhanced postexercise AAT expression, transport into muscle, and myofibrillar protein synthesis, postexercise ingestion of a protein blend results in a slightly prolonged net amino acid balance across the leg compared with whey protein. PMID:24699854

  16. Acyl homoserine lactone changes the abundance of proteins and the levels of organic acids associated with stationary phase in Salmonella Enteritidis.

    PubMed

    de Almeida, Felipe Alves; Pimentel-Filho, Natan de Jesus; Carrijo, Lanna Clícia; Bento, Cláudia Braga Pereira; Baracat-Pereira, Maria Cristina; Pinto, Uelinton Manoel; de Oliveira, Leandro Licursi; Vanetti, Maria Cristina Dantas

    2017-01-01

    Quorum sensing (QS) is cell-cell communication mechanism mediated by signaling molecules known as autoinducers (AIs) that lead to differential gene expression. Salmonella is unable to synthesize the AI-1 acyl homoserine lactone (AHL), but is able to recognize AHLs produced by other microorganisms through SdiA protein. Our study aimed to evaluate the influence of AI-1 on the abundance of proteins and the levels of organic acids of Salmonella Enteritidis. The presence of N-dodecyl-homoserine lactone (C12-HSL) did not interfere on the growth or the total amount of extracted proteins of Salmonella. However, the abundance of the proteins PheT, HtpG, PtsI, Adi, TalB, PmgI (or GpmI), Eno, and PykF enhanced while the abundance of the proteins RplB, RplE, RpsB, Tsf, OmpA, OmpC, OmpD, and GapA decreased when Salmonella Enteritidis was anaerobically cultivated in the presence of C12-HSL. Additionally, the bacterium produced less succinic, lactic, and acetic acids in the presence of C12-HSL. However, the concentration of extracellular formic acid reached 20.46 mM after 24 h and was not detected when the growth was in the absence of AI-1. Considering the cultivation period for protein extraction, their abundance, process and function, as well as the levels of organic acids, we observed in cells cultivated in presence of C12-HSL a correlation with what is described in the literature as entry into the stationary phase of growth, mainly related to nitrogen and amino acid starvation and acid stress. Further studies are needed in order to determine the specific role of the differentially abundant proteins and extracellular organic acids secreted by Salmonella in the presence of quorum sensing signaling molecules. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. A differential protein solubility approach for the depletion of highly abundant proteins in plasma using ammonium sulfate.

    PubMed

    Bollineni, Ravi Chand; Guldvik, Ingrid J; Grönberg, Henrik; Wiklund, Fredrik; Mills, Ian G; Thiede, Bernd

    2015-12-21

    Depletion of highly abundant proteins is an approved step in blood plasma analysis by mass spectrometry (MS). In this study, we explored a precipitation and differential protein solubility approach as a fractionation strategy for abundant protein removal from plasma. Total proteins from plasma were precipitated with 90% saturated ammonium sulfate, followed by differential solubilization in 55% and 35% saturated ammonium sulfate solutions. Using a four hour liquid chromatography (LC) gradient and an LTQ-Orbitrap XL mass spectrometer, a total of 167 and 224 proteins were identified from the 55% and 35% ammonium sulfate fractions, whereas 235 proteins were found in the remaining protein fractions with at least two unique peptides. SDS-PAGE and exclusive total spectrum counts from LC-MS/MS analyses clearly showed that majority of the abundant plasma proteins were solubilized in 55% and 35% ammonium sulfate solutions, indicating that the remaining protein fraction is of potential interest for identification of less abundant plasma proteins. Serum albumin, serotransferrin, alpha-1-antitrypsin and transthyretin were the abundant proteins that were highly enriched in 55% ammonium sulfate fractions. Immunoglobulins, complement system proteins, and apolipoproteins were among other abundant plasma proteins that were enriched in 35% ammonium sulfate fractions. In the remaining protein fractions a total of 40 unique proteins were identified of which, 32 proteins were identified with at least 10 exclusive spectrum counts. According to PeptideAtlas, 9 of these 32 proteins were estimated to be present at low μg ml(-1) (0.12-1.9 μg ml(-1)) concentrations in the plasma, and 17 at low ng ml(-1) (0.1-55 ng ml(-1)) range.

  18. A cherry protein and its gene, abundantly expressed in ripening fruit, have been identified as thaumatin-like.

    PubMed

    Fils-Lycaon, B R; Wiersma, P A; Eastwell, K C; Sautiere, P

    1996-05-01

    A 29-kD polypeptide is the most abundant soluble protein in ripe cherry fruit (Prunus avium L); accumulation begins at the onset of ripening as the fruit turns from yellow to red. This protein was extracted from ripe cherries and purified by size-exclusion and ion-exchange chromatography. Antibodies to the purified protein were used to screen a cDNA library from ripe cherries. Numerous recombinant plaques reacted positively with the antibodies; the DNA sequence of representative clones encoded a polypeptide of 245 amino acid residues. A signal peptide was indicated, and the predicted mature protein corresponded to the purified protein in size (23.3 kD, by mass spectrometry) and isoelectric point (4.2). A search of known protein sequences revealed a strong similarity between this polypeptide and the thaumatin family of pathogenesis-related proteins. The cherry thaumatin-like protein does not have a sweet taste, and no antifungal activity was seen in preliminary assays. Expression of the protein appears to be regulated at the gene level, with mRNA levels at their highest in the ripe fruit.

  19. A cherry protein and its gene, abundantly expressed in ripening fruit, have been identified as thaumatin-like.

    PubMed Central

    Fils-Lycaon, B R; Wiersma, P A; Eastwell, K C; Sautiere, P

    1996-01-01

    A 29-kD polypeptide is the most abundant soluble protein in ripe cherry fruit (Prunus avium L); accumulation begins at the onset of ripening as the fruit turns from yellow to red. This protein was extracted from ripe cherries and purified by size-exclusion and ion-exchange chromatography. Antibodies to the purified protein were used to screen a cDNA library from ripe cherries. Numerous recombinant plaques reacted positively with the antibodies; the DNA sequence of representative clones encoded a polypeptide of 245 amino acid residues. A signal peptide was indicated, and the predicted mature protein corresponded to the purified protein in size (23.3 kD, by mass spectrometry) and isoelectric point (4.2). A search of known protein sequences revealed a strong similarity between this polypeptide and the thaumatin family of pathogenesis-related proteins. The cherry thaumatin-like protein does not have a sweet taste, and no antifungal activity was seen in preliminary assays. Expression of the protein appears to be regulated at the gene level, with mRNA levels at their highest in the ripe fruit. PMID:8685266

  20. Support Vector Machine-based classification of protein folds using the structural properties of amino acid residues and amino acid residue pairs.

    PubMed

    Shamim, Mohammad Tabrez Anwar; Anwaruddin, Mohammad; Nagarajaram, H A

    2007-12-15

    Fold recognition is a key step in the protein structure discovery process, especially when traditional sequence comparison methods fail to yield convincing structural homologies. Although many methods have been developed for protein fold recognition, their accuracies remain low. This can be attributed to insufficient exploitation of fold discriminatory features. We have developed a new method for protein fold recognition using structural information of amino acid residues and amino acid residue pairs. Since protein fold recognition can be treated as a protein fold classification problem, we have developed a Support Vector Machine (SVM) based classifier approach that uses secondary structural state and solvent accessibility state frequencies of amino acids and amino acid pairs as feature vectors. Among the individual properties examined secondary structural state frequencies of amino acids gave an overall accuracy of 65.2% for fold discrimination, which is better than the accuracy by any method reported so far in the literature. Combination of secondary structural state frequencies with solvent accessibility state frequencies of amino acids and amino acid pairs further improved the fold discrimination accuracy to more than 70%, which is approximately 8% higher than the best available method. In this study we have also tested, for the first time, an all-together multi-class method known as Crammer and Singer method for protein fold classification. Our studies reveal that the three multi-class classification methods, namely one versus all, one versus one and Crammer and Singer method, yield similar predictions. Dataset and stand-alone program are available upon request.

  1. Determination of amino acid contents of manketti seeds (Schinziophyton rautanenii) by pre-column derivatisation with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and RP-HPLC.

    PubMed

    Gwatidzo, Luke; Botha, Ben M; McCrindle, Rob I

    2013-12-01

    Defatted kernel flour from manketti seed kernels (Schinziophyton rautanenii) is an underutilised natural product. The plant grows in the wild, on sandy soils little used for agriculture in Southern Africa. The kernels are rich in protein and have a great potential for improving nutrition. The protein content and amino acid profile of manketti seed kernel were studied, using a new analytical method, in order to evaluate the nutritional value. The crude protein content of the press cake and defatted kernel flour was 29.0% and 67.5%, respectively. Leucine and arginine were found to be the most abundant essential and non-essential amino acids, respectively. The seed kernel contained 4.77 g leucine and 12.34 g arginine/100 g of defatted seed kernel flour. Methionine and proline were the least abundant essential and non-essential amino acids to with 0.23 g methionine and 0.36 g proline/100 g of defatted seed kernel flour, respectively. Validation of the pre-column derivatisation procedure with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) for the determination of amino acids was carried out. The analytical parameters were determined: linearity (0.0025-0.20 mM), accuracy of the derivatisation procedure: 86.7-109.8%, precision (method: 0.72-5.04%, instrumental: 0.14-1.88% and derivatisation: 0.15-2.94% and 0.41-4.32% for intraday and interday, respectively). Limits of detection and quantification were 6.80-157 mg/100 g and 22.7-523 mg/100 g kernel flour, respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Designing Light-Activated Charge-Separating Proteins with a Naphthoquinone Amino Acid

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lichtenstein, Bruce R.; Bialas, Chris; Cerda, José F.

    2015-09-14

    The first principles design of manmade redox-protein maquettes is used to clarify the physical/chemical engineering supporting the mechanisms of natural enzymes with a view to recapitulate and surpass natural performance. Herein, we use intein-based protein semisynthesis to pair a synthetic naphthoquinone amino acid (Naq) with histidine-ligated photoactive metal–tetrapyrrole cofactors, creating a 100 μs photochemical charge separation unit akin to photosynthetic reaction centers. By using propargyl groups to protect the redox-active para-quinone during synthesis and assembly while permitting selective activation, we gain the ability to employ the quinone amino acid redox cofactor with the full set of natural amino acids inmore » protein design. Direct anchoring of quinone to the protein backbone permits secure and adaptable control of intraprotein electron-tunneling distances and rates.« less

  3. The content of protein and of amino acids in Jerusalem artichoke tubers (Helianthus tuberosus L.) of red variety Rote Zonenkugel.

    PubMed

    Cieślik, Ewa; Gębusia, Agnieszka; Florkiewicz, Adam; Mickowska, Barbara

    2011-01-01

    Jerusalem artichoke (Helianthus tuberosus L.) is grown primarily for its edible tubers, which were first cultivated by native Americans before the arrival of the Europeans. Unlike most tubers, but in common with other members of the Asteraceae, the tubers store fructans instead of starch. Fructans are non-digestible carbohydrates considered functional food ingredients because they affect body processes in ways that result in better health and in many diseases prevention. However, the Jerusalem artichoke deserves attention not only because of the content of fructans, recent studies also indicate a high protein content, including essential amino acids. The aim of the work was to establish the content of protein and amino acids in Jerusalem artichoke tubers (Helianthus tuberosus L.) of red variety--Rote Zonenkugel. The content of protein was estimated by Dumas method. The amino acids composition was analysed with ion-change chromatography with postcolumn derivatisation and detection of ninhydryn reaction with automatic amino acids analyser. The assessed liophylisate was characterised by high protein content (6.36%) in comparison to chicory (which is the main industrial source of fructans) and to commonly consumed potatoes. There was shown a few times higher content of essential amino acids (also of methionine) in comparison to chicory and potato. The examined essential amino acids were present in very advantagenous proportions. In Jerusalem artichoke tubers of Rote Zonenkugel variety of the high content of protein was established in comparison to other plant sources. The high content was found of amino acids with special stress on essential amino acids (esp. sulphur ones).

  4. Separomics applied to the proteomics and peptidomics of low-abundance proteins: Choice of methods and challenges – A review

    PubMed Central

    Baracat-Pereira, Maria Cristina; de Oliveira Barbosa, Meire; Magalhães, Marcos Jorge; Carrijo, Lanna Clicia; Games, Patrícia Dias; Almeida, Hebréia Oliveira; Sena Netto, José Fabiano; Pereira, Matheus Rodrigues; de Barros, Everaldo Gonçalves

    2012-01-01

    The enrichment and isolation of proteins are considered limiting steps in proteomic studies. Identification of proteins whose expression is transient, those that are of low-abundance, and of natural peptides not described in databases, is still a great challenge. Plant extracts are in general complex, and contaminants interfere with the identification of proteins involved in important physiological processes, such as plant defense against pathogens. This review discusses the challenges and strategies of separomics applied to the identification of low-abundance proteins and peptides in plants, especially in plants challenged by pathogens. Separomics is described as a group of methodological strategies for the separation of protein molecules for proteomics. Several tools have been used to remove highly abundant proteins from samples and also non-protein contaminants. The use of chromatographic techniques, the partition of the proteome into subproteomes, and an effort to isolate proteins in their native form have allowed the isolation and identification of rare proteins involved in different processes. PMID:22802713

  5. Prediction of mitochondrial proteins of malaria parasite using split amino acid composition and PSSM profile.

    PubMed

    Verma, Ruchi; Varshney, Grish C; Raghava, G P S

    2010-06-01

    The rate of human death due to malaria is increasing day-by-day. Thus the malaria causing parasite Plasmodium falciparum (PF) remains the cause of concern. With the wealth of data now available, it is imperative to understand protein localization in order to gain deeper insight into their functional roles. In this manuscript, an attempt has been made to develop prediction method for the localization of mitochondrial proteins. In this study, we describe a method for predicting mitochondrial proteins of malaria parasite using machine-learning technique. All models were trained and tested on 175 proteins (40 mitochondrial and 135 non-mitochondrial proteins) and evaluated using five-fold cross validation. We developed a Support Vector Machine (SVM) model for predicting mitochondrial proteins of P. falciparum, using amino acids and dipeptides composition and achieved maximum MCC 0.38 and 0.51, respectively. In this study, split amino acid composition (SAAC) is used where composition of N-termini, C-termini, and rest of protein is computed separately. The performance of SVM model improved significantly from MCC 0.38 to 0.73 when SAAC instead of simple amino acid composition was used as input. In addition, SVM model has been developed using composition of PSSM profile with MCC 0.75 and accuracy 91.38%. We achieved maximum MCC 0.81 with accuracy 92% using a hybrid model, which combines PSSM profile and SAAC. When evaluated on an independent dataset our method performs better than existing methods. A web server PFMpred has been developed for predicting mitochondrial proteins of malaria parasites ( http://www.imtech.res.in/raghava/pfmpred/).

  6. Unusual glycosylation of proteins: Beyond the universal sequon and other amino acids.

    PubMed

    Dutta, Devawati; Mandal, Chhabinath; Mandal, Chitra

    2017-12-01

    Glycosylation of proteins is the most common, multifaceted co- and post-translational modification responsible for many biological processes and cellular functions. Significant alterations and aberrations of these processes are related to various pathological conditions, and often turn out to be disease biomarkers. Conventional N-glycosylation occurs through the recognition of the consensus sequon, asparagine (Asn)-X-serine (Ser)/threonine (Thr), where X is any amino acid except for proline, with N-acetylglucosamine (GlcNAc) as the first glycosidic linkage. Usually, O-glycosylation adds a glycan to the hydroxyl group of Ser or Thr beginning with N-acetylgalactosamine (GalNAc). Protein glycosylation is further governed by additional diversifications in sequon and structure, which are yet to be fully explored. This review mainly focuses on the occurrence of N-glycosylation in non-consensus motifs, where Ser/Thr at the +2 position is substituted by other amino acids. Additionally, N-glycosylation is also observed in other amide/amine group-containing amino acids. Similarly, O-glycosylation occurs at hydroxyl group-containing amino acids other than serine/threonine. The neighbouring amino acids and local structural features around the potential glycosylation site also play a significant role in determining the extent of glycosylation. All of these phenomena that yield glycosylation at the atypical sites are reported in a variety of biological systems, including different pathological conditions. Therefore, the discovery of more novel sequence patterns for N- and O-glycosylation may help in understanding the functions of complex biological processes and cellular functions. Taken together, all these information provided in this review would be helpful for the biological readers. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. The adsorption of amino acids and cations onto goethite: a prebiotic chemistry experiment.

    PubMed

    Farias, Ana Paula S F; Carneiro, Cristine E A; de Batista Fonseca, Inês C; Zaia, Cássia T B V; Zaia, Dimas A M

    2016-06-01

    Few prebiotic chemistry experiments have assessed the adsorption of biomolecules by iron oxide-hydroxides. The present work investigated the effects of cations in artificial seawaters on the adsorption of Gly, α-Ala and β-Ala onto goethite, and vice versa. Goethite served to concentrate K and Mg cations from solution; these effects could have played important roles in peptide nucleoside formation. Goethite showed low adsorption of Gly and α-Ala. On the other hand, β-Ala (a non-protein amino acid) was highly adsorbed by goethite. Because Gly and α-Ala are the most common amino acids in living beings, and iron oxide-hydroxides are widespread on Earth, additional iron oxides should be studied. Increased ionic strength in artificial seawaters decreased the adsorption of amino acids by goethite. Because Na was highly abundant in the artificial seawater, it showed the highest effect on amino acid adsorption. β-Ala increased the adsorption of K and Ca by goethite, this effect could have been important for peptide synthesis.

  8. Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids.

    PubMed

    Nickling, Jessica H; Baumann, Tobias; Schmitt, Franz-Josef; Bartholomae, Maike; Kuipers, Oscar P; Friedrich, Thomas; Budisa, Nediljko

    2018-05-04

    Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) → ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell's resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants

  9. Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

    DOEpatents

    Wang, Jiangyun; Schultz, Peter G.

    2010-09-07

    The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

  10. Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

    DOEpatents

    Wang, Jiangyun; Schultz, Peter G.

    2012-07-10

    The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

  11. [Analysis of proteins, amino acids and inorganic elements in Holotrichia diomphalia from different areas].

    PubMed

    Cao, Wei; Liu, Dan; Zhang, Yi-Kai; Wang, Xiao-Yu; Chang, Yan-Rong; Yang, Qian; Wang, Si-Wang

    2010-10-01

    To analyze the content of proteins,amino acids and inorganic elements of Holotrichia diomphalia in different growing areas as the references for quality evaluation and reasonable application of them. The contents of proteins were determined using semi-micro Kjeldahl method. The contents of seventeen amino acids and inorganic elements were determined with amino acid analyzer and atomic absorption spectrometer and elemental analyzer, respectively. The contents of protein were 33.4%-44.4%, and that in Jiangxi were the highest in five different areas. There were seventeen kinds of amino acids in Holotrichia diomphalia. Among them, seven amino acids were essential to human life. The content of glutamic acid was the highest in seventeen amino acids. In inorganic elements, the content of Mg, Ca was higher in macroelements and Fe, Zn was higher in microelements. There are many kinds of necessary amino acids and inorganic elements for man kind in Holotrichia diomphalia. The contents of proteins, amino acids and inorganic elements have some difference in Holotrichia diomphalia of different growing areas.

  12. AFAL: a web service for profiling amino acids surrounding ligands in proteins

    NASA Astrophysics Data System (ADS)

    Arenas-Salinas, Mauricio; Ortega-Salazar, Samuel; Gonzales-Nilo, Fernando; Pohl, Ehmke; Holmes, David S.; Quatrini, Raquel

    2014-11-01

    With advancements in crystallographic technology and the increasing wealth of information populating structural databases, there is an increasing need for prediction tools based on spatial information that will support the characterization of proteins and protein-ligand interactions. Herein, a new web service is presented termed amino acid frequency around ligand (AFAL) for determining amino acids type and frequencies surrounding ligands within proteins deposited in the Protein Data Bank and for assessing the atoms and atom-ligand distances involved in each interaction (availability: http://structuralbio.utalca.cl/AFAL/index.html). AFAL allows the user to define a wide variety of filtering criteria (protein family, source organism, resolution, sequence redundancy and distance) in order to uncover trends and evolutionary differences in amino acid preferences that define interactions with particular ligands. Results obtained from AFAL provide valuable statistical information about amino acids that may be responsible for establishing particular ligand-protein interactions. The analysis will enable investigators to compare ligand-binding sites of different proteins and to uncover general as well as specific interaction patterns from existing data. Such patterns can be used subsequently to predict ligand binding in proteins that currently have no structural information and to refine the interpretation of existing protein models. The application of AFAL is illustrated by the analysis of proteins interacting with adenosine-5'-triphosphate.

  13. AFAL: a web service for profiling amino acids surrounding ligands in proteins.

    PubMed

    Arenas-Salinas, Mauricio; Ortega-Salazar, Samuel; Gonzales-Nilo, Fernando; Pohl, Ehmke; Holmes, David S; Quatrini, Raquel

    2014-11-01

    With advancements in crystallographic technology and the increasing wealth of information populating structural databases, there is an increasing need for prediction tools based on spatial information that will support the characterization of proteins and protein-ligand interactions. Herein, a new web service is presented termed amino acid frequency around ligand (AFAL) for determining amino acids type and frequencies surrounding ligands within proteins deposited in the Protein Data Bank and for assessing the atoms and atom-ligand distances involved in each interaction (availability: http://structuralbio.utalca.cl/AFAL/index.html ). AFAL allows the user to define a wide variety of filtering criteria (protein family, source organism, resolution, sequence redundancy and distance) in order to uncover trends and evolutionary differences in amino acid preferences that define interactions with particular ligands. Results obtained from AFAL provide valuable statistical information about amino acids that may be responsible for establishing particular ligand-protein interactions. The analysis will enable investigators to compare ligand-binding sites of different proteins and to uncover general as well as specific interaction patterns from existing data. Such patterns can be used subsequently to predict ligand binding in proteins that currently have no structural information and to refine the interpretation of existing protein models. The application of AFAL is illustrated by the analysis of proteins interacting with adenosine-5'-triphosphate.

  14. A Complex Prime Numerical Representation of Amino Acids for Protein Function Comparison.

    PubMed

    Chen, Duo; Wang, Jiasong; Yan, Ming; Bao, Forrest Sheng

    2016-08-01

    Computationally assessing the functional similarity between proteins is an important task of bioinformatics research. It can help molecular biologists transfer knowledge on certain proteins to others and hence reduce the amount of tedious and costly benchwork. Representation of amino acids, the building blocks of proteins, plays an important role in achieving this goal. Compared with symbolic representation, representing amino acids numerically can expand our ability to analyze proteins, including comparing the functional similarity of them. Among the state-of-the-art methods, electro-ion interaction pseudopotential (EIIP) is widely adopted for the numerical representation of amino acids. However, it could suffer from degeneracy that two different amino acid sequences have the same numerical representation, due to the design of EIIP. In light of this challenge, we propose a complex prime numerical representation (CPNR) of amino acids, inspired by the similarity between a pattern among prime numbers and the number of codons of amino acids. To empirically assess the effectiveness of the proposed method, we compare CPNR against EIIP. Experimental results demonstrate that the proposed method CPNR always achieves better performance than EIIP. We also develop a framework to combine the advantages of CPNR and EIIP, which enables us to improve the performance and study the unique characteristics of different representations.

  15. Advances in protein-amino acid nutrition of poultry.

    PubMed

    Baker, David H

    2009-05-01

    The ideal protein concept has allowed progress in defining requirements as well as the limiting order of amino acids in corn, soybean meal, and a corn-soybean meal mixture for growth of young chicks. Recent evidence suggests that glycine (or serine) is a key limiting amino acid in reduced protein [23% crude protein (CP) reduced to 16% CP] corn-soybean meal diets for broiler chicks. Research with sulfur amino acids has revealed that small excesses of cysteine are growth depressing in chicks fed methionine-deficient diets. Moreover, high ratios of cysteine:methionine impair utilization of the hydroxy analog of methionine, but not of methionine itself. A high level of dietary L: -cysteine (2.5% or higher) is lethal for young chicks, but a similar level of DL: -methionine, L: -cystine or N-acetyl-L: -cysteine causes no mortality. A supplemental dietary level of 3.0% L: -cysteine (7x requirement) causes acute metabolic acidosis that is characterized by a striking increase in plasma sulfate and decrease in plasma bicarbonate. S-Methylmethionine, an analog of S-adenosylmethionine, has been shown to have choline-sparing activity, but it only spares methionine when diets are deficient in choline and(or) betaine. Creatine, or its precursor guanidinoacetic acid, can spare dietary arginine in chicks.

  16. Hydrogen production using amino acids obtained by protein degradation in waste biomass by combined dark- and photo-fermentation.

    PubMed

    Cheng, Jun; Ding, Lingkan; Xia, Ao; Lin, Richen; Li, Yuyou; Zhou, Junhu; Cen, Kefa

    2015-03-01

    The biological hydrogen production from amino acids obtained by protein degradation was comprehensively investigated to increase heating value conversion efficiency. The five amino acids (i.e., alanine, serine, aspartic acid, arginine, and leucine) produced limited hydrogen (0.2-16.2 mL/g) but abundant soluble metabolic products (40.1-84.0 mM) during dark-fermentation. The carbon conversion efficiencies of alanine (85.3%) and serine (94.1%) during dark-fermentation were significantly higher than those of other amino acids. Residual dark-fermentation solutions treated with zeolite for NH4(+) removal were inoculated with photosynthetic bacteria to further produce hydrogen during photo-fermentation. The hydrogen yields of alanine and serine through combined dark- and photo-fermentation were 418.6 and 270.2 mL/g, respectively. The heating value conversion efficiency of alanine to hydrogen was 25.1%, which was higher than that of serine (21.2%). Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids

    PubMed Central

    Hesse, Almut

    2016-01-01

    Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports. PMID:27559481

  18. Biochemical studies of some non-conventional sources of proteins. Part 7. Effect of detoxification treatments on the nutritional quality of apricot kernels.

    PubMed

    el-Adawy, T A; Rahma, E H; el-Badawey, A A; Gomaa, M A; Lásztity, R; Sarkadi, L

    1994-01-01

    Detoxification of apricot kernels by soaking in distilled water and ammonium hydroxide for 30 h at 47 degrees C decreased the total protein, non-protein nitrogen, total ash, glucose, sucrose, minerals, non-essential amino acids, polar amino acids, acidic amino acids, aromatic amino acids, antinutritional factors, hydrocyanic acid, tannins and phytic acid. On the other hand, removal of toxic and bitter compounds from apricot kernels increased the relative content of crude fibre, starch, total essential amino acids. Higher in-vitro protein digestibility and biological value was also observed. Generally, the detoxified apricot kernels were nutritionally well balanced. Utilization and incorporation of detoxified apricot kernel flours in food products is completely safe from the toxicity point of view.

  19. Mitochondrial genetic codes evolve to match amino acid requirements of proteins.

    PubMed

    Swire, Jonathan; Judson, Olivia P; Burt, Austin

    2005-01-01

    Mitochondria often use genetic codes different from the standard genetic code. Now that many mitochondrial genomes have been sequenced, these variant codes provide the first opportunity to examine empirically the processes that produce new genetic codes. The key question is: Are codon reassignments the sole result of mutation and genetic drift? Or are they the result of natural selection? Here we present an analysis of 24 phylogenetically independent codon reassignments in mitochondria. Although the mutation-drift hypothesis can explain reassignments from stop to an amino acid, we found that it cannot explain reassignments from one amino acid to another. In particular--and contrary to the predictions of the mutation-drift hypothesis--the codon involved in such a reassignment was not rare in the ancestral genome. Instead, such reassignments appear to take place while the codon is in use at an appreciable frequency. Moreover, the comparison of inferred amino acid usage in the ancestral genome with the neutral expectation shows that the amino acid gaining the codon was selectively favored over the amino acid losing the codon. These results are consistent with a simple model of weak selection on the amino acid composition of proteins in which codon reassignments are selected because they compensate for multiple slightly deleterious mutations throughout the mitochondrial genome. We propose that the selection pressure is for reduced protein synthesis cost: most reassignments give amino acids that are less expensive to synthesize. Taken together, our results strongly suggest that mitochondrial genetic codes evolve to match the amino acid requirements of proteins.

  20. Probing Protein Structure by Amino Acid-Specific Covalent Labeling and Mass Spectrometry

    PubMed Central

    Mendoza, Vanessa Leah; Vachet, Richard W.

    2009-01-01

    For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies. PMID:19016300

  1. Gross and true ileal digestible amino acid contents of several animal body proteins and their hydrolysates.

    PubMed

    Cui, J; Chong, B; Rutherfurd, S M; Wilkinson, B; Singh, H; Moughan, P J

    2013-07-01

    Amino acid compositions of ovine muscle, ovine myofibrillar protein, ovine spleen, ovine liver, bovine blood plasma, bovine blood globulins and bovine serum albumin and the amino acid compositions and in vivo (laboratory rat) true ileal amino acid digestibilities of hydrolysates (sequential hydrolysis with Neutrase, Alcalase and Flavourzyme) of these protein sources were determined. True ileal amino acid digestibility differed (P<0.05) among the seven protein hydrolysates. The ovine myofibrillar protein and liver hydrolysates were the most digestible, with a mean true ileal digestibility across all amino acids of 99%. The least digestible protein hydrolysate was bovine serum albumin with a comparable mean true ileal digestibility of 93%. When the digestible amino acid contents were expressed as proportions relative to lysine, considerable differences, across the diverse protein sources, were found in the pattern of predicted absorbed amino acids. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Role of the Tomato Non-Ripening Mutation in Regulating Fruit Quality Elucidated Using iTRAQ Protein Profile Analysis

    PubMed Central

    Yuan, Xin-Yu; Wang, Rui-Heng; Zhao, Xiao-Dan; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Natural mutants of the Non-ripening (Nor) gene repress the normal ripening of tomato fruit. The molecular mechanism of fruit ripening regulation by the Nor gene is unclear. To elucidate how the Nor gene can affect ripening and fruit quality at the protein level, we used the fruits of Nor mutants and wild-type Ailsa Craig (AC) to perform iTRAQ (isobaric tags for relative and absolute quantitation) analysis. The Nor mutation altered tomato fruit ripening and affected quality in various respects, including ethylene biosynthesis by down-regulating the abundance of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), pigment biosynthesis by repressing phytoene synthase 1 (PSY1), ζ-carotene isomerase (Z-ISO), chalcone synthase 1 (CHS1) and other proteins, enhancing fruit firmness by increasing the abundance of cellulose synthase protein, while reducing those of polygalacturonase 2 (PG2) and pectate lyase (PL), altering biosynthesis of nutrients such as carbohydrates, amino acids, and anthocyanins. Conversely, Nor mutation also enhanced the fruit’s resistance to some pathogens by up-regulating the expression of several genes associated with stress and defense. Therefore, the Nor gene is involved in the regulation of fruit ripening and quality. It is useful in the future as a means to improve fruit quality in tomato. PMID:27732677

  3. Influence of the Amino Acid Sequence on Protein-Mineral Interactions in Soil

    NASA Astrophysics Data System (ADS)

    Chacon, S. S.; Reardon, P. N.; Purvine, S.; Lipton, M. S.; Washton, N.; Kleber, M.

    2017-12-01

    The intimate associations between protein and mineral surfaces have profound impacts on nutrient cycling in soil. Proteins are an important source of organic C and N, and a subset of proteins, extracellular enzymes (EE), can catalyze the depolymerization of soil organic matter (SOM). Our goal was to determine how variation in the amino acid sequence could influence a protein's susceptibility to become chemically altered by mineral surfaces to infer the fate of adsorbed EE function in soil. We hypothesized that (1) addition of charged amino acids would enhance the adsorption onto oppositely charged mineral surfaces (2) addition of aromatic amino acids would increase adsorption onto zero charged surfaces (3) Increase adsorption of modified proteins would enhance their susceptibility to alterations by redox active minerals. To test these hypotheses, we generated three engineered proxies of a model protein Gb1 (IEP 4.0, 6.2 kDA) by inserting either negatively charged, positively charged or aromatic amino acids in the second loop. These modified proteins were allowed to interact with functionally different mineral surfaces (goethite, montmorillonite, kaolinite and birnessite) at pH 5 and 7. We used LC-MS/MS and solution-state Heteronuclear Single Quantum Coherence Spectroscopy NMR to observe modifications on engineered proteins as a consequence to mineral interactions. Preliminary results indicate that addition of any amino acids to a protein increase its susceptibility to fragmentation and oxidation by redox active mineral surfaces, and alter adsorption to the other mineral surfaces. This suggest that not all mineral surfaces in soil may act as sorbents for EEs and chemical modification of their structure should also be considered as an explanation for decrease in EE activity. Fragmentation of proteins by minerals can bypass the need to produce proteases, but microbial acquisition of other nutrients that require enzymes such as cellulases, ligninases or phosphatases

  4. Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

    DOEpatents

    Wang, Jiangyun; Schultz, Peter G.

    2013-03-12

    The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetase that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel sythetases molecules, methods for identifying and making the novel synthetases, methods for producing containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lapidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

  5. Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa.

    PubMed

    Trevino, Saul R; Scholtz, J Martin; Pace, C Nick

    2007-02-16

    Poor protein solubility is a common problem in high-resolution structural studies, formulation of protein pharmaceuticals, and biochemical characterization of proteins. One popular strategy to improve protein solubility is to use site-directed mutagenesis to make hydrophobic to hydrophilic mutations on the protein surface. However, a systematic investigation of the relative contributions of all 20 amino acids to protein solubility has not been done. Here, 20 variants at the completely solvent-exposed position 76 of ribonuclease (RNase) Sa are made to compare the contributions of each amino acid. Stability measurements were also made for these variants, which occur at the i+1 position of a type II beta-turn. Solubility measurements in ammonium sulfate solutions were made at high positive net charge, low net charge, and high negative net charge. Surprisingly, there was a wide range of contributions to protein solubility even among the hydrophilic amino acids. The results suggest that aspartic acid, glutamic acid, and serine contribute significantly more favorably than the other hydrophilic amino acids especially at high net charge. Therefore, to increase protein solubility, asparagine, glutamine, or threonine should be replaced with aspartic acid, glutamic acid or serine.

  6. Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa

    PubMed Central

    Trevino, Saul R.; Scholtz, J. Martin; Pace, C. Nick

    2009-01-01

    SUMMARY Poor protein solubility is a common problem in high resolution structural studies, formulation of protein pharmaceuticals, and biochemical characterization of proteins. One popular strategy to improve protein solubility is to use site-directed mutagenesis to make hydrophobic to hydrophilic mutations on the protein surface. However, a systematic investigation of the relative contributions of all twenty amino acids to protein solubility has not been done. Here, twenty variants at the completely solvent-exposed position 76 of Ribonuclease (RNase) Sa are made to compare the contributions of each amino acid. Stability measurements were also made for these variants, which occur at the i+1 position of a type II β-turn. Solubility measurements in ammonium sulfate solutions were made at high positive net charge, low net charge, and high negative net charge. Surprisingly, there was a wide range of contributions to protein solubility even among the hydrophilic amino acids. The results suggest that aspartic acid, glutamic acid, and serine contribute significantly more favorably than the other hydrophilic amino acids especially at high net charge. Therefore, to increase protein solubility, asparagine, glutamine, or threonine should be replaced with aspartic acid, glutamic acid or serine. PMID:17174328

  7. A nine-country study of the protein content and amino acid composition of mature human milk

    PubMed Central

    Feng, Ping; Gao, Ming; Burgher, Anita; Zhou, Tian Hui; Pramuk, Kathryn

    2016-01-01

    Background Numerous studies have evaluated protein and amino acid levels in human milk. However, research in this area has been limited by small sample sizes and study populations with little ethnic or racial diversity. Objective Evaluate the protein and amino acid composition of mature (≥30 days) human milk samples collected from a large, multinational study using highly standardized methods for sample collection, storage, and analysis. Design Using a single, centralized laboratory, human milk samples from 220 women (30–188 days postpartum) from nine countries were analyzed for amino acid composition using Waters AccQ-Tag high-performance liquid chromatography and total nitrogen content using the LECO FP-528 nitrogen analyzer. Total protein was calculated as total nitrogen×6.25. True protein, which includes protein, free amino acids, and peptides, was calculated from the total amino acids. Results Mean total protein from individual countries (standard deviation [SD]) ranged from 1,133 (125.5) to 1,366 (341.4) mg/dL; the mean across all countries (SD) was 1,192 (200.9) mg/dL. Total protein, true protein, and amino acid composition were not significantly different across countries except Chile, which had higher total and true protein. Amino acid profiles (percent of total amino acids) did not differ across countries. Total and true protein concentrations and 16 of 18 amino acid concentrations declined with the stage of lactation. Conclusions Total protein, true protein, and individual amino acid concentrations in human milk steadily decline from 30 to 151 days of lactation, and are significantly higher in the second month of lactation compared with the following 4 months. There is a high level of consistency in the protein content and amino acid composition of human milk across geographic locations. The size and diversity of the study population and highly standardized procedures for the collection, storage, and analysis of human milk support the validity and

  8. Prey-induced changes in the accumulation of amino acids and phenolic metabolites in the leaves of Drosera capensis L.

    PubMed

    Kováčik, Jozef; Klejdus, Bořivoj; Stork, František; Hedbavny, Josef

    2012-04-01

    Effect of prey feeding (ants Formica fusca) on the quantitative changes in the accumulation of free amino acids, soluble proteins, phenolic metabolites and mineral nutrients in the leaves of carnivorous plant Drosera capensis was studied. Arginine was the most abundant compound in Drosera leaves, while proline was abundant in ants. The amount of the majority of amino acids and their sum were elevated in the fed leaves after 3 and 21 days, and the same, but with further enhancement after 21 days, was observed in ants. Accumulation of amino acids also increased in young non-fed leaves of fed plants. Soluble proteins decreased in ants, but were not enhanced in fed leaves. This confirms the effectiveness of sundew's enzymatic machinery in digestion of prey and suggests that amino acids are not in situ deposited, but rather are allocated within the plant. The content of total soluble phenols, flavonoids and two selected flavonols (quercetin and kaempferol) was not affected by feeding in Drosera leaves, indicating that their high basal level was sufficient for the plant's metabolism and prey-induced changes were mainly N based. The prey also showed to be an important source of other nutrients besides N, and a stimulation of root uptake of some mineral nutrients is assumed (Mg, Cu, Zn). Accumulation of Ca and Na was not affected by feeding.

  9. Differentially abundant proteins associated with heterosis in the primary roots of popcorn.

    PubMed

    Rockenbach, Mathias F; Corrêa, Caio C G; Heringer, Angelo S; Freitas, Ismael L J; Santa-Catarina, Claudete; do Amaral-Júnior, Antônio T; Silveira, Vanildo

    2018-01-01

    Although heterosis has significantly contributed to increases in worldwide crop production, the molecular mechanisms regulating this phenomenon are still unknown. In the present study, we used a comparative proteomic approach to explore hybrid vigor via the proteome of both the popcorn L54 ♀ and P8 ♂ genotypes and the resultant UENF/UEM01 hybrid cross. To analyze the differentially abundant proteins involved in heterosis, we used the primary roots of these genotypes to analyze growth parameters and extract proteins. The results of the growth parameter analysis showed that the mid- and best-parent heterosis were positive for root length and root dry matter but negative for root fresh matter, seedling fresh matter, and protein content. The comparative proteomic analysis identified 1343 proteins in the primary roots of hybrid UENF/UEM01 and its parental lines; 220 proteins were differentially regulated in terms of protein abundance. The mass spectrometry proteomic data are available via ProteomeXchange with identifier "PXD009436". A total of 62 regulated proteins were classified as nonadditive, of which 53.2% were classified as high parent abundance (+), 17.8% as above-high parent abundance (+ +), 16.1% as below-low parent abundance (- -), and 12.9% as low parent abundance (-). A total of 22 biological processes were associated with nonadditive proteins; processes involving translation, ribosome biogenesis, and energy-related metabolism represented 45.2% of the nonadditive proteins. Our results suggest that heterosis in the popcorn hybrid UENF/UEM01 at an early stage of plant development is associated with an up-regulation of proteins related to synthesis and energy metabolism.

  10. Free amino acids in spider hemolymph.

    PubMed

    Tillinghast, Edward K; Townley, Mark A

    2008-11-01

    We examined the free amino acid composition of hemolymph from representatives of five spider families with an interest in knowing if the amino acid profile in the hemolymph of orb-web-building spiders reflects the high demands for small organic compounds in the sticky droplets of their webs. In nearly all analyses, on both orb and non-orb builders, glutamine was the most abundant free amino acid. Glycine, taurine, proline, histidine, and alanine also tended to be well-represented in orb and non-orb builders. While indications of taxon-specific differences in amino acid composition were observed, it was not apparent that two presumptive precursors (glutamine, taurine) of orb web sticky droplet compounds were uniquely enriched in araneids (orb builders). However, total amino acid concentrations were invariably highest in the araneids and especially so in overwintering juveniles, even as several of the essential amino acids declined during this winter diapause. Comparing the data from this study with those from earlier studies revealed a number of discrepancies. The possible origins of these differences are discussed.

  11. Meteoritic Amino Acids: Diversity in Compositions Reflects Parent Body Histories

    NASA Technical Reports Server (NTRS)

    Elsila, Jamie E.; Aponte, Jose C.; Blackmond, Donna G.; Burton, Aaron S.; Dworkin, Jason P.; Glavin, Daniel P.

    2016-01-01

    The analysis of amino acids in meteorites dates back over 50 years; however, it is only in recent years that research has expanded beyond investigations of a narrow set of meteorite groups (exemplied by the Murchison meteorite) into meteorites of other types and classes. These new studies have shown a wide diversity in the abundance and distribution of amino acids across carbonaceous chondrite groups, highlighting the role of parent body processes and composition in the creation, preservation, or alteration of amino acids. Although most chiral amino acids are racemic in meteorites, the enantiomeric distribution of some amino acids, particularly of the nonprotein amino acid isovaline, has also been shown to vary both within certain meteorites and across carbonaceous meteorite groups. Large -enantiomeric excesses of some extraterrestrial protein amino acids (up to 60) have also been observed in rare cases and point to nonbiological enantiomeric enrichment processes prior to the emergence of life. In this Outlook, we review these recent meteoritic analyses, focusing on variations in abundance, structural distributions, and enantiomeric distributions of amino acids and discussing possible explanations for these observations and the potential for future work.

  12. Meteoritic Amino Acids: Diversity in Compositions Reflects Parent Body Histories

    PubMed Central

    2016-01-01

    The analysis of amino acids in meteorites dates back over 50 years; however, it is only in recent years that research has expanded beyond investigations of a narrow set of meteorite groups (exemplified by the Murchison meteorite) into meteorites of other types and classes. These new studies have shown a wide diversity in the abundance and distribution of amino acids across carbonaceous chondrite groups, highlighting the role of parent body processes and composition in the creation, preservation, or alteration of amino acids. Although most chiral amino acids are racemic in meteorites, the enantiomeric distribution of some amino acids, particularly of the nonprotein amino acid isovaline, has also been shown to vary both within certain meteorites and across carbonaceous meteorite groups. Large l-enantiomeric excesses of some extraterrestrial protein amino acids (up to ∼60%) have also been observed in rare cases and point to nonbiological enantiomeric enrichment processes prior to the emergence of life. In this Outlook, we review these recent meteoritic analyses, focusing on variations in abundance, structural distributions, and enantiomeric distributions of amino acids and discussing possible explanations for these observations and the potential for future work. PMID:27413780

  13. Structural Insight into Amino Group-carrier Protein-mediated Lysine Biosynthesis

    PubMed Central

    Yoshida, Ayako; Tomita, Takeo; Fujimura, Tsutomu; Nishiyama, Chiharu; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2015-01-01

    In the biosynthesis of lysine by Thermus thermophilus, the metabolite α-ketoglutarate is converted to the intermediate α-aminoadipate (AAA), which is protected by the 54-amino acid acidic protein LysW. In this study, we determined the crystal structure of LysZ from T. thermophilus (TtLysZ), an amino acid kinase that catalyzes the second step in the AAA to lysine conversion, which was in a complex with LysW at a resolution of 1.85 Å. A crystal analysis coupled with isothermal titration calorimetry of the TtLysZ mutants for TtLysW revealed tight interactions between LysZ and the globular and C-terminal extension domains of the LysW protein, which were mainly attributed to electrostatic forces. These results provided structural evidence for LysW acting as a protecting molecule for the α-amino group of AAA and also as a carrier protein to guarantee better recognition by biosynthetic enzymes for the efficient biosynthesis of lysine. PMID:25392000

  14. Conversion of proteins from a non-polarized to an apical secretory pattern in MDCK cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vogel, Lotte K.; Larsen, Jakob E.; Hansen, Martin

    2005-05-13

    Previously it was shown that fusion proteins containing the amino terminus of an apical targeted member of the serpin family fused to the corresponding carboxyl terminus of the non-polarized secreted serpin, antithrombin, are secreted mainly to the apical side of MDCK cells. The present study shows that this is neither due to the transfer of an apical sorting signal from the apically expressed proteins, since a sequence of random amino acids acts the same, nor is it due to the deletion of a conserved signal for correct targeting from the non-polarized secreted protein. Our results suggest that the polarity ofmore » secretion is determined by conformational sensitive sorting signals.« less

  15. Fluorescence Modulation of Green Fluorescent Protein Using Fluorinated Unnatural Amino Acids.

    PubMed

    Villa, Jordan K; Tran, Hong-Anh; Vipani, Megha; Gianturco, Stephanie; Bhasin, Konark; Russell, Brent L; Harbron, Elizabeth J; Young, Douglas D

    2017-07-16

    The ability to modulate protein function through minimal perturbations to amino acid structure represents an ideal mechanism to engineer optimized proteins. Due to the novel spectroscopic properties of green fluorescent protein, it has found widespread application as a reporter protein throughout the fields of biology and chemistry. Using site-specific amino acid mutagenesis, we have incorporated various fluorotyrosine residues directly into the fluorophore of the protein, altering the fluorescence and shifting the pKa of the phenolic proton associated with the fluorophore. Relative to wild type GFP, the fluorescence spectrum of the protein is altered with each additional fluorine atom, and the mutant GFPs have the potential to be employed as pH sensors due to the altered electronic properties of the fluorine atoms.

  16. Diverse characteristics of the urinary excretion of amino acids in humans and the use of amino acid supplementation to reduce fatigue and sub-health in adults.

    PubMed

    Dunstan, R H; Sparkes, D L; Macdonald, M M; De Jonge, X Janse; Dascombe, B J; Gottfries, J; Gottfries, C-G; Roberts, T K

    2017-03-23

    The excretion of amino acids in urine represents an important avenue for the loss of key nutrients. Some amino acids such as glycine and histidine are lost in higher abundance than others. These two amino acids perform important physiological functions and are required for the synthesis of key proteins such as haemoglobin and collagen. Stage 1 of this study involved healthy subjects (n = 151) who provided first of the morning urine samples and completed symptom questionnaires. Urine was analysed for amino acid composition by gas chromatography. Stage 2 involved a subset of the initial cohort (n = 37) who completed a 30 day trial of an amino acid supplement and subsequent symptom profile evaluation. Analyses of urinary amino acid profiles revealed that three groups could be objectively defined from the 151 participants using k-means clustering. The amino acid profiles were significantly different between each of the clusters (Wilks' Lambda = 0.13, p < 0.0001). Cluster 1 had the highest loss of amino acids with histidine being the most abundant component. Cluster 2 had glycine present as the most abundant urinary amino acid and cluster 3 had equivalent abundances of glycine and histidine. Strong associations were observed between urinary proline concentrations and fatigue/pain scores (r = .56 to .83) for females in cluster 1, with several other differential sets of associations observed for the other clusters. Different phenotypic subsets exist in the population based on amino acid excretion characteristics found in urine. Provision of the supplement resulted in significant improvements in reported fatigue and sleep for 81% of the trial cohort with all females reporting improvements in fatigue. The study was registered on the 18th April 2011 with the Australian New Zealand Clinical Trials Registry ( ACTRN12611000403932 ).

  17. Differentially abundant proteins associated with heterosis in the primary roots of popcorn

    PubMed Central

    Heringer, Angelo S.; Freitas, Ismael L. J.; Santa-Catarina, Claudete; do Amaral-Júnior, Antônio T.

    2018-01-01

    Although heterosis has significantly contributed to increases in worldwide crop production, the molecular mechanisms regulating this phenomenon are still unknown. In the present study, we used a comparative proteomic approach to explore hybrid vigor via the proteome of both the popcorn L54 ♀ and P8 ♂ genotypes and the resultant UENF/UEM01 hybrid cross. To analyze the differentially abundant proteins involved in heterosis, we used the primary roots of these genotypes to analyze growth parameters and extract proteins. The results of the growth parameter analysis showed that the mid- and best-parent heterosis were positive for root length and root dry matter but negative for root fresh matter, seedling fresh matter, and protein content. The comparative proteomic analysis identified 1343 proteins in the primary roots of hybrid UENF/UEM01 and its parental lines; 220 proteins were differentially regulated in terms of protein abundance. The mass spectrometry proteomic data are available via ProteomeXchange with identifier “PXD009436”. A total of 62 regulated proteins were classified as nonadditive, of which 53.2% were classified as high parent abundance (+), 17.8% as above-high parent abundance (+ +), 16.1% as below-low parent abundance (− −), and 12.9% as low parent abundance (-). A total of 22 biological processes were associated with nonadditive proteins; processes involving translation, ribosome biogenesis, and energy-related metabolism represented 45.2% of the nonadditive proteins. Our results suggest that heterosis in the popcorn hybrid UENF/UEM01 at an early stage of plant development is associated with an up-regulation of proteins related to synthesis and energy metabolism. PMID:29758068

  18. [Comparison of acetonitrile, ethanol and chromatographic column to eliminate high-abundance proteins in human serum].

    PubMed

    Li, Yin; Liao, Ming; He, Xiao; Zhou, Yi; Luo, Rong; Li, Hongtao; Wang, Yun; He, Min

    2012-11-01

    To compare the effects of acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal to eliminate high-abundance proteins in human serum. Elimination of serum high-abundance proteins performed with acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal. Bis-Tris Mini Gels electrophoresis and two-dimensional gel electrophoresis to detect the effect. Grey value analysis from 1-DE figure showed that after serum processed by acetonitrile method, multiple affinity chromatography column Human 14 removal method and ethanol method, the grey value of albumin changed into 157.2, 40.8 and 8.2 respectively from the original value of 19. 2-DE analysis results indicated that using multiple affinity chromatography column Human 14 method, the protein points noticeable increased by 137 compared to the original serum. After processed by acetonitrile method and ethanol method, the protein point reduced, but the low abundance protein point emerged. The acetonitrile precipitation could eliminate the vast majority of high abundance proteins in serum and gain more proteins of low molecular weight, ethanol precipitation could eliminate part of high abundance proteins in serum, but low abundance proteins less harvested, and multiple affinity chromatography column Human 14 method could effectively removed the high abundance proteins, and keep a large number of low abundance proteins.

  19. Correlating Mineralogy and Amino Acid Contents of Milligram-Scale Murchison Carbonaceous Chondrite Samples

    NASA Technical Reports Server (NTRS)

    Burton, Aaron, S.; Berger, Eve L.; Locke, Darren R.; Elsila, Jamie E.; Glavin, Daniel P.; Dworkin, Jason P.

    2015-01-01

    Amino acids, the building blocks of proteins, have been found to be indigenous in most of the carbonaceous chondrite groups. The abundances of amino acids, as well as their structural, enantiomeric and isotopic compositions differ significantly among meteorites of different groups and petrologic types. This suggests that there is a link between parent-body conditions, mineralogy and the synthesis and preservation of amino acids (and likely other organic molecules). However, elucidating specific causes for the observed differences in amino acid composition has proven extremely challenging because samples analyzed for amino acids are typically much larger ((is) approximately 100 mg powders) than the scale at which meteorite heterogeneity is observed (sub mm-scale differences, (is) approximately 1-mg or smaller samples). Thus, the effects of differences in mineralogy on amino acid abundances could not be easily discerned. Recent advances in the sensitivity of instrumentation have made possible the analysis of smaller samples for amino acids, enabling a new approach to investigate the link between mineralogical con-text and amino acid compositions/abundances in meteorites. Through coordinated mineral separation, mineral characterization and highly sensitive amino acid analyses, we have performed preliminary investigations into the relationship between meteorite mineralogy and amino acid composition. By linking amino acid data to mineralogy, we have started to identify amino acid-bearing mineral phases in different carbonaceous meteorites. The methodology and results of analyses performed on the Murchison meteorite are presented here.

  20. A dominant conformational role for amino acid diversity in minimalist protein–protein interfaces

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gilbreth, Ryan N.; Esaki, Kaori; Koide, Akiko

    Recent studies have shown that highly simplified interaction surfaces consisting of combinations of just two amino acids, Tyr and Ser, exhibit high affinity and specificity. The high functional levels of such minimalist interfaces might thus indicate small contributions of greater amino acid diversity seen in natural interfaces. Toward addressing this issue, we have produced a pair of binding proteins built on the fibronectin type III scaffold, termed “monobodies.” One monobody contains the Tyr/Ser binary-code interface (termed YS) and the other contains an expanded amino acid diversity interface (YSX), but both bind to an identical target, maltose-binding protein. The YSX monobodymore » bound with higher affinity, a slower off rate and a more favorable enthalpic contribution than the YS monobody. High-resolution X-ray crystal structures revealed that both proteins bound to an essentially identical epitope, providing a unique opportunity to directly investigate the role of amino acid diversity in a protein interaction interface. Surprisingly, Tyr still dominates the YSX paratope and the additional amino acid types are primarily used to conformationally optimize contacts made by tyrosines. Scanning mutagenesis showed that while all contacting Tyr side chains are essential in the YS monobody, the YSX interface was more tolerant to mutations. These results suggest that the conformational, not chemical, diversity of additional types of amino acids provided higher functionality and evolutionary robustness, supporting the dominant role of Tyr and the importance of conformational diversity in forming protein interaction interfaces.« less

  1. A sensitive method for measuring protein turnover based on the measurement of 2-3H-labelled amino acids in protein.

    PubMed Central

    Humphrey, T J; Davies, D D

    1976-01-01

    A method for measuring the rate of protein degradation is described. The method measures the change in 2-3H content of protein with time by racemization of the protein hydrolysate with acetic anhydride. The 3H on C-2 of amino acids is stable in proteins but becomes labile, owing to the action of transaminases, once the amino acids are released by proteolysis. The specific measurement of 2-3H in amino acids largely overcomes problems due to compartmentation and isotope recycling and evidence to support this claim is presented. Values for the half-life of Lemna minor (duckweed) protein determined by the new method are compared with values obtained by other methods. PMID:949338

  2. Analysis of Protein Thermostability Enhancing Factors in Industrially Important Thermus Bacteria Species

    PubMed Central

    Kumwenda, Benjamin; Litthauer, Derek; Bishop, Özlem Tastan; Reva, Oleg

    2013-01-01

    Elucidation of evolutionary factors that enhance protein thermostability is a critical problem and was the focus of this work on Thermus species. Pairs of orthologous sequences of T. scotoductus SA-01 and T. thermophilus HB27, with the largest negative minimum folding energy (MFE) as predicted by the UNAFold algorithm, were statistically analyzed. Favored substitutions of amino acids residues and their properties were determined. Substitutions were analyzed in modeled protein structures to determine their locations and contribution to energy differences using PyMOL and FoldX programs respectively. Dominant trends in amino acid substitutions consistent with differences in thermostability between orthologous sequences were observed. T. thermophilus thermophilic proteins showed an increase in non-polar, tiny, and charged amino acids. An abundance of alanine substituted by serine and threonine, as well as arginine substituted by glutamine and lysine was observed in T. thermophilus HB27. Structural comparison showed that stabilizing mutations occurred on surfaces and loops in protein structures. PMID:24023508

  3. Neuromuscular electrical stimulation prior to presleep protein feeding stimulates the use of protein-derived amino acids for overnight muscle protein synthesis.

    PubMed

    Dirks, Marlou L; Groen, Bart B L; Franssen, Rinske; van Kranenburg, Janneau; van Loon, Luc J C

    2017-01-01

    Short periods of muscle disuse result in substantial skeletal muscle atrophy. Recently, we showed that both neuromuscular electrical stimulation (NMES) as well as presleep dietary protein ingestion represent effective strategies to stimulate muscle protein synthesis rates. In this study, we test our hypothesis that NMES can augment the use of presleep protein-derived amino acids for overnight muscle protein synthesis in older men. Twenty healthy, older [69 ± 1 (SE) yr] men were subjected to 24 h of bed rest, starting at 8:00 AM. In the evening, volunteers were subjected to 70-min 1-legged NMES, while the other leg served as nonstimulated control (CON). Immediately following NMES, 40 g of intrinsically l-[1- 13 C]-phenylalanine labeled protein was ingested prior to sleep. Blood samples were taken throughout the night, and muscle biopsies were obtained from both legs in the evening and the following morning (8 h after protein ingestion) to assess dietary protein-derived l-[1- 13 C]-phenylalanine enrichments in myofibrillar protein. Plasma phenylalanine concentrations and plasma l-[1- 13 C]-phenylalanine enrichments increased significantly following protein ingestion and remained elevated for up to 6 h after protein ingestion (P < 0.05). During overnight sleep, myofibrillar protein-bound l-[1- 13 C]-phenylalanine enrichments (MPE) increased to a greater extent in the stimulated compared with the control leg (0.0344 ± 0.0019 vs. 0.0297 ± 0.0016 MPE, respectively; P < 0.01), representing 18 ± 6% greater incorporation of presleep protein-derived amino acids in the NMES compared with CON leg. In conclusion, application of NMES prior to presleep protein feeding stimulates the use of dietary protein-derived amino acids for overnight muscle protein synthesis in older men. Neuromuscular electrical stimulation (NMES) as well as presleep dietary protein ingestion represent effective strategies to stimulate muscle protein synthesis rates. Here we demonstrate that in older

  4. Dissolved and Particulate Amino Acids in the Lower Mississippi and Pearl Rivers (USA)

    NASA Astrophysics Data System (ADS)

    Duan, S.; Bianchi, T. S.

    2006-12-01

    Seasonal changes (monthly samples) in abundance and composition of dissolved and particulate amino acids were observed at one station in the lower Mississippi and Pearl Rivers (MS, USA) from September 2001 to August 2003. Spatial variability was also observed during a 4 day transmit from river-mile 225 to river mouth (Head of Passes, LA) in the Mississippi River, and a two-day downstream sampling from Jackson (MS) to Stennis Space Center (MS). Temporal data in the lower Mississippi River showed significantly lower concentrations of dissolved combined amino acids (DCAA, 0.45-1.4 μ M) and dissolved amino acids in high molecular weight fraction (HMW DAA, 0.13-0.27 μ M) than in the Pearl River (DCAA, 0.91-2.8 μ M; HMW DAA, 0.25-0.95 μ M). DCAA and HMW DAA in both rivers were generally higher during high-flow periods. DFAA was significantly lower than DCAA in both rivers (0.05-0.08 μ M), and displayed minimal seasonal variability. Total particulate amino acids (PAA) in both rivers were in the same range (0.7-1.4 μ M). A C- normalized yield of PAA (PAA-C/POC) was negatively correlated with suspended particulate matter and positively with chl-a in both rivers. No significant difference in PAA composition was observed in the two rivers. However, PAA in both rivers was relatively enriched in arginine, alanine, methionine and leucine, and depleted in aspartic acid, serine, and non-protein amino acids, compared to DCAA. While DCAA spatial variability in the lower Mississippi River was minimal, decreases in PAA (from 1.06 to 0.43 μ M) were consistent with particulate organic carbon (POC) and particulate nitrogen (PN). Frequent variations in the PAA-C/POC ratio were inversely correlated with suspended particulate matter and PAA (R = -0.7, n = 48), suggesting short- scale sedimentation and resuspension events. A gradual increase in % non-protein AA along with a loss of phytoplankton biomass along the river, suggested was indicative of bacterial utilization of labile

  5. Specific Non-Local Interactions Are Not Necessary for Recovering Native Protein Dynamics

    PubMed Central

    Dasgupta, Bhaskar; Kasahara, Kota; Kamiya, Narutoshi; Nakamura, Haruki; Kinjo, Akira R.

    2014-01-01

    The elastic network model (ENM) is a widely used method to study native protein dynamics by normal mode analysis (NMA). In ENM we need information about all pairwise distances, and the distance between contacting atoms is restrained to the native value. Therefore ENM requires O(N2) information to realize its dynamics for a protein consisting of N amino acid residues. To see if (or to what extent) such a large amount of specific structural information is required to realize native protein dynamics, here we introduce a novel model based on only O(N) restraints. This model, named the ‘contact number diffusion’ model (CND), includes specific distance restraints for only local (along the amino acid sequence) atom pairs, and semi-specific non-local restraints imposed on each atom, rather than atom pairs. The semi-specific non-local restraints are defined in terms of the non-local contact numbers of atoms. The CND model exhibits the dynamic characteristics comparable to ENM and more correlated with the explicit-solvent molecular dynamics simulation than ENM. Moreover, unrealistic surface fluctuations often observed in ENM were suppressed in CND. On the other hand, in some ligand-bound structures CND showed larger fluctuations of buried protein atoms interacting with the ligand compared to ENM. In addition, fluctuations from CND and ENM show comparable correlations with the experimental B-factor. Although there are some indications of the importance of some specific non-local interactions, the semi-specific non-local interactions are mostly sufficient for reproducing the native protein dynamics. PMID:24625758

  6. Aromatic D-amino acids act as chemoattractant factors for human leukocytes through a G protein-coupled receptor, GPR109B.

    PubMed

    Irukayama-Tomobe, Yoko; Tanaka, Hirokazu; Yokomizo, Takehiko; Hashidate-Yoshida, Tomomi; Yanagisawa, Masashi; Sakurai, Takeshi

    2009-03-10

    GPR109B (HM74) is a putative G protein-coupled receptor (GPCR) whose cognate ligands have yet to be characterized. GPR109B shows a high degree of sequence similarity to GPR109A, another GPCR that was identified as a high-affinity nicotinic acid (niacin) receptor. However, the affinity of nicotinic acid to GPR109B is very low. In this study, we found that certain aromatic D-amino acids, including D-phenylalanine, D-tryptophan, and the metabolite of the latter, D-kynurenine, decreased the activity of adenylate cyclase in cells transfected with GPR109B cDNA through activation of pertussis toxin (PTX)-sensitive G proteins. These D-amino acids also elicited a transient rise of intracellular Ca(2+) level in cells expressing GPR109B in a PTX-sensitive manner. In contrast, these D-amino acids did not show any effects on cells expressing GPR109A. We found that the GPR109B mRNA is abundantly expressed in human neutrophils. D-phenylalanine and D-tryptophan induced a transient increase of intracellular Ca(2+) level and a reduction of cAMP levels in human neutrophils. Furthermore, knockdown of GPR109B by RNA interference inhibited the D-amino acids-induced decrease of cellular cAMP levels in human neutrophils. These D-amino acids induced chemotactic activity of freshly prepared human neutrophils. We also found that D-phenylalanine and D-tryptophan induced chemotactic responses in Jurkat cells transfected with the GPR109B cDNA but not in mock-transfected Jurkat cells. These results suggest that these aromatic D-amino acids elicit a chemotactic response in human neutrophils via activation of GPR109B.

  7. Expression pattern of peptide and amino acid genes in digestive tract of transporter juvenile turbot ( Scophthalmus maximus L.)

    NASA Astrophysics Data System (ADS)

    Xu, Dandan; He, Gen; Mai, Kangsen; Zhou, Huihui; Xu, Wei; Song, Fei

    2016-04-01

    Turbot ( Scophthalmus maximus L.), a carnivorous fish species with high dietary protein requirement, was chosen to examine the expression pattern of peptide and amino acid transporter genes along its digestive tract which was divided into six segments including stomach, pyloric caeca, rectum, and three equal parts of the remainder of the intestine. The results showed that the expression of two peptide and eleven amino acid transporters genes exhibited distinct patterns. Peptide transporter 1 (PepT1) was rich in proximal intestine while peptide transporter 2 (PepT2) was abundant in distal intestine. A number of neutral and cationic amino acid transporters expressed richly in whole intestine including B0-type amino acid transporter 1 (B0AT1), L-type amino acid transporter 2 (LAT2), T-type amino acid transporter 1 (TAT1), proton-coupled amino acid transporter 1 (PAT1), y+L-type amino acid transporter 1 (y+LAT1), and cationic amino acid transporter 2 (CAT2) while ASC amino acid transporter 2 (ASCT2), sodium-coupled neutral amino acid transporter 2 (SNAT2), and y+L-type amino acid transporter 2 (y+LAT2) abundantly expressed in stomach. In addition, system b0,+ transporters (rBAT and b0,+AT) existed richly in distal intestine. These findings comprehensively characterized the distribution of solute carrier family proteins, which revealed the relative importance of peptide and amino acid absorption through luminal membrane. Our findings are helpful to understand the mechanism of the utilization of dietary protein in fish with a short digestive tract.

  8. Imbalance of plasma amino acids, metabolites and lipids in patients with lysinuric protein intolerance (LPI).

    PubMed

    Kurko, Johanna; Tringham, Maaria; Tanner, Laura; Näntö-Salonen, Kirsti; Vähä-Mäkilä, Mari; Nygren, Heli; Pöhö, Päivi; Lietzen, Niina; Mattila, Ismo; Olkku, Anu; Hyötyläinen, Tuulia; Orešič, Matej; Simell, Olli; Niinikoski, Harri; Mykkänen, Juha

    2016-09-01

    Lysinuric protein intolerance (LPI [MIM 222700]) is an aminoaciduria with defective transport of cationic amino acids in epithelial cells in the small intestine and proximal kidney tubules due to mutations in the SLC7A7 gene. LPI is characterized by protein malnutrition, failure to thrive and hyperammonemia. Many patients also suffer from combined hyperlipidemia and chronic kidney disease (CKD) with an unknown etiology. Here, we studied the plasma metabolomes of the Finnish LPI patients (n=26) and healthy control individuals (n=19) using a targeted platform for analysis of amino acids as well as two analytical platforms with comprehensive coverage of molecular lipids and polar metabolites. Our results demonstrated that LPI patients have a dichotomy of amino acid profiles, with both decreased essential and increased non-essential amino acids. Altered levels of metabolites participating in pathways such as sugar, energy, amino acid and lipid metabolism were observed. Furthermore, of these metabolites, myo-inositol, threonic acid, 2,5-furandicarboxylic acid, galactaric acid, 4-hydroxyphenylacetic acid, indole-3-acetic acid and beta-aminoisobutyric acid associated significantly (P<0.001) with the CKD status. Lipid analysis showed reduced levels of phosphatidylcholines and elevated levels of triacylglycerols, of which long-chain triacylglycerols associated (P<0.01) with CKD. This study revealed an amino acid imbalance affecting the basic cellular metabolism, disturbances in plasma lipid composition suggesting hepatic steatosis and fibrosis and novel metabolites correlating with CKD in LPI. In addition, the CKD-associated metabolite profile along with increased nitrite plasma levels suggests that LPI may be characterized by increased oxidative stress and apoptosis, altered microbial metabolism in the intestine and uremic toxicity. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Amino acid sequence of the smaller basic protein from rat brain myelin

    PubMed Central

    Dunkley, Peter R.; Carnegie, Patrick R.

    1974-01-01

    1. The complete amino acid sequence of the smaller basic protein from rat brain myelin was determined. This protein differs from myelin basic proteins of other species in having a deletion of a polypeptide of 40 amino acid residues from the centre of the molecule. 2. A detailed comparison is made of the constant and variable regions in a group of myelin basic proteins from six species. 3. An arginine residue in the rat protein was found to be partially methylated. The ratio of methylated to unmethylated arginine at this position differed from that found for the human basic protein. 4. Three tryptic peptides were isolated in more than one form. The differences between the two forms of each peptide are discussed in relation to the electrophoretic heterogeneity of myelin basic proteins, which is known to occur at alkaline pH values. 5. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50029 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5. PMID:4141893

  10. Molecular Characterization of Aquaporin 1 and Aquaporin 3 from the Gills of the African Lungfish, Protopterus annectens, and Changes in Their Branchial mRNA Expression Levels and Protein Abundance during Three Phases of Aestivation.

    PubMed

    Chng, You R; Ong, Jasmine L Y; Ching, Biyun; Chen, Xiu L; Hiong, Kum C; Wong, Wai P; Chew, Shit F; Lam, Siew H; Ip, Yuen K

    2016-01-01

    African lungfishes can undergo long periods of aestivation on land during drought. During aestivation, lungfishes are confronted with desiccation and dehydration, and their gills become non-functional and covered with a thick layer of dried mucus. Aquaporins (Aqps) are a superfamily of integral membrane proteins which generally facilitate the permeation of water through plasma membranes. This study aimed to obtain the complete cDNA coding sequences of aqp1 and aqp3 from the gills of Protopterus annectens , and to determine their branchial mRNA and protein expression levels during the induction, maintenance and arousal phases of aestivation. Dendrogramic analyses of the deduced Aqp1 and Aqp3 amino acid sequences of P. annectens revealed their close relationships with those of Latimeria chalumnae and tetrapods. During the induction phase, there were significant decreases in the transcript levels of aqp1 and aqp3 in the gills of P. annectens , but the branchial Aqp1 and Aqp3 protein abundance remained unchanged. As changes in transcription might precede changes in translation, this could be regarded as an adaptive response to decrease the protein abundance of Aqp1 and Aqp3 in the subsequent maintenance phase of aestivation. As expected, the branchial transcript levels and protein abundance of aqp1 /Aqp1 and aqp3 /Aqp3 were significantly down-regulated during the maintenance phase, probably attributable to the shutdown of branchial functions and the cessation of volume regulation of branchial epithelial cells. Additionally, these changes could reduce the loss of water through branchial epithelial surfaces, supplementing the anti-desiccating property of the dried mucus. Upon arousal, it was essential for the lungfish to restore branchial functions. Indeed, the protein abundance of Aqp1 recovered partially, with complete recovery of mRNA expression level and protein abundance of Aqp3, in the gills of P. annectens after 3 days of arousal. These results provide insights

  11. Effects of transgenic cry1Ie maize on non-lepidopteran pest abundance, diversity and community composition.

    PubMed

    Guo, Jingfei; He, Kanglai; Bai, Shuxiong; Zhang, Tiantao; Liu, Yunjun; Wang, Fuxin; Wang, Zhenying

    2016-12-01

    Non-lepidopteran pests are exposed to, and may be influenced by, Bt toxins when feeding on Bt maize that express insecticidal Cry proteins derived from Bacillus thuringiensis (Bt). In order to assess the potential effects of transgenic cry1Ie maize on non-lepidopteran pest species and ecological communities, a 2-year field study was conducted to compare the non-lepidopteran pest abundance, diversity and community composition between transgenic cry1Ie maize (Event IE09S034, Bt maize) and its near isoline (Zong 31, non-Bt maize) by whole plant inspections. Results showed that Bt maize had no effects on non-lepidopteran pest abundance and diversity (Shannon-Wiener diversity index, Simpson's diversity index, species richness, and Pielou's index). There was a significant effect of year and sampling time on those indices analyzed. Redundancy analysis indicated maize type, sampling time and year totally explained 20.43 % of the variance in the non-lepidopteran pest community composition, but no association was presented between maize type (Bt maize and non-Bt maize) and the variance. Nonmetric multidimensional scaling analysis showed that sampling time and year, rather than maize type had close relationship with the non-lepidopteran pest community composition. These results corroborated the hypothesis that, at least in the short-term, the transgenic cry1Ie maize had negligible effects on the non-lepidopteran pest abundance, diversity and community composition.

  12. Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade.

    PubMed

    Yamaguchi, Ayami; Tanaka, Sae; Yamaguchi, Shiho; Kuwahara, Hirokazu; Takamura, Chizuko; Imajoh-Ohmi, Shinobu; Horikawa, Daiki D; Toyoda, Atsushi; Katayama, Toshiaki; Arakawa, Kazuharu; Fujiyama, Asao; Kubo, Takeo; Kunieda, Takekazu

    2012-01-01

    Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals.

  13. Integrating the intrinsic conformational preferences of non-coded α-amino acids modified at the peptide bond into the NCAD database

    PubMed Central

    Revilla-López, Guillem; Rodríguez-Ropero, Francisco; Curcó, David; Torras, Juan; Calaza, M. Isabel; Zanuy, David; Jiménez, Ana I.; Cativiela, Carlos; Nussinov, Ruth; Alemán, Carlos

    2011-01-01

    Recently, we reported a database (NCAD, Non-Coded Amino acids Database; http://recerca.upc.edu/imem/index.htm) that was built to compile information about the intrinsic conformational preferences of non-proteinogenic residues determined by quantum mechanical calculations, as well as bibliographic information about their synthesis, physical and spectroscopic characterization, the experimentally-established conformational propensities, and applications (J. Phys. Chem. B 2010, 114, 7413). The database initially contained the information available for α-tetrasubstituted α-amino acids. In this work, we extend NCAD to three families of compounds, which can be used to engineer peptides and proteins incorporating modifications at the –NHCO– peptide bond. Such families are: N-substituted α-amino acids, thio-α-amino acids, and diamines and diacids used to build retropeptides. The conformational preferences of these compounds have been analyzed and described based on the information captured in the database. In addition, we provide an example of the utility of the database and of the compounds it compiles in protein and peptide engineering. Specifically, the symmetry of a sequence engineered to stabilize the 310-helix with respect to the α-helix has been broken without perturbing significantly the secondary structure through targeted replacements using the information contained in the database. PMID:21491493

  14. Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis.

    PubMed

    Wilson, Fiona A; Suryawan, Agus; Orellana, Renán A; Nguyen, Hanh V; Jeyapalan, Asumthia S; Gazzaneo, Maria C; Davis, Teresa A

    2008-10-01

    Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 microg x kg(-1) x day(-1)) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) (P<0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level (P<0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1.eIF4E complex association, and increased active eIF4E.eIF4G complex formation (P<0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex.

  15. Protein Thermostability Is Owing to Their Preferences to Non-Polar Smaller Volume Amino Acids, Variations in Residual Physico-Chemical Properties and More Salt-Bridges

    PubMed Central

    Panja, Anindya Sundar; Bandopadhyay, Bidyut; Maiti, Smarajit

    2015-01-01

    Introduction Protein thermostability is an important field for its evolutionary perspective of mesophilic versus thermophilic relationship and for its industrial/ therapeutic applications. Methods Presently, a total 400 (200 thermophilic and 200 mesophilic homologue) proteins were studied utilizing several software/databases to evaluate their amino acid preferences. Randomly selected 50 homologous proteins with available PDB-structure of each group were explored for the understanding of the protein charges, isoelectric-points, hydrophilicity, hydrophobicity, tyrosine phosphorylation and salt-bridge occurrences. These 100 proteins were further probed to generate Ramachandran plot/data for the gross secondary structure prediction in and comparison between the thermophilic and mesophilic proteins. Results Present results strongly suggest that nonpolar smaller volume amino acids Ala (χ 2 = 238.54, p<0.001) and Gly (χ 2 = 73.35, p<0.001) are highly and Val moderately (χ 2 = 144.43, p<0.001) occurring in the 85% of thermophilic proteins. Phospho-regulated Tyr and redox-sensitive Cys are also moderately distributed (χ 2~20.0, p<0.01) in a larger number of thermophilic proteins. A consistent lower distribution of thermophilicity and discretely higher distribution of hydrophobicity is noticed in a large number of thermophilic versus their mesophilic protein homolog. The mean differences of isoelectric points and charges are found to be significantly less (7.11 vs. 6.39, p<0.05 and 1 vs. -0.6, p<0.01, respectively) in thermophilic proteins compared to their mesophilic counterpart. The possible sites for Tyr phosphorylation are noticed to be 25% higher (p<0.05) in thermophilic proteins. The 60% thermophiles are found with higher number of salt bridges in this study. The average percentage of salt-bridge of thermophiles is found to be higher by 20% than their mesophilic homologue. The GLU-HIS and GLU-LYS salt-bridge dyads are calculated to be significantly higher (p<0

  16. Protein Thermostability Is Owing to Their Preferences to Non-Polar Smaller Volume Amino Acids, Variations in Residual Physico-Chemical Properties and More Salt-Bridges.

    PubMed

    Panja, Anindya Sundar; Bandopadhyay, Bidyut; Maiti, Smarajit

    2015-01-01

    Protein thermostability is an important field for its evolutionary perspective of mesophilic versus thermophilic relationship and for its industrial/ therapeutic applications. Presently, a total 400 (200 thermophilic and 200 mesophilic homologue) proteins were studied utilizing several software/databases to evaluate their amino acid preferences. Randomly selected 50 homologous proteins with available PDB-structure of each group were explored for the understanding of the protein charges, isoelectric-points, hydrophilicity, hydrophobicity, tyrosine phosphorylation and salt-bridge occurrences. These 100 proteins were further probed to generate Ramachandran plot/data for the gross secondary structure prediction in and comparison between the thermophilic and mesophilic proteins. Present results strongly suggest that nonpolar smaller volume amino acids Ala (χ2 = 238.54, p<0.001) and Gly (χ2 = 73.35, p<0.001) are highly and Val moderately (χ2 = 144.43, p<0.001) occurring in the 85% of thermophilic proteins. Phospho-regulated Tyr and redox-sensitive Cys are also moderately distributed (χ2~20.0, p<0.01) in a larger number of thermophilic proteins. A consistent lower distribution of thermophilicity and discretely higher distribution of hydrophobicity is noticed in a large number of thermophilic versus their mesophilic protein homolog. The mean differences of isoelectric points and charges are found to be significantly less (7.11 vs. 6.39, p<0.05 and 1 vs. -0.6, p<0.01, respectively) in thermophilic proteins compared to their mesophilic counterpart. The possible sites for Tyr phosphorylation are noticed to be 25% higher (p<0.05) in thermophilic proteins. The 60% thermophiles are found with higher number of salt bridges in this study. The average percentage of salt-bridge of thermophiles is found to be higher by 20% than their mesophilic homologue. The GLU-HIS and GLU-LYS salt-bridge dyads are calculated to be significantly higher (p<0.05 and p<0.001, respectively) in

  17. Trends of amino acid usage in the proteins from the unicellular parasite Giardia lamblia.

    PubMed

    Garat, B; Musto, H

    2000-12-29

    Correspondence analysis of amino acid frequencies was applied to 75 complete coding sequences from the unicellular parasite Giardia lamblia, and it was found that three major factors influence the variability of amino acidic composition of proteins. The first trend strongly correlated with (a) the cysteine content and (b) the mean weight of the amino acids used in each protein. The second trend correlated with the global levels of hydropathy and aromaticity of each protein. Both axes might be related with the defense of the parasite to oxygen free radicals. Finally, the third trend correlated with the expressivity of each gene, indicating that in G. lamblia highly expressed sequences display a tendency to preferentially use a subset of the total amino acids.

  18. Characterization of the amino acid contribution to the folding degree of proteins.

    PubMed

    Estrada, Ernesto

    2004-03-01

    The folding degree index (Estrada, Bioinformatics 2002;18:697-704) is extended to account for the contribution of amino acids to folding. First, the mathematical formalism for extending the folding degree index is presented. Then, the amino acid contributions to folding degree of several proteins are used to analyze its relation to secondary structure. The possibilities of using these contributions in helping or checking the assignation of secondary structure to amino acids are also introduced. The influence of external factors to the amino acids contribution to folding degree is studied through the temperature effect on ribonuclease A. Finally, the analysis of 3D protein similarity through the use of amino acid contributions to folding degree is studied by selecting a series of lysozymes. These results are compared to that obtained by sequence alignment (2D similarity) and 3D superposition of the structures, showing the uniqueness of the current approach. Copyright 2004 Wiley-Liss, Inc.

  19. Differential regulation of protein synthesis by amino acids and insulin in peripheral and visceral tissues of neonatal pigs

    PubMed Central

    Suryawan, Agus; O’Connor, Pamela M. J.; Bush, Jill A.; Nguyen, Hanh V.

    2009-01-01

    The high efficiency of protein deposition during the neonatal period is driven by high rates of protein synthesis, which are maximally stimulated after feeding. In the current study, we examined the individual roles of amino acids and insulin in the regulation of protein synthesis in peripheral and visceral tissues of the neonate by performing pancreatic glucose–amino acid clamps in overnight-fasted 7-day-old pigs. We infused pigs (n = 8–12/group) with insulin at 0, 10, 22, and 110 ng kg−0.66 min−1 to achieve ~0, 2, 6 and 30 μU ml−1 insulin so as to simulate below fasting, fasting, intermediate, and fed insulin levels, respectively. At each insulin dose, amino acids were maintained at the fasting or fed level. In conjunction with the highest insulin dose, amino acids were also allowed to fall below the fasting level. Tissue protein synthesis was measured using a flooding dose of L-[4-3H] phenylalanine. Both insulin and amino acids increased fractional rates of protein synthesis in longissimus dorsi, gastrocnemius, masseter, and diaphragm muscles. Insulin, but not amino acids, increased protein synthesis in the skin. Amino acids, but not insulin, increased protein synthesis in the liver, pancreas, spleen, and lung and tended to increase protein synthesis in the jejunum and kidney. Neither insulin nor amino acids altered protein synthesis in the stomach. The results suggest that the stimulation of protein synthesis by feeding in most tissues of the neonate is regulated by the post-prandial rise in amino acids. However, the feeding-induced stimulation of protein synthesis in skeletal muscles is independently mediated by insulin as well as amino acids. PMID:18683020

  20. Values for digestible indispensable amino acid scores (DIAAS) for some dairy and plant proteins may better describe protein quality than values calculated using the concept for protein digestibility-corrected amino acid scores (PDCAAS).

    PubMed

    Mathai, John K; Liu, Yanhong; Stein, Hans H

    2017-02-01

    An experiment was conducted to compare values for digestible indispensable amino acid scores (DIAAS) for four animal proteins and four plant proteins with values calculated as recommended for protein digestibility-corrected amino acid scores (PDCAAS), but determined in pigs instead of in rats. Values for standardised total tract digestibility (STTD) of crude protein (CP) and standardised ileal digestibility (SID) of amino acids (AA) were calculated for whey protein isolate (WPI), whey protein concentrate (WPC), milk protein concentrate (MPC), skimmed milk powder (SMP), pea protein concentrate (PPC), soya protein isolate (SPI), soya flour and whole-grain wheat. The PDCAAS-like values were calculated using the STTD of CP to estimate AA digestibility and values for DIAAS were calculated from values for SID of AA. Results indicated that values for SID of most indispensable AA in WPI, WPC and MPC were greater (P<0·05) than for SMP, PPC, SPI, soya flour and wheat. With the exception of arginine and tryptophan, the SID of all indispensable AA in SPI was greater (P<0·05) than in soya flour, and with the exception of threonine, the SID of all indispensable AA in wheat was less (P<0·05) than in all other ingredients. If the same scoring pattern for children between 6 and 36 months was used to calculate PDCAAS-like values and DIAAS, PDCAAS-like values were greater (P<0·05) than DIAAS values for SMP, PPC, SPI, soya flour and wheat indicating that PDCAAS-like values estimated in pigs may overestimate the quality of these proteins.

  1. Brain–blood amino acid correlates following protein restriction in murine maple syrup urine disease

    PubMed Central

    2014-01-01

    Background Conventional therapy for patients with maple syrup urine disease (MSUD) entails restriction of protein intake to maintain acceptable levels of the branched chain amino acid, leucine (LEU), monitored in blood. However, no data exists on the correlation between brain and blood LEU with protein restriction, and whether correction in blood is reflected in brain. Methods To address this question, we fed intermediate MSUD mice diets of 19% (standard) and 6% protein, with collection of sera (SE), striata (STR), cerebellum (CE) and cortex (CTX) for quantitative amino acid analyses. Results LEU and valine (VAL) levels in all brain regions improved on average 28% when shifting from 19% to 6% protein, whereas the same improvements in SE were on average 60%. Isoleucine (ILE) in brain regions did not improve, while the SE level improved 24% with low-protein consumption. Blood-branched chain amino acids (LEU, ILE, and VAL in sera (SE)) were 362-434 μM, consistent with human values considered within control. Nonetheless, numerous amino acids in brain regions remained abnormal despite protein restriction, including glutamine (GLN), aspartate (ASP), glutamate (GLU), gamma-aminobutyric acid (GABA), asparagine (ASN), citrulline (CIT) and serine (SER). To assess the specificity of these anomalies, we piloted preliminary studies in hyperphenylalaninemic mice, modeling another large neutral aminoacidopathy. Employing an identical dietary regimen, we found remarkably consistent abnormalities in GLN, ASP, and GLU. Conclusions Our results suggest that blood amino acid analysis may be a poor surrogate for assessing the outcomes of protein restriction in the large neutral amino acidopathies, and further indicate that chronic neurotransmitter disruptions (GLU, GABA, ASP) may contribute to long-term neurocognitive dysfunction in these disorders. PMID:24886632

  2. Brain-blood amino acid correlates following protein restriction in murine maple syrup urine disease.

    PubMed

    Vogel, Kara R; Arning, Erland; Wasek, Brandi L; McPherson, Sterling; Bottiglieri, Teodoro; Gibson, K Michael

    2014-05-08

    Conventional therapy for patients with maple syrup urine disease (MSUD) entails restriction of protein intake to maintain acceptable levels of the branched chain amino acid, leucine (LEU), monitored in blood. However, no data exists on the correlation between brain and blood LEU with protein restriction, and whether correction in blood is reflected in brain. To address this question, we fed intermediate MSUD mice diets of 19% (standard) and 6% protein, with collection of sera (SE), striata (STR), cerebellum (CE) and cortex (CTX) for quantitative amino acid analyses. LEU and valine (VAL) levels in all brain regions improved on average 28% when shifting from 19% to 6% protein, whereas the same improvements in SE were on average 60%. Isoleucine (ILE) in brain regions did not improve, while the SE level improved 24% with low-protein consumption. Blood-branched chain amino acids (LEU, ILE, and VAL in sera (SE)) were 362-434 μM, consistent with human values considered within control. Nonetheless, numerous amino acids in brain regions remained abnormal despite protein restriction, including glutamine (GLN), aspartate (ASP), glutamate (GLU), gamma-aminobutyric acid (GABA), asparagine (ASN), citrulline (CIT) and serine (SER). To assess the specificity of these anomalies, we piloted preliminary studies in hyperphenylalaninemic mice, modeling another large neutral aminoacidopathy. Employing an identical dietary regimen, we found remarkably consistent abnormalities in GLN, ASP, and GLU. Our results suggest that blood amino acid analysis may be a poor surrogate for assessing the outcomes of protein restriction in the large neutral amino acidopathies, and further indicate that chronic neurotransmitter disruptions (GLU, GABA, ASP) may contribute to long-term neurocognitive dysfunction in these disorders.

  3. Pro-Inflammatory Flagellin Proteins of Prevalent Motile Commensal Bacteria Are Variably Abundant in the Intestinal Microbiome of Elderly Humans

    PubMed Central

    Neville, B. Anne; Sheridan, Paul O.; Harris, Hugh M. B.; Coughlan, Simone; Flint, Harry J.; Duncan, Sylvia H.; Jeffery, Ian B.; Claesson, Marcus J.; Ross, R. Paul; Scott, Karen P.; O'Toole, Paul W.

    2013-01-01

    Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute “cell motility” category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the β- and γ- proteobacteria. Many fliC genes were deduced to be under the control of σ28. The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13–4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (≤1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved

  4. Statistical potential-based amino acid similarity matrices for aligning distantly related protein sequences.

    PubMed

    Tan, Yen Hock; Huang, He; Kihara, Daisuke

    2006-08-15

    Aligning distantly related protein sequences is a long-standing problem in bioinformatics, and a key for successful protein structure prediction. Its importance is increasing recently in the context of structural genomics projects because more and more experimentally solved structures are available as templates for protein structure modeling. Toward this end, recent structure prediction methods employ profile-profile alignments, and various ways of aligning two profiles have been developed. More fundamentally, a better amino acid similarity matrix can improve a profile itself; thereby resulting in more accurate profile-profile alignments. Here we have developed novel amino acid similarity matrices from knowledge-based amino acid contact potentials. Contact potentials are used because the contact propensity to the other amino acids would be one of the most conserved features of each position of a protein structure. The derived amino acid similarity matrices are tested on benchmark alignments at three different levels, namely, the family, the superfamily, and the fold level. Compared to BLOSUM45 and the other existing matrices, the contact potential-based matrices perform comparably in the family level alignments, but clearly outperform in the fold level alignments. The contact potential-based matrices perform even better when suboptimal alignments are considered. Comparing the matrices themselves with each other revealed that the contact potential-based matrices are very different from BLOSUM45 and the other matrices, indicating that they are located in a different basin in the amino acid similarity matrix space.

  5. Indigenous Amino Acids in Iron Meteorites

    NASA Technical Reports Server (NTRS)

    Elsila, J. E.; Dworkin, J. P.; Glavin, D. P.; Johnson, N. M.

    2018-01-01

    Understanding the organic content of meteorites and the potential delivery of molecules relevant to the origin of life on Earth is an important area of study in astrobiology. There have been many studies of meteoritic organics, with much focus on amino acids as monomers of proteins and enzymes essential to terrestrial life. The majority of these studies have involved analysis of carbonaceous chondrites, primitive meteorites containing approx. 3-5 wt% carbon. Amino acids have been observed in varying abundances and distributions in representatives of all eight carbonaceous chondrite groups, as well as in ungrouped carbonaceous chondrites, ordinary and R chondrites, ureilites, and planetary achondrites [1 and references therein].

  6. Identification of non-zein proteins in BR473 maize protein bodies by LC-nanoESI-MS/MS.

    PubMed

    Bicudo, Rogério Campos; Bicudo, Tatiana Campos; Forato, Lucimara A; Titato, Guilherme M; Colnago, Luiz A; Lanças, Fernando M

    2009-11-01

    The nutritional value of maize seed is limited due to its high content of storage proteins (zeins), which are deficient in essential amino acids such as lysine and tryptophan. In a previous paper, we showed that protein bodies obtained from BR473 maize variety, developed by Embrapa (Brazilian Agricultural Research Corporation), were mainly constituted by Z27 and a smaller quantity of Z50 gamma-zeins. Besides zein proteins, other not identified protein band in the SDS/PAGE was also observed, which could indicate the presence of non-zein proteins additionally to gamma-zeins. In the present paper, we have demonstrated the presence of non-zein proteins in BR473 maize protein bodies by LC-nanoESI-MS/MS and database searching. This fact could be related to the excellent energetic value and higher protein quality of BR473 maize grains, since high lysine concentration in some maize varieties has been related to the presence of cytoskeleton proteins that are non-zeins. We have identified the following proteins: Brittle-1 protein (chloroplast precursor), Legumin-1, glyceroldehyde-3-phosphate dehydrogenase, and elongation factor 1-alpha.

  7. Profiling Amino Acids of Jordanian Scalp Hair as a Tool for Diabetes Mellitus Diagnosis: A Pilot Study.

    PubMed

    Rashaid, Ayat H B; Harrington, Peter de B; Jackson, Glen P

    2015-07-21

    Hair analysis is an area of increasing interest in the fields of medical and forensic sciences. Human scalp hair has attractive features in clinical studies because hair can be sampled easily and noninvasively from human subjects, and unlike blood and urine samples, it contains a chronological record of medication use. Keratin protein is the major component of scalp hair shaft material and it is composed of 21 amino acids. The method used herein for the amino acid determination in hair included keratin protein acid hydrolysis using 6 M hydrochloric acid (HCl), followed by amino acids derivatization using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and the determination of derivatized amino acids by gas chromatography/mass spectrometry (GC/MS). Amino acid profiles of scalp hair of 27 Jordanian subjects (15 diabetes mellitus (DM) type 2 patients and 12 control subjects) were analyzed. A fuzzy rule-building expert system (FuRES) classified the amino acid profiles into diabetic and control groups based on multivariate analyses of the abundance of 14 amino acids. The sensitivity and specificity were 100% for diabetes detection using leave-one-individual-out cross-validation. The areas under the receiver operative characteristics (ROC) curves were 1.0, which represents a highly sensitive and specific diabetes test. The nonessential amino acids Gly and Glu, and the essential amino acid Ile were more abundant in the scalp hair of diabetic patients compared to the hair of control subjects. The associations between the abundance of amino acids of human hair and health status may have clinical applications in providing diagnostic indicator or predicting other chronic or acute diseases.

  8. Noncoded amino acids in protein engineering: Structure-activity relationship studies of hirudin-thrombin interaction.

    PubMed

    De Filippis, Vincenzo; Acquasaliente, Laura; Pontarollo, Giulia; Peterle, Daniele

    2018-01-01

    The advent of recombinant DNA technology allowed to site-specifically insert, delete, or mutate almost any amino acid in a given protein, significantly improving our knowledge of protein structure, stability, and function. Nevertheless, a quantitative description of the physical and chemical basis that makes a polypeptide chain to efficiently fold into a stable and functionally active conformation is still elusive. This mainly originates from the fact that nature combined, in a yet unknown manner, different properties (i.e., hydrophobicity, conformational propensity, polarizability, and hydrogen bonding capability) into the 20 standard natural amino acids, thus making difficult, if not impossible, to univocally relate the change in protein stability or function to the alteration of physicochemical properties caused by amino acid exchange(s). In this view, incorporation of noncoded amino acids with tailored side chains, allowing to finely tune the structure at a protein site, would facilitate to dissect the effects of a given mutation in terms of one or a few physicochemical properties, thus much expanding the scope of physical organic chemistry in the study of proteins. In this review, relevant applications from our laboratory will be presented on the use of noncoded amino acids in structure-activity relationships studies of hirudin binding to thrombin. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  9. Region-Specific Protein Abundance Changes in the Brain of MPTP-induced Parkinson’s Disease Mouse Model

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Xu; Zhou, Jianying; Chin, Mark H

    2010-02-15

    Parkinson’s disease (PD) is characterized by dopaminergic neurodegeneration in the nigrostriatal region of the brain; however, the neurodegeneration extends well beyond dopaminergic neurons. To gain a better understanding of the molecular changes relevant to PD, we applied two-dimensional LC-MS/MS to comparatively analyze the proteome changes in four brain regions (striatum, cerebellum, cortex, and the rest of brain) using a MPTP-induced PD mouse model with the objective to identify nigrostriatal-specific and other region-specific protein abundance changes. The combined analyses resulted in the identification of 4,895 non-redundant proteins with at least two unique peptides per protein. The relative abundance changes in eachmore » analyzed brain region were estimated based on the spectral count information. A total of 518 proteins were observed with significant MPTP-induced changes across different brain regions. 270 of these proteins were observed with specific changes occurring either only in the striatum and/or in the rest of the brain region that contains substantia nigra, suggesting that these proteins are associated with the underlying nigrostriatal pathways. Many of the proteins that exhibit significant abundance changes were associated with dopamine signaling, mitochondrial dysfunction, the ubiquitin system, calcium signaling, the oxidative stress response, and apoptosis. A set of proteins with either consistent change across all brain regions or with changes specific to the cortex and cerebellum regions were also detected. One of the interesting proteins is ubiquitin specific protease (USP9X), a deubiquination enzyme involved in the protection of proteins from degradation and promotion of the TGF-β pathway, which exhibited altered abundances in all brain regions. Western blot validation showed similar spatial changes, suggesting that USP9X is potentially associated with neurodegeneration. Together, this study for the first time presents an overall

  10. Amino acid selective unlabeling for sequence specific resonance assignments in proteins

    PubMed Central

    Krishnarjuna, B.; Jaipuria, Garima; Thakur, Anushikha

    2010-01-01

    Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative approach is the method of amino acid selective ‘unlabeling’ or reverse labeling, which involves selective unlabeling of specific amino acid types against a uniformly 13C/15N labeled background. Based on this method, we present a novel approach for sequential assignments in proteins. The method involves a new NMR experiment named, {12COi–15Ni+1}-filtered HSQC, which aids in linking the 1HN/15N resonances of the selectively unlabeled residue, i, and its C-terminal neighbor, i + 1, in HN-detected double and triple resonance spectra. This leads to the assignment of a tri-peptide segment from the knowledge of the amino acid types of residues: i − 1, i and i + 1, thereby speeding up the sequential assignment process. The method has the advantage of being relatively inexpensive, applicable to 2H labeled protein and can be coupled with cell-free synthesis and/or automated assignment approaches. A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of 14N at undesired sites. Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies. Electronic supplementary material The online version of this article (doi:10.1007/s10858-010-9459-z) contains supplementary material, which is available to authorized users. PMID:21153044

  11. Increasing CO2 differentially affects essential and non-essential amino acid concentration of rice grains grown in cadmium-contaminated soils.

    PubMed

    Wu, Huibin; Song, Zhengguo; Wang, Xiao; Liu, Zhongqi; Tang, Shirong

    2016-09-01

    Environmental pollution by both ambient CO2 and heavy metals has been steadily increasing, but we do not know how fluctuating CO2 concentrations influence plant nutrients under high Cd pollution, especially in crops. Here, we studied the effects of elevated CO2 and Cd accumulation on proteins and amino acids in rice under Cd stress. In this pot experiment, we analyzed the amino-acid profile of 20 rice cultivars that accumulate Cd differently; the plants were grown in Cd-containing soils under ambient conditions and elevated CO2 levels. We found that although Cd concentrations appeared to be higher in most cultivars under elevated CO2 than under ambient CO2, the effect was significant only in seven cultivars. Combined exposure to Cd and elevated CO2 strongly decreased rice protein and amino acid profiles, including essential and non-essential amino acids. Under elevated CO2, the ratios of specific amino acids were either higher or lower than the optimal ratios provided by FAO/WHO, suggesting that CO2 may flatten the overall amino-acid profile, leading to an excess in some amino acids and deficiencies in others when the rice is consumed. Thus, Cd-tainted rice limits the concentration of essential amino acids in rice-based diets, and the combination with elevated CO2 further exacerbates the problem. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Stabilization of Proteins and Noncovalent Protein Complexes during Electrospray Ionization by Amino Acid Additives.

    PubMed

    Zhang, Hua; Lu, Haiyan; Chingin, Konstantin; Chen, Huanwen

    2015-07-21

    Ionization of proteins and noncovalent protein complexes with minimal disturbance to their native structure presents a great challenge for biological mass spectrometry (MS). In living organisms, the native structure of intracellular proteins is commonly stabilized by solute amino acids (AAs) accumulated in cells at very high concentrations. Inspired by nature, we hypothesized that AAs could also pose a stabilizing effect on the native structure of proteins and noncovalent protein complexes during ionization. To test this hypothesis, here we explored MS response for various protein complexes upon the addition of free AAs at mM concentrations into the electrospray ionization (ESI) solution. Thermal activation of ESI droplets in the MS inlet capillary was employed as a model destabilizing factor during ionization. Our results indicate that certain AAs, in particular proline (Pro), pose considerable positive effect on the stability of noncovalent protein complexes in ESI-MS without affecting the signal intensity of protein ions and original protein-ligand equilibrium, even when added at the 20 mM concentration. The data suggest that the degree of protein stabilization is primarily determined by the osmolytic and ampholytic characteristics of AA solutes. The highest stability and visibility of noncovalent protein complexes in ESI-MS are achieved using AA additives with neutral isoelectric point, moderate proton affinity, and unfavorable interaction with the native protein state. Overall, our results indicate that the simple addition of free amino acids into the working solution can notably improve the stability and accuracy of protein analysis by native ESI-MS.

  13. Incorporation of Amino Acids with Long-Chain Terminal Olefins into Proteins.

    PubMed

    Exner, Matthias P; Köhling, Sebastian; Rivollier, Julie; Gosling, Sandrine; Srivastava, Puneet; Palyancheva, Zheni I; Herdewijn, Piet; Heck, Marie-Pierre; Rademann, Jörg; Budisa, Nediljko

    2016-02-29

    The increasing need for site-specific protein decorations that mimic natural posttranslational modifications requires access to a variety of noncanonical amino acids with moieties enabling bioorthogonal conjugation chemistry. Here we present the incorporation of long-chain olefinic amino acids into model proteins with rational variants of pyrrolysyl-tRNA synthetase (PylRS). Nε-heptenoyl lysine was incorporated for the first time using the known promiscuous variant PylRS(Y306A/Y384F), and Nε-pentenoyl lysine was incorporated in significant yields with the novel variant PylRS(C348A/Y384F). This is the only example of rational modification at position C348 to enlarge the enzyme's binding pocket. Furthermore, we demonstrate the feasibility of our chosen amino acids in the thiol-ene conjugation reaction with a thiolated polysaccharide.

  14. Double mimicry evades tRNA synthetase editing by toxic vegetable-sourced non-proteinogenic amino acid.

    PubMed

    Song, Youngzee; Zhou, Huihao; Vo, My-Nuong; Shi, Yi; Nawaz, Mir Hussain; Vargas-Rodriguez, Oscar; Diedrich, Jolene K; Yates, John R; Kishi, Shuji; Musier-Forsyth, Karin; Schimmel, Paul

    2017-12-22

    Hundreds of non-proteinogenic (np) amino acids (AA) are found in plants and can in principle enter human protein synthesis through foods. While aminoacyl-tRNA synthetase (AARS) editing potentially provides a mechanism to reject np AAs, some have pathological associations. Co-crystal structures show that vegetable-sourced azetidine-2-carboxylic acid (Aze), a dual mimic of proline and alanine, is activated by both human prolyl- and alanyl-tRNA synthetases. However, it inserts into proteins as proline, with toxic consequences in vivo. Thus, dual mimicry increases odds for mistranslation through evasion of one but not both tRNA synthetase editing systems.

  15. Support vector machine with a Pearson VII function kernel for discriminating halophilic and non-halophilic proteins.

    PubMed

    Zhang, Guangya; Ge, Huihua

    2013-10-01

    Understanding of proteins adaptive to hypersaline environment and identifying them is a challenging task and would help to design stable proteins. Here, we have systematically analyzed the normalized amino acid compositions of 2121 halophilic and 2400 non-halophilic proteins. The results showed that halophilic protein contained more Asp at the expense of Lys, Ile, Cys and Met, fewer small and hydrophobic residues, and showed a large excess of acidic over basic amino acids. Then, we introduce a support vector machine method to discriminate the halophilic and non-halophilic proteins, by using a novel Pearson VII universal function based kernel. In the three validation check methods, it achieved an overall accuracy of 97.7%, 91.7% and 86.9% and outperformed other machine learning algorithms. We also address the influence of protein size on prediction accuracy and found the worse performance for small size proteins might be some significant residues (Cys and Lys) were missing in the proteins. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mTOR-dependent translation initiation.

    PubMed

    Orellana, Renán A; Jeyapalan, Asumthia; Escobar, Jeffery; Frank, Jason W; Nguyen, Hanh V; Suryawan, Agus; Davis, Teresa A

    2007-11-01

    In skeletal muscle of adults, sepsis reduces protein synthesis by depressing translation initiation and induces resistance to branched-chain amino acid stimulation. Normal neonates maintain a high basal muscle protein synthesis rate that is sensitive to amino acid stimulation. In the present study, we determined the effect of amino acids on protein synthesis in skeletal muscle and other tissues in septic neonates. Overnight-fasted neonatal pigs were infused with endotoxin (LPS, 0 and 10 microg.kg(-1).h(-1)), whereas glucose and insulin were maintained at fasting levels; amino acids were clamped at fasting or fed levels. In the presence of fasting insulin and amino acids, LPS reduced protein synthesis in longissimus dorsi (LD) and gastrocnemius muscles and increased protein synthesis in the diaphragm, but had no effect in masseter and heart muscles. Increasing amino acids to fed levels accelerated muscle protein synthesis in LD, gastrocnemius, masseter, and diaphragm. LPS stimulated protein synthesis in liver, lung, spleen, pancreas, and kidney in fasted animals. Raising amino acids to fed levels increased protein synthesis in liver of controls, but not LPS-treated animals. The increase in muscle protein synthesis in response to amino acids was associated with increased mTOR, 4E-BP1, and S6K1 phosphorylation and eIF4G-eIF4E association in control and LPS-infused animals. These findings suggest that amino acids stimulate skeletal muscle protein synthesis during acute endotoxemia via mTOR-dependent ribosomal assembly despite reduced basal protein synthesis rates in neonatal pigs. However, provision of amino acids does not further enhance the LPS-induced increase in liver protein synthesis.

  17. Designing medical foods for inherited metabolic disorders: why intact protein is superior to amino acids.

    PubMed

    Ney, Denise Marie; Etzel, Mark Raymond

    2017-04-01

    Phenylketonuria and tyrosinemia are inherited metabolic disorders characterized by high blood levels of phenylalanine (Phe) or tyrosine (Tyr), due to mutations in genes affecting Phe and Tyr metabolism, respectively. The primary management is a lifelong diet restricted in protein from natural foods in combination with medical foods comprised mixtures of synthetic amino acids. Compliance is often poor after childhood leading to neuropsychological sequela. Glycomacropeptide, an intact 64 amino acid glycophosphopeptide isolated from cheese whey, provides a new paradigm for the management of phenylketonuria and tyrosinemia because glycomacropeptide contains no Phe and Tyr in its pure form, and is also a prebiotic. Medical foods made from glycomacropeptide have been used successfully for the management of phenylketonuria and tyrosinemia. Preclinical and clinical studies demonstrate that intact protein from glycomacropeptide provides a more acceptable and physiologic source of defined protein compared to amino acids in medical foods. For example, harmful gut bacteria were reduced, beneficial short chain fatty acids increased, renal workload decreased, protein utilization increased, and bone fragility decreased using intact protein versus amino acids. Advances in biotechnology will propel the transition from synthetic amino acids to intact proteins for the management of inherited metabolic disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis

    PubMed Central

    Wilson, Fiona A.; Suryawan, Agus; Orellana, Renán A.; Nguyen, Hanh V.; Jeyapalan, Asumthia S.; Gazzaneo, Maria C.; Davis, Teresa A.

    2008-01-01

    Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 μg·kg−1·day−1) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) (P < 0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level (P < 0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1·eIF4E complex association, and increased active eIF4E·eIF4G complex formation (P < 0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex. PMID:18682537

  19. Amino acid alphabet reduction preserves fold information contained in contact interactions in proteins.

    PubMed

    Solis, Armando D

    2015-12-01

    To reduce complexity, understand generalized rules of protein folding, and facilitate de novo protein design, the 20-letter amino acid alphabet is commonly reduced to a smaller alphabet by clustering amino acids based on some measure of similarity. In this work, we seek the optimal alphabet that preserves as much of the structural information found in long-range (contact) interactions among amino acids in natively-folded proteins. We employ the Information Maximization Device, based on information theory, to partition the amino acids into well-defined clusters. Numbering from 2 to 19 groups, these optimal clusters of amino acids, while generated automatically, embody well-known properties of amino acids such as hydrophobicity/polarity, charge, size, and aromaticity, and are demonstrated to maintain the discriminative power of long-range interactions with minimal loss of mutual information. Our measurements suggest that reduced alphabets (of less than 10) are able to capture virtually all of the information residing in native contacts and may be sufficient for fold recognition, as demonstrated by extensive threading tests. In an expansive survey of the literature, we observe that alphabets derived from various approaches-including those derived from physicochemical intuition, local structure considerations, and sequence alignments of remote homologs-fare consistently well in preserving contact interaction information, highlighting a convergence in the various factors thought to be relevant to the folding code. Moreover, we find that alphabets commonly used in experimental protein design are nearly optimal and are largely coherent with observations that have arisen in this work. © 2015 Wiley Periodicals, Inc.

  20. Release of free amino acids upon oxidation of peptides and proteins by hydroxyl radicals.

    PubMed

    Liu, Fobang; Lai, Senchao; Tong, Haijie; Lakey, Pascale S J; Shiraiwa, Manabu; Weller, Michael G; Pöschl, Ulrich; Kampf, Christopher J

    2017-03-01

    Hydroxyl radical-induced oxidation of proteins and peptides can lead to the cleavage of the peptide, leading to a release of fragments. Here, we used high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and pre-column online ortho-phthalaldehyde (OPA) derivatization-based amino acid analysis by HPLC with diode array detection and fluorescence detection to identify and quantify free amino acids released upon oxidation of proteins and peptides by hydroxyl radicals. Bovine serum albumin (BSA), ovalbumin (OVA) as model proteins, and synthetic tripeptides (comprised of varying compositions of the amino acids Gly, Ala, Ser, and Met) were used for reactions with hydroxyl radicals, which were generated by the Fenton reaction of iron ions and hydrogen peroxide. The molar yields of free glycine, aspartic acid, asparagine, and alanine per peptide or protein varied between 4 and 55%. For protein oxidation reactions, the molar yields of Gly (∼32-55% for BSA, ∼10-21% for OVA) were substantially higher than those for the other identified amino acids (∼5-12% for BSA, ∼4-6% for OVA). Upon oxidation of tripeptides with Gly in C-terminal, mid-chain, or N-terminal positions, Gly was preferentially released when it was located at the C-terminal site. Overall, we observe evidence for a site-selective formation of free amino acids in the OH radical-induced oxidation of peptides and proteins, which may be due to a reaction pathway involving nitrogen-centered radicals.

  1. Measurement of the incorporation rates of four amino acids into proteins for estimating bacterial production.

    PubMed

    Servais, P

    1995-03-01

    In aquatic ecosystems, [(3)H]thymidine incorporation into bacterial DNA and [(3)H]leucine incorporation into proteins are usually used to estimate bacterial production. The incorporation rates of four amino acids (leucine, tyrosine, lysine, alanine) into proteins of bacteria were measured in parallel on natural freshwater samples from the basin of the river Meuse (Belgium). Comparison of the incorporation into proteins and into the total macromolecular fraction showed that these different amino acids were incorporated at more than 90% into proteins. From incorporation measurements at four subsaturated concentrations (range, 2-77 nm), the maximum incorporation rates were determined. Strong correlations (r > 0.91 for all the calculated correlations) were found between the maximum incorporation rates of the different tested amino acids over a range of two orders of magnitude of bacterial activity. Bacterial production estimates were calculated using theoretical and experimental conversion factors. The productions calculated from the incorporation rates of the four amino acids were in good concordance, especially when the experimental conversion factors were used (slope range, 0.91-1.11, and r > 0.91). This study suggests that the incorporation of various amino acids into proteins can be used to estimate bacterial production.

  2. Comparative evolutionary genomics of Corynebacterium with special reference to codon and amino acid usage diversities.

    PubMed

    Pal, Shilpee; Sarkar, Indrani; Roy, Ayan; Mohapatra, Pradeep K Das; Mondal, Keshab C; Sen, Arnab

    2018-02-01

    The present study has been aimed to the comparative analysis of high GC composition containing Corynebacterium genomes and their evolutionary study by exploring codon and amino acid usage patterns. Phylogenetic study by MLSA approach, indel analysis and BLAST matrix differentiated Corynebacterium species in pathogenic and non-pathogenic clusters. Correspondence analysis on synonymous codon usage reveals that, gene length, optimal codon frequencies and tRNA abundance affect the gene expression of Corynebacterium. Most of the optimal codons as well as translationally optimal codons are C ending i.e. RNY (R-purine, N-any nucleotide base, and Y-pyrimidine) and reveal translational selection pressure on codon bias of Corynebacterium. Amino acid usage is affected by hydrophobicity, aromaticity, protein energy cost, etc. Highly expressed genes followed the cost minimization hypothesis and are less diverged at their synonymous positions of codons. Functional analysis of core genes shows significant difference in pathogenic and non-pathogenic Corynebacterium. The study reveals close relationship between non-pathogenic and opportunistic pathogenic Corynebaterium as well as between molecular evolution and survival niches of the organism.

  3. Identify Secretory Protein of Malaria Parasite with Modified Quadratic Discriminant Algorithm and Amino Acid Composition.

    PubMed

    Feng, Yong-E

    2016-06-01

    Malaria parasite secretes various proteins in infected red blood cell for its growth and survival. Thus identification of these secretory proteins is important for developing vaccine or drug against malaria. In this study, the modified method of quadratic discriminant analysis is presented for predicting the secretory proteins. Firstly, 20 amino acids are divided into five types according to the physical and chemical characteristics of amino acids. Then, we used five types of amino acids compositions as inputs of the modified quadratic discriminant algorithm. Finally, the best prediction performance is obtained by using 20 amino acid compositions, the sensitivity of 96 %, the specificity of 92 % with 0.88 of Mathew's correlation coefficient in fivefold cross-validation test. The results are also compared with those of existing prediction methods. The compared results shown our method are prominent in the prediction of secretory proteins.

  4. Site specific incorporation of heavy atom-containing unnatural amino acids into proteins for structure determination

    DOEpatents

    Xie, Jianming [San Diego, CA; Wang, Lei [San Diego, CA; Wu, Ning [Boston, MA; Schultz, Peter G [La Jolla, CA

    2008-07-15

    Translation systems and other compositions including orthogonal aminoacyl tRNA-synthetases that preferentially charge an orthogonal tRNA with an iodinated or brominated amino acid are provided. Nucleic acids encoding such synthetases are also described, as are methods and kits for producing proteins including heavy atom-containing amino acids, e.g., brominated or iodinated amino acids. Methods of determining the structure of a protein, e.g., a protein into which a heavy atom has been site-specifically incorporated through use of an orthogonal tRNA/aminoacyl tRNA-synthetase pair, are also described.

  5. Amino acid δ13C analysis shows flexibility in the routing of dietary protein and lipids to the tissue of an omnivore.

    PubMed

    Newsome, Seth D; Wolf, Nathan; Peters, Jacob; Fogel, Marilyn L

    2014-11-01

    Stable-isotope analysis (SIA) has revolutionized animal ecology by providing time-integrated estimates of the use of resources and/or habitats. SIA is based on the premise that the isotopic composition of a consumer's tissues originates from its food, but is offset by trophic-discrimination (enrichment) factors controlled by metabolic processes associated with the assimilation of nutrients and the biosynthesis of tissues. Laboratory preparation protocols dictate that tissues both of consumers and of their potential prey be lipid-extracted prior to analysis, because (1) lipids have carbon isotope (δ(13)C) values that are lower by approximately 3-8‰ than associated proteins and (2) amino acids in consumers' proteinaceous tissues are assumed to be completely routed from dietary protein. In contrast, models of stable-isotope mixing assume that dietary macromolecules are broken into their elemental constituents from which non-essential amino acids are resynthesized to build tissues. Here, we show that carbon from non-protein dietary macromolecules, namely lipids, was used to synthesize muscle tissue in an omnivorous rodent (Mus musculus). We traced the influence of dietary lipids on the synthesis of consumers' tissues by inversely varying the dietary proportion of C4-based lipids and C3-based protein while keeping carbohydrate content constant in four dietary treatments, and analyzing the δ(13)C values of amino acids in mouse muscle after 4 months of feeding. The influence of dietary lipids on non-essential amino acids varied as function of biosynthetic pathway. The source of carbon in ketogenic amino acids synthesized through the Krebs cycle was highly sensitive to dietary lipid content, with significant increases of approximately 2-4‰ in Glutamate and Aspartate δ(13)C values from the 5% to 15% dietary lipid treatment. Glucogenic amino acids (Glycine and Serine) were less sensitive to dietary lipid, but increased by approximately 3-4‰ from the 25% to 40% lipid

  6. When Can Species Abundance Data Reveal Non-neutrality?

    PubMed Central

    Al Hammal, Omar; Alonso, David; Etienne, Rampal S.; Cornell, Stephen J.

    2015-01-01

    Species abundance distributions (SAD) are probably ecology’s most well-known empirical pattern, and over the last decades many models have been proposed to explain their shape. There is no consensus over which model is correct, because the degree to which different processes can be discerned from SAD patterns has not yet been rigorously quantified. We present a power calculation to quantify our ability to detect deviations from neutrality using species abundance data. We study non-neutral stochastic community models, and show that the presence of non-neutral processes is detectable if sample size is large enough and/or the amplitude of the effect is strong enough. Our framework can be used for any candidate community model that can be simulated on a computer, and determines both the sampling effort required to distinguish between alternative processes, and a range for the strength of non-neutral processes in communities whose patterns are statistically consistent with neutral theory. We find that even data sets of the scale of the 50 Ha forest plot on Barro Colorado Island, Panama, are unlikely to be large enough to detect deviations from neutrality caused by competitive interactions alone, though the presence of multiple non-neutral processes with contrasting effects on abundance distributions may be detectable. PMID:25793889

  7. [Evaluation of the adjusted amino acid score by digestibility for estimating the protein quality and protein available in food and diets].

    PubMed

    Pak, N; Vera, G; Araya, H

    1985-03-01

    The purpose of the present study was to evaluate the amino acid score adjusted by digestibility to estimate protein quality and utilizable protein in foods and diets, considering net protein utilization (NPU) as a biological reference method. Ten foods of vegetable origin and ten of animal origin, as well as eight mixtures of foods of vegetable and animal origin were studied. When all the foods were considered, a positive (r = 0.83) and highly significant correlation (p less than 0.001) between NPU and the amino acid score adjusted by digestibility was found. When the foods were separated according to their origin, this correlation was positive only for the foods of vegetable origin (r = 0.93) and statistically significant (p less than 0.001). Also, only in those foods were similar values found between NPU and amino acid score adjusted by digestibility, as well as in utilizable protein estimated considering both methods. Caution is required to interpret protein quality and utilizable protein values of foods of animal origin and mixtures of foods of vegetable and animal origin when the amino acid score method adjusted by digestibility, or NPU, are utilized.

  8. Evaluation of three high abundance protein depletion kits for umbilical cord serum proteomics

    PubMed Central

    2011-01-01

    Background High abundance protein depletion is a major challenge in the study of serum/plasma proteomics. Prior to this study, most commercially available kits for depletion of highly abundant proteins had only been tested and evaluated in adult serum/plasma, while the depletion efficiency on umbilical cord serum/plasma had not been clarified. Structural differences between some adult and fetal proteins (such as albumin) make it likely that depletion approaches for adult and umbilical cord serum/plasma will be variable. Therefore, the primary purposes of the present study are to investigate the efficiencies of several commonly-used commercial kits during high abundance protein depletion from umbilical cord serum and to determine which kit yields the most effective and reproducible results for further proteomics research on umbilical cord serum. Results The immunoaffinity based kits (PROTIA-Sigma and 5185-Agilent) displayed higher depletion efficiency than the immobilized dye based kit (PROTBA-Sigma) in umbilical cord serum samples. Both the PROTIA-Sigma and 5185-Agilent kit maintained high depletion efficiency when used three consecutive times. Depletion by the PROTIA-Sigma Kit improved 2DE gel quality by reducing smeared bands produced by the presence of high abundance proteins and increasing the intensity of other protein spots. During image analysis using the identical detection parameters, 411 ± 18 spots were detected in crude serum gels, while 757 ± 43 spots were detected in depleted serum gels. Eight spots unique to depleted serum gels were identified by MALDI- TOF/TOF MS, seven of which were low abundance proteins. Conclusions The immunoaffinity based kits exceeded the immobilized dye based kit in high abundance protein depletion of umbilical cord serum samples and dramatically improved 2DE gel quality for detection of trace biomarkers. PMID:21554704

  9. On the reported optical activity of amino acids in the Murchison meteorite

    USGS Publications Warehouse

    Bada, J.L.; Cronin, J.R.; Ho, M.-S.; Kvenvolden, K.A.; Lawless, J.G.; Miller, S.L.; Oro, John; Steinberg, S.

    1983-01-01

    In analyses of extracts from the Murchison meteorite (a carbonaceous chondrite), Engel and Nagy1 reported an excess of L-enantiomers for several protein amino acids but found that the non-protein amino acids were racemic. They suggested that the excess of L-isomers might have resulted from an asymmetric synthesis or decomposition. Their results disagree with those obtained previously2-4 and they claim this is due to improved methodology. In fact, their extraction method and analytical procedure (gas chromatography-mass spectrometry, GC-MS) was similar to those used in the original report2 of amino acids in the Murchison meteorite except that they used specific ion monitoring in the GC-MS measurements. We found the results of Engel and Nagy odd in that likely contaminants (the protein amino acids ala, leu, glu, asp and pro) were nonracemic while unlikely contaminants (isovaline and ??-amino-n-butyric acid) were racemic. For example, Engel and Nagy report that the leucine is ???90% L-enantiomer in the water-extracted sample whereas isovaline (??-methyl-??-aminobutyric acid) is racemic. It would be most unusual for an abiotic stereoselective decomposition or synthesis of amino acids to occur with protein amino acids but not with non-protein amino acids. We now show here that the explanation of terrestrial contamination is consistent with their results and is much more probable. ?? 1983 Nature Publishing Group.

  10. Computational mining for hypothetical patterns of amino acid side chains in protein data bank (PDB)

    NASA Astrophysics Data System (ADS)

    Ghani, Nur Syatila Ab; Firdaus-Raih, Mohd

    2018-04-01

    The three-dimensional structure of a protein can provide insights regarding its function. Functional relationship between proteins can be inferred from fold and sequence similarities. In certain cases, sequence or fold comparison fails to conclude homology between proteins with similar mechanism. Since the structure is more conserved than the sequence, a constellation of functional residues can be similarly arranged among proteins of similar mechanism. Local structural similarity searches are able to detect such constellation of amino acids among distinct proteins, which can be useful to annotate proteins of unknown function. Detection of such patterns of amino acids on a large scale can increase the repertoire of important 3D motifs since available known 3D motifs currently, could not compensate the ever-increasing numbers of uncharacterized proteins to be annotated. Here, a computational platform for an automated detection of 3D motifs is described. A fuzzy-pattern searching algorithm derived from IMagine an Amino Acid 3D Arrangement search EnGINE (IMAAAGINE) was implemented to develop an automated method for searching of hypothetical patterns of amino acid side chains in Protein Data Bank (PDB), without the need for prior knowledge on related sequence or structure of pattern of interest. We present an example of the searches, which is the detection of a hypothetical pattern derived from known structural motif of C2H2 structural pattern from zinc fingers. The conservation of particular patterns of amino acid side chains in unrelated proteins is highlighted. This approach can act as a complementary method for available structure- and sequence-based platforms and may contribute in improving functional association between proteins.

  11. Effect of Maillard browning reaction on protein utilization and plasma amino acid response by rainbow trout (Salmo gairdneri).

    PubMed

    Plakas, S M; Lee, T C; Wolke, R E; Meade, T L

    1985-12-01

    The effect of the Maillard browning reaction in the diet of rainbow trout (Salmo gairdneri) on growth and amino acid availability was investigated. Chemical and enzymatic hydrolysis methods were applied for the detection of the losses of amino acids in a model protein browning system. Arginine and lysine exhibited the greatest losses in the mixture of fish protein isolate and glucose stored for 40 d at 37 degrees C. The apparent digestibility and absorption of individual amino acids, particularly lysine, was lower in trout fed browned protein than in those fed the control protein. Plasma lysine levels were significantly depressed, while the plasma levels of glucose and most other amino acids were elevated in relation to the loss in nutritive value of dietary protein after browning. The early Maillard reaction derivative of lysine, epsilon-deoxy-fructosyl-lysine, was recovered from browned protein (by using the in vitro enzymatic hydrolysis procedure) and from the plasma of trout fed browned protein. Analysis of plasma free amino acids provided an indication of lysine bioavailability and identified lysine as the first-limiting amino acid in the diets containing browned protein.

  12. Natural Genetic Variation Influences Protein Abundances in C. elegans Developmental Signalling Pathways

    PubMed Central

    Singh, Kapil Dev; Roschitzki, Bernd; Snoek, L. Basten; Grossmann, Jonas; Zheng, Xue; Elvin, Mark; Kamkina, Polina; Schrimpf, Sabine P.; Poulin, Gino B.; Kammenga, Jan E.; Hengartner, Michael O.

    2016-01-01

    Complex traits, including common disease-related traits, are affected by many different genes that function in multiple pathways and networks. The apoptosis, MAPK, Notch, and Wnt signalling pathways play important roles in development and disease progression. At the moment we have a poor understanding of how allelic variation affects gene expression in these pathways at the level of translation. Here we report the effect of natural genetic variation on transcript and protein abundance involved in developmental signalling pathways in Caenorhabditis elegans. We used selected reaction monitoring to analyse proteins from the abovementioned four pathways in a set of recombinant inbred lines (RILs) generated from the wild-type strains N2 (Bristol) and CB4856 (Hawaii) to enable quantitative trait locus (QTL) mapping. About half of the cases from the 44 genes tested showed a statistically significant change in protein abundance between various strains, most of these were however very weak (below 1.3-fold change). We detected a distant QTL on the left arm of chromosome II that affected protein abundance of the phosphatidylserine receptor protein PSR-1, and two separate QTLs that influenced embryonic and ionizing radiation-induced apoptosis on chromosome IV. Our results demonstrate that natural variation in C. elegans is sufficient to cause significant changes in signalling pathways both at the gene expression (transcript and protein abundance) and phenotypic levels. PMID:26985669

  13. Pendrin protein abundance in the kidney is regulated by nitric oxide and cAMP.

    PubMed

    Thumova, Monika; Pech, Vladimir; Froehlich, Otto; Agazatian, Diana; Wang, Xiaonan; Verlander, Jill W; Kim, Young Hee; Wall, Susan M

    2012-09-15

    Pendrin is a Cl(-)/HCO(3)(-) exchanger, expressed in the apical regions of some intercalated cell subtypes, and is critical in the pressor response to angiotensin II. Since angiotensin type 1 receptor inhibitors reduce renal pendrin protein abundance in mice in vivo through a mechanism that is dependent on nitric oxide (NO), we asked if NO modulates renal pendrin expression in vitro and explored the mechanism by which it occurs. Thus we quantified pendrin protein abundance by confocal fluorescent microscopy in cultured mouse cortical collecting ducts (CCDs) and connecting tubules (CNTs). After overnight culture, CCDs maintain their tubular structure and maintain a solute gradient when perfused in vitro. Pendrin protein abundance increased 67% in CNT and 53% in CCD when NO synthase was inhibited (N(G)-nitro-L-arginine methyl ester, 100 μM), while NO donor (DETA NONOate, 200 μM) application reduced pendrin protein by ∼33% in the CCD and CNT. When CNTs were cultured in the presence of the guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (10 μM), NO donors did not alter pendrin abundance. Conversely, pendrin protein abundance rose when cAMP content was increased by the application of an adenylyl cyclase agonist (forskolin, 10 μM), a cAMP analog (8-bromo-cAMP, 1 mM), or a phosphodiesterase inhibitor (BAY60-7550, 50 μM). Since NO reduces cellular cAMP in the CNT, we asked if NO reduces pendrin abundance by reducing cAMP. With blockade of cGMP-stimulated phosphodiesterase II, NO did not alter pendrin protein abundance. We conclude that NO acts through cAMP to reduce pendrin total protein abundance by enhancing cAMP degradation.

  14. Pendrin protein abundance in the kidney is regulated by nitric oxide and cAMP

    PubMed Central

    Thumova, Monika; Pech, Vladimir; Froehlich, Otto; Agazatian, Diana; Wang, Xiaonan; Verlander, Jill W.; Kim, Young Hee

    2012-01-01

    Pendrin is a Cl−/HCO3− exchanger, expressed in the apical regions of some intercalated cell subtypes, and is critical in the pressor response to angiotensin II. Since angiotensin type 1 receptor inhibitors reduce renal pendrin protein abundance in mice in vivo through a mechanism that is dependent on nitric oxide (NO), we asked if NO modulates renal pendrin expression in vitro and explored the mechanism by which it occurs. Thus we quantified pendrin protein abundance by confocal fluorescent microscopy in cultured mouse cortical collecting ducts (CCDs) and connecting tubules (CNTs). After overnight culture, CCDs maintain their tubular structure and maintain a solute gradient when perfused in vitro. Pendrin protein abundance increased 67% in CNT and 53% in CCD when NO synthase was inhibited (NG-nitro-l-arginine methyl ester, 100 μM), while NO donor (DETA NONOate, 200 μM) application reduced pendrin protein by ∼33% in the CCD and CNT. When CNTs were cultured in the presence of the guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (10 μM), NO donors did not alter pendrin abundance. Conversely, pendrin protein abundance rose when cAMP content was increased by the application of an adenylyl cyclase agonist (forskolin, 10 μM), a cAMP analog (8-bromo-cAMP, 1 mM), or a phosphodiesterase inhibitor (BAY60-7550, 50 μM). Since NO reduces cellular cAMP in the CNT, we asked if NO reduces pendrin abundance by reducing cAMP. With blockade of cGMP-stimulated phosphodiesterase II, NO did not alter pendrin protein abundance. We conclude that NO acts through cAMP to reduce pendrin total protein abundance by enhancing cAMP degradation. PMID:22811483

  15. Distinctive Roles of D-Amino Acids in the Homochiral World: Chirality of Amino Acids Modulates Mammalian Physiology and Pathology.

    PubMed

    Sasabe, Jumpei; Suzuki, Masataka

    2018-05-22

    Living organisms enantioselectively employ L-amino acids as the molecular architecture of protein synthesized in the ribosome. Although L-amino acids are dominantly utilized in most biological processes, accumulating evidence points to the distinctive roles of D-amino acids in non-ribosomal physiology. Among the three domains of life, bacteria have the greatest capacity to produce a wide variety of D-amino acids. In contrast, archaea and eukaryotes are thought generally to synthesize only two kinds of D-amino acids: D-serine and D-aspartate. In mammals, D-serine is critical for neurotransmission as an endogenous coagonist of N-methyl D-aspartate receptors. Additionally, D-aspartate is associated with neurogenesis and endocrine systems. Furthermore, recognition of D-amino acids originating in bacteria is linked to systemic and mucosal innate immunity. Among the roles played by D-amino acids in human pathology, the dysfunction of neurotransmission mediated by D-serine is implicated in psychiatric and neurological disorders. Non-enzymatic conversion of L-aspartate or L-serine residues to their D-configurations is involved in age-associated protein degeneration. Moreover, the measurement of plasma or urinary D-/L-serine or D-/L-aspartate levels may have diagnostic or prognostic value in the treatment of kidney diseases. This review aims to summarize current understanding of D-amino-acid-associated biology with a major focus on mammalian physiology and pathology.

  16. The effects of non-synonymous single nucleotide polymorphisms (nsSNPs) on protein-protein interactions.

    PubMed

    Yates, Christopher M; Sternberg, Michael J E

    2013-11-01

    Non-synonymous single nucleotide polymorphisms (nsSNPs) are single base changes leading to a change to the amino acid sequence of the encoded protein. Many of these variants are associated with disease, so nsSNPs have been well studied, with studies looking at the effects of nsSNPs on individual proteins, for example, on stability and enzyme active sites. In recent years, the impact of nsSNPs upon protein-protein interactions has also been investigated, giving a greater insight into the mechanisms by which nsSNPs can lead to disease. In this review, we summarize these studies, looking at the various mechanisms by which nsSNPs can affect protein-protein interactions. We focus on structural changes that can impair interaction, changes to disorder, gain of interaction, and post-translational modifications before looking at some examples of nsSNPs at human-pathogen protein-protein interfaces and the analysis of nsSNPs from a network perspective. © 2013.

  17. Simultaneous determination of natural-abundance δ15N values and quantities of individual amino acids in proteins from milk of lactating women and from infant hair using gas chromatography/isotope ratio mass spectrometry.

    PubMed

    Tea, Illa; Le Guennec, Adrien; Frasquet-Darrieux, Marine; Julien, Maxime; Romek, Katarzyna; Antheaume, Ingrid; Hankard, Régis; Robins, Richard J

    2013-06-30

    In isotope tracer experiments used in nutritional studies, it is frequently desirable both to determine the (15)N/(14)N ratios of target compounds and to quantify these compounds. This report shows how this can be achieved in a single chromatographic run for protein amino acids using an isotope ratio mass spectrometer. Protein hydrolysis by acidic digestion was used to release amino acids, which were then derivatized as their N-pivaloyl-O-isopropyl esters. Suitable conditions for sample preparation were established for both hair and milk proteins. The N-pivaloyl-O-isopropyl esters of amino acids were separated by gas chromatography (GC) on a 60 m ZB-WAX column linked via a combustion interface to an isotope ratio mass spectrometer. The (15)N/(14)N ratios were obtained from the m/z 28, 29 and 30 peak intensities and the quantities from the Area All (Vs) integrated peak areas. It is shown from a five-point calibration curve that both parameters can be measured reliably within the range of 1.0 to 2.0 mg/mL for the major amino acids derived from the hydrolysis of human maternal milk or hair samples. The method was validated in terms of inter-day and inter-user repeatability for both parameters for 14 amino acids. The amino acid percentage composition showed a good correlation with literature values. The method was applied to determine the variability in a population of lactating mothers and their infants. It has been established that δ(15)N values can be simultaneously determined for a complex mixture of amino acids at variable concentrations. It is shown that the percentage composition obtained correlates well with that obtained by calculation from the protein composition or from literature values. This procedure should provide a significant saving in analysis time, especially in those cases where the GC run-time is necessarily long. It allows the satisfactory determination of the variation within a sample population. Copyright © 2013 John Wiley & Sons, Ltd.

  18. Increased reactive oxygen species production and lower abundance of complex I subunits and carnitine palmitoyltransferase 1B protein despite normal mitochondrial respiration in insulin-resistant human skeletal muscle.

    PubMed

    Lefort, Natalie; Glancy, Brian; Bowen, Benjamin; Willis, Wayne T; Bailowitz, Zachary; De Filippis, Elena A; Brophy, Colleen; Meyer, Christian; Højlund, Kurt; Yi, Zhengping; Mandarino, Lawrence J

    2010-10-01

    The contribution of mitochondrial dysfunction to skeletal muscle insulin resistance remains elusive. Comparative proteomics are being applied to generate new hypotheses in human biology and were applied here to isolated mitochondria to identify novel changes in mitochondrial protein abundance present in insulin-resistant muscle. Mitochondria were isolated from vastus lateralis muscle from lean and insulin-sensitive individuals and from obese and insulin-resistant individuals who were otherwise healthy. Respiration and reactive oxygen species (ROS) production rates were measured in vitro. Relative abundances of proteins detected by mass spectrometry were determined using a normalized spectral abundance factor method. NADH- and FADH(2)-linked maximal respiration rates were similar between lean and obese individuals. Rates of pyruvate and palmitoyl-DL-carnitine (both including malate) ROS production were significantly higher in obesity. Mitochondria from obese individuals maintained higher (more negative) extramitochondrial ATP free energy at low metabolic flux, suggesting that stronger mitochondrial thermodynamic driving forces may underlie the higher ROS production. Tandem mass spectrometry identified protein abundance differences per mitochondrial mass in insulin resistance, including lower abundance of complex I subunits and enzymes involved in the oxidation of branched-chain amino acids (BCAA) and fatty acids (e.g., carnitine palmitoyltransferase 1B). We provide data suggesting normal oxidative capacity of mitochondria in insulin-resistant skeletal muscle in parallel with high rates of ROS production. Furthermore, we show specific abundance differences in proteins involved in fat and BCAA oxidation that might contribute to the accumulation of lipid and BCAA frequently associated with the pathogenesis of insulin resistance.

  19. Mapping and mutagenesis of the amino-terminal transcriptional repression domain of the Drosophila Krüppel protein.

    PubMed Central

    Licht, J D; Hanna-Rose, W; Reddy, J C; English, M A; Ro, M; Grossel, M; Shaknovich, R; Hansen, U

    1994-01-01

    We previously demonstrated that the Drosophila Krüppel protein is a transcriptional repressor with separable DNA-binding and transcriptional repression activities. In this study, the minimal amino (N)-terminal repression region of the Krüppel protein was defined by transferring regions of the Krüppel protein to a heterologous DNA-binding protein, the lacI protein. Fusion of a predicted alpha-helical region from amino acids 62 to 92 in the N terminus of the Krüppel protein was sufficient to transfer repression activity. This putative alpha-helix has several hydrophobic surfaces, as well as a glutamine-rich surface. Mutants containing multiple amino acid substitutions of the glutamine residues demonstrated that this putative alpha-helical region is essential for repression activity of a Krüppel protein containing the entire N-terminal and DNA-binding regions. Furthermore, one point mutant with only a single glutamine on this surface altered to lysine abolished the ability of the Krüppel protein to repress, indicating the importance of the amino acid at residue 86 for repression. The N terminus also contained an adjacent activation region localized between amino acids 86 and 117. Finally, in accordance with predictions from primary amino acid sequence similarity, a repression region from the Drosophila even-skipped protein, which was six times more potent than that of the Krüppel protein in the mammalian cells, was characterized. This segment included a hydrophobic stretch of 11 consecutive alanine residues and a proline-rich region. Images PMID:8196644

  20. Protein Hydrolysates as Promoters of Non-Haem Iron Absorption

    PubMed Central

    Li, Yanan; Jiang, Han; Huang, Guangrong

    2017-01-01

    Iron (Fe) is an essential micronutrient for human growth and health. Organic iron is an excellent iron supplement due to its bioavailability. Both amino acids and peptides improve iron bioavailability and absorption and are therefore valuable components of iron supplements. This review focuses on protein hydrolysates as potential promoters of iron absorption. The ability of protein hydrolysates to chelate iron is thought to be a key attribute for the promotion of iron absorption. Iron-chelatable protein hydrolysates are categorized by their absorption forms: amino acids, di- and tri-peptides and polypeptides. Their structural characteristics, including their size and amino acid sequence, as well as the presence of special amino acids, influence their iron chelation abilities and bioavailabilities. Protein hydrolysates promote iron absorption by keeping iron soluble, reducing ferric iron to ferrous iron, and promoting transport across cell membranes into the gut. We also discuss the use and relative merits of protein hydrolysates as iron supplements. PMID:28617327

  1. Amino acids in the Martian meteorite Nakhla

    NASA Technical Reports Server (NTRS)

    Glavin, D. P.; Bada, J. L.; Brinton, K. L.; McDonald, G. D.

    1999-01-01

    A suite of protein and nonprotein amino acids were detected with high-performance liquid chromatography in the water- and acid-soluble components of an interior fragment of the Martian meteorite Nakhla, which fell in Egypt in 1911. Aspartic and glutamic acids, glycine, alanine, beta-alanine, and gamma-amino-n-butyric acid (gamma-ABA) were the most abundant amino acids detected and were found primarily in the 6 M HCl-hydrolyzed, hot water extract. The concentrations ranged from 20 to 330 parts per billion of bulk meteorite. The amino acid distribution in Nakhla, including the D/L ratios (values range from <0.1 to 0.5), is similar to what is found in bacterially degraded organic matter. The amino acids in Nakhla appear to be derived from terrestrial organic matter that infiltrated the meteorite soon after its fall to Earth, although it is possible that some of the amino acids are endogenous to the meteorite. The rapid amino acid contamination of Martian meteorites after direct exposure to the terrestrial environment has important implications for Mars sample-return missions and the curation of the samples from the time of their delivery to Earth.

  2. Amino acids in the Martian meteorite Nakhla.

    PubMed

    Glavin, D P; Bada, J L; Brinton, K L; McDonald, G D

    1999-08-03

    A suite of protein and nonprotein amino acids were detected with high-performance liquid chromatography in the water- and acid-soluble components of an interior fragment of the Martian meteorite Nakhla, which fell in Egypt in 1911. Aspartic and glutamic acids, glycine, alanine, beta-alanine, and gamma-amino-n-butyric acid (gamma-ABA) were the most abundant amino acids detected and were found primarily in the 6 M HCl-hydrolyzed, hot water extract. The concentrations ranged from 20 to 330 parts per billion of bulk meteorite. The amino acid distribution in Nakhla, including the D/L ratios (values range from <0.1 to 0.5), is similar to what is found in bacterially degraded organic matter. The amino acids in Nakhla appear to be derived from terrestrial organic matter that infiltrated the meteorite soon after its fall to Earth, although it is possible that some of the amino acids are endogenous to the meteorite. The rapid amino acid contamination of Martian meteorites after direct exposure to the terrestrial environment has important implications for Mars sample-return missions and the curation of the samples from the time of their delivery to Earth.

  3. Amino acids in the Martian meteorite Nakhla

    PubMed Central

    Glavin, Daniel P.; Bada, Jeffrey L.; Brinton, Karen L. F.; McDonald, Gene D.

    1999-01-01

    A suite of protein and nonprotein amino acids were detected with high-performance liquid chromatography in the water- and acid-soluble components of an interior fragment of the Martian meteorite Nakhla, which fell in Egypt in 1911. Aspartic and glutamic acids, glycine, alanine, β-alanine, and γ-amino-n-butyric acid (γ-ABA) were the most abundant amino acids detected and were found primarily in the 6 M HCl-hydrolyzed, hot water extract. The concentrations ranged from 20 to 330 parts per billion of bulk meteorite. The amino acid distribution in Nakhla, including the d/l ratios (values range from <0.1 to 0.5), is similar to what is found in bacterially degraded organic matter. The amino acids in Nakhla appear to be derived from terrestrial organic matter that infiltrated the meteorite soon after its fall to Earth, although it is possible that some of the amino acids are endogenous to the meteorite. The rapid amino acid contamination of Martian meteorites after direct exposure to the terrestrial environment has important implications for Mars sample-return missions and the curation of the samples from the time of their delivery to Earth. PMID:10430856

  4. Non-LTE aluminium abundances in late-type stars

    NASA Astrophysics Data System (ADS)

    Nordlander, T.; Lind, K.

    2017-11-01

    Aims: Aluminium plays a key role in studies of the chemical enrichment of the Galaxy and of globular clusters. However, strong deviations from LTE (non-LTE) are known to significantly affect the inferred abundances in giant and metal-poor stars. Methods: We present non-local thermodynamic equilibrium (NLTE) modeling of aluminium using recent and accurate atomic data, in particular utilizing new transition rates for collisions with hydrogen atoms, without the need for any astrophysically calibrated parameters. For the first time, we perform 3D NLTE modeling of aluminium lines in the solar spectrum. We also compute and make available extensive grids of abundance corrections for lines in the optical and near-infrared using one-dimensional model atmospheres, and apply grids of precomputed departure coefficients to direct line synthesis for a set of benchmark stars with accurately known stellar parameters. Results: Our 3D NLTE modeling of the solar spectrum reproduces observed center-to-limb variations in the solar spectrum of the 7835 Å line as well as the mid-infrared photospheric emission line at 12.33 μm. We infer a 3D NLTE solar photospheric abundance of A(Al) = 6.43 ± 0.03, in exact agreement with the meteoritic abundance. We find that abundance corrections vary rapidly with stellar parameters; for the 3961 Å resonance line, corrections are positive and may be as large as +1 dex, while corrections for subordinate lines generally have positive sign for warm stars but negative for cool stars. Our modeling reproduces the observed line profiles of benchmark K-giants, and we find abundance corrections as large as -0.3 dex for Arcturus. Our analyses of four metal-poor benchmark stars yield consistent abundances between the 3961 Å resonance line and lines in the UV, optical and near-infrared regions. Finally, we discuss implications for the galactic chemical evolution of aluminium.

  5. Applications of hydrophilic interaction chromatography to amino acids, peptides, and proteins.

    PubMed

    Periat, Aurélie; Krull, Ira S; Guillarme, Davy

    2015-02-01

    This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity. Hydrophilic interaction chromatography is also recognized as a powerful technique for peptide analysis, and there are a lot of papers showing its applicability for proteomic applications (peptide mapping). It is expected that its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed-phase liquid chromatography, in a two-dimensional setup, to reach very high resolving power. Finally, the interest in hydrophilic interaction chromatography for intact proteins analysis is less evident due to possible solubility issues and a lack of suitable hydrophilic interaction chromatography stationary phases. To date, it has been successfully employed only for the characterization of membrane proteins, histones, and the separation of glycosylated isoforms of an intact glycoprotein. From our point of view, the number of hydrophilic interaction chromatography columns compatible with intact proteins (higher upper temperature limit, large pore size, etc.) is still too limited. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Automated protein hydrolysis delivering sample to a solid acid catalyst for amino acid analysis.

    PubMed

    Masuda, Akiko; Dohmae, Naoshi

    2010-11-01

    In this study, we developed an automatic protein hydrolysis system using strong cation-exchange resins as solid acid catalysts. Examining several kinds of inorganic solid acids and cation-exchange resins, we found that a few cation-exchange resins worked as acid catalysts for protein hydrolysis when heated in the presence of water. The most efficient resin yielded amounts of amino acids that were over 70% of those recovered after conventional hydrolysis with hydrochloric acid and resulted in amino acid compositions matching the theoretical values. The solid-acid hydrolysis was automated by packing the resin into columns, combining the columns with a high-performance liquid chromatography system, and heating them. The amino acids that constitute a protein can thereby be determined, minimizing contamination from the environment.

  7. Temperature effects on protein depolymerization and amino acid immobilization rates in soils.

    NASA Astrophysics Data System (ADS)

    Noll, Lisa; Hu, Yuntao; Zhang, Shasha; Zheng, Qing; Wanek, Wolfgang

    2017-04-01

    Increasing N deposition, land use change, elevated atmospheric CO2 concentrations and global warming have altered soil nitrogen (N) cycling during the last decades. Those changes affected ecosystem services, such as C and N sequestration in soils, which calls for a better understanding of soil N transformation processes. The cleavage of macromolecular organic N by extracellular enzymes maintains an ongoing flow of new bioavailable organic N into biotic systems and is considered to be the bottle neck of terrestrial N cycling in litter and soils. Recent studies showed that protein depolymerization is susceptible to changes in C and N availabilities. Based on general biological observations the temperature sensitivity of soil organic N processes is expected to depend on whether they are rather enzyme limited (i.e. Q10=2) or diffusion limited (i.e. Q10= 1.0 - 1.3). However, temperature sensitivities of protein depolymerization and amino acid immobilization are still unknown. We therefore here report short-term temperature effects on organic N transformation rates in soils differing in physicochemical parameters but not in climate. Soil samples were collected from two geologically distinct sites close to the LFZ Raumberg-Gumpenstein, Styria, Austria, each from three different management types (arable land, grassland, forest). Four replicates of mineral soil were taken from every site and management type. The area provides a unique opportunity to study geological and management controls in soils without confounding effects of climate and elevation. The soils differ in several soil chemical parameters, such as soil pH, base saturation, soil C: N ratio and SOM content as well as in soil physical parameters, such as soil texture, bulk density and water holding capacity. Soils were pre-incubated at 5, 15 and 25˚ C for one day. Protein depolymerization rates and amino acid immobilization rates were assessed by an isotope pool dilution assay with 15N labeled amino acids at

  8. Mussel-inspired hyperbranched poly(amino ester) polymer as strong wet tissue adhesive.

    PubMed

    Zhang, Hong; Bré, Lígia P; Zhao, Tianyu; Zheng, Yu; Newland, Ben; Wang, Wenxin

    2014-01-01

    Current medical adhesives based on cyanoacrylates typically exhibit cellular toxicity. In contrast, fibrin adhesives are non-toxic but have poor adhesive properties. To overcome these drawbacks we designed a simple and scalable adhesive precursor inspired by marine mussel adhesion that functioned with strong adhesion in wet conditions and with low cytotoxicity. Dopamine, an-amine derivative of an amino acid abundantly present in mussel adhesive proteins, was co-polymerised with a tri-functional vinyl monomer, to form a hyperbranched poly(β-amino ester) polymer termed poly(dopamine-co-acrylate) (PDA). A variety of molecular weights and crosslinking methods were analysed using an ex vivo porcine skin model and an almost 4 fold increase in wet adhesion strength was observed compared to TISSEEL(®) fibrin sealant. With a fast curing time, degradable properties and low cytotoxicity, PDA is highly attractive for medical purposes and could have a broad impact on surgeries where surgical tissue adhesives, sealants, and haemostatic agents are used. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. A Group 6 Late Embryogenesis Abundant Protein from Common Bean Is a Disordered Protein with Extended Helical Structure and Oligomer-forming Properties*

    PubMed Central

    Rivera-Najera, Lucero Y.; Saab-Rincón, Gloria; Battaglia, Marina; Amero, Carlos; Pulido, Nancy O.; García-Hernández, Enrique; Solórzano, Rosa M.; Reyes, José L.; Covarrubias, Alejandra A.

    2014-01-01

    Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association. PMID:25271167

  10. Constraints and consequences of the emergence of amino acid repeats in eukaryotic proteins.

    PubMed

    Chavali, Sreenivas; Chavali, Pavithra L; Chalancon, Guilhem; de Groot, Natalia Sanchez; Gemayel, Rita; Latysheva, Natasha S; Ing-Simmons, Elizabeth; Verstrepen, Kevin J; Balaji, Santhanam; Babu, M Madan

    2017-09-01

    Proteins with amino acid homorepeats have the potential to be detrimental to cells and are often associated with human diseases. Why, then, are homorepeats prevalent in eukaryotic proteomes? In yeast, homorepeats are enriched in proteins that are essential and pleiotropic and that buffer environmental insults. The presence of homorepeats increases the functional versatility of proteins by mediating protein interactions and facilitating spatial organization in a repeat-dependent manner. During evolution, homorepeats are preferentially retained in proteins with stringent proteostasis, which might minimize repeat-associated detrimental effects such as unregulated phase separation and protein aggregation. Their presence facilitates rapid protein divergence through accumulation of amino acid substitutions, which often affect linear motifs and post-translational-modification sites. These substitutions may result in rewiring protein interaction and signaling networks. Thus, homorepeats are distinct modules that are often retained in stringently regulated proteins. Their presence facilitates rapid exploration of the genotype-phenotype landscape of a population, thereby contributing to adaptation and fitness.

  11. Quantitative Non-canonical Amino Acid Tagging (QuaNCAT) Proteomics Identifies Distinct Patterns of Protein Synthesis Rapidly Induced by Hypertrophic Agents in Cardiomyocytes, Revealing New Aspects of Metabolic Remodeling*

    PubMed Central

    Liu, Rui; Kenney, Justin W.; Manousopoulou, Antigoni; Johnston, Harvey E.; Kamei, Makoto; Woelk, Christopher H.; Xie, Jianling; Schwarzer, Michael; Proud, Christopher G.

    2016-01-01

    Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. In the former, myocardial growth is a risk factor for cardiac failure and faster protein synthesis is a major factor driving cardiomyocyte growth. Our goal was to quantify the rapid effects of different pro-hypertrophic stimuli on the synthesis of specific proteins in ARVC and to determine whether such effects are caused by alterations on mRNA abundance or the translation of specific mRNAs. Cardiomyocytes have very low rates of protein synthesis, posing a challenging problem in terms of studying changes in the synthesis of specific proteins, which also applies to other nondividing primary cells. To study the rates of accumulation of specific proteins in these cells, we developed an optimized version of the Quantitative Noncanonical Amino acid Tagging LC/MS proteomic method to label and selectively enrich newly synthesized proteins in these primary cells while eliminating the suppressive effects of pre-existing and highly abundant nonisotope-tagged polypeptides. Our data revealed that a classical pathologic (phenylephrine; PE) and the recently identified insulin stimulus that also contributes to the development of pathological cardiac hypertrophy (insulin), both increased the synthesis of proteins involved in, e.g. glycolysis, the Krebs cycle and beta-oxidation, and sarcomeric components. However, insulin increased synthesis of many metabolic enzymes to a greater extent than PE. Using a novel validation method, we confirmed that synthesis of selected candidates is indeed up-regulated by PE and insulin. Synthesis of all proteins studied was up-regulated by signaling through mammalian target of rapamycin complex 1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was up-regulated in rat hearts following TAC. This isoform possesses specific regulatory

  12. A capillary electrophoresis-tandem mass spectrometry methodology for the determination of non-protein amino acids in vegetable oils as novel markers for the detection of adulterations in olive oils.

    PubMed

    Sánchez-Hernández, Laura; Marina, Maria Luisa; Crego, Antonio L

    2011-07-29

    A new analytical methodology based on capillary electrophoresis-mass spectrometry (CE-MS(2)) is presented in this work, enabling the identification and determination of six non-protein amino acids (ornithine, β-alanine, GABA, alloisoleucine, citrulline and pyroglutamic acid) in vegetable oils. This methodology is based on a previous derivatization with butanol and subsequent separation using acidic conditions followed by on-line coupling to an ion trap analyzer for MS(2) detection established through an electrospray-coaxial sheath flow interface. The electrophoretic and interface parameters were optimized obtaining the separation of all compounds in less than 15 min and with resolutions higher than 5. The proposed method was validated by assessing its accuracy, precision (RSD<7% for corrected peak areas), LODs and LOQs (between 0.04-0.19 ng/g and 0.06-0.31 ng/g, respectively) and linearity range (R(2)>0.99), and it was used in order to identify the selected non-protein amino acids in soybean oils, sunflower oils, corn oils and extra virgin olive oils. MS(2) experiments performed the fingerprint fragmentation of these compounds allowing to corroborate ornithine and alloisoleucine in seed oils but not in olive oils. The method was applied to identify and quantify olive oil adulterations with soybean oil detecting in a single run the amino acids in mixtures up to 2% (w/w). The results showed a high potential in using these compounds as novel markers for the detection of adulterations of extra virgin olive oils with seed oils. Thus, the developed method could be considered a simple, rapid and reliable method for the quality evaluation of extra virgin olive oil permitting its authentication. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. How the folding rates of two- and multistate proteins depend on the amino acid properties.

    PubMed

    Huang, Jitao T; Huang, Wei; Huang, Shanran R; Li, Xin

    2014-10-01

    Proteins fold by either two-state or multistate kinetic mechanism. We observe that amino acids play different roles in different mechanism. Many residues that are easy to form regular secondary structures (α helices, β sheets and turns) can promote the two-state folding reactions of small proteins. Most of hydrophilic residues can speed up the multistate folding reactions of large proteins. Folding rates of large proteins are equally responsive to the flexibility of partial amino acids. Other properties of amino acids (including volume, polarity, accessible surface, exposure degree, isoelectric point, and phase transfer energy) have contributed little to folding kinetics of the proteins. Cysteine is a special residue, it triggers two-state folding reaction and but inhibits multistate folding reaction. These findings not only provide a new insight into protein structure prediction, but also could be used to direct the point mutations that can change folding rate. © 2014 Wiley Periodicals, Inc.

  14. Protein-bound D-amino acids, and to a lesser extent lysinoalanine, decrease true ileal protein digestibility in minipigs as determined with (15)N-labeling.

    PubMed

    de Vrese, M; Frik, R; Roos, N; Hagemeister, H

    2000-08-01

    Heat and alkali treatment of food may increase the concentrations of protein-bound D-amino acids and cross-links such as lysinoalanine (LAL). To examine how protein treatment affects digestibility, purified test meals [total protein 150 g/kg dry matter (DM), 0.44 MJ/(kg BW(0.75). d)] were prepared, containing (g/kg DM) casein, 75; beta-lactoglobulin, 50; or wheat protein, 40. Each was (15)N-labeled. Test proteins were used either in their native form or after treatment for 6 or 24 h at 65 degrees C, pH 10.5-11.5. Each meal was fed to nine adult miniature pigs (twofold complete cross-classification). Chyme was collected continuously over 33 h postprandially via T-fistulas in the terminal ileum, and digestibilities of test proteins and individual L- and D-amino acids were calculated on the basis of recovery of (15)N and the respective amino acids in the chyme. Treatment of casein, beta-lactoglobulin or wheat protein for 24 h increased levels of D-amino acid residues. L-Asparagine and aspartate (L-Asx) were particularly susceptible; 14. 7 +/- 0.4, 11.7 +/- 0.2 and 11.0 +/- 0.9%, respectively, underwent racemization. LAL levels increased in parallel; 11.7 +/- 0.3, 13.6 +/- 0 and 14.8 +/- 0.0%, respectively, of total lysine was converted to LAL. At the same time, prececal protein digestibility was decreased by 13.4 +/- 2.3, 15.3 +/- 1.4 and 17.8 +/- 1.2% units, respectively (P < 0.05; mean +/- SEM, n = 9). Digestibility of individual L-amino acids decreased by 10-15%, but L-amino acids prone to peptic cleavage, such as L-phenylalanine and L-tyrosine, were not affected. Digestibilities of D-amino acids and LAL were approximately 35%. It seems that mainly D-amino acids, and to a lesser extent LAL, were responsible for lower digestibility by interfering with peptic cleavage.

  15. "De-novo" amino acid sequence elucidation of protein G'e by combined "top-down" and "bottom-up" mass spectrometry.

    PubMed

    Yefremova, Yelena; Al-Majdoub, Mahmoud; Opuni, Kwabena F M; Koy, Cornelia; Cui, Weidong; Yan, Yuetian; Gross, Michael L; Glocker, Michael O

    2015-03-01

    Mass spectrometric de-novo sequencing was applied to review the amino acid sequence of a commercially available recombinant protein G´ with great scientific and economic importance. Substantial deviations to the published amino acid sequence (Uniprot Q54181) were found by the presence of 46 additional amino acids at the N-terminus, including a so-called "His-tag" as well as an N-terminal partial α-N-gluconoylation and α-N-phosphogluconoylation, respectively. The unexpected amino acid sequence of the commercial protein G' comprised 241 amino acids and resulted in a molecular mass of 25,998.9 ± 0.2 Da for the unmodified protein. Due to the higher mass that is caused by its extended amino acid sequence compared with the original protein G' (185 amino acids), we named this protein "protein G'e." By means of mass spectrometric peptide mapping, the suggested amino acid sequence, as well as the N-terminal partial α-N-gluconoylations, was confirmed with 100% sequence coverage. After the protein G'e sequence was determined, we were able to determine the expression vector pET-28b from Novagen with the Xho I restriction enzyme cleavage site as the best option that was used for cloning and expressing the recombinant protein G'e in E. coli. A dissociation constant (K(d)) value of 9.4 nM for protein G'e was determined thermophoretically, showing that the N-terminal flanking sequence extension did not cause significant changes in the binding affinity to immunoglobulins.

  16. Effects of preparation methods on protein and amino acid contents of various eggs available in Malaysian local markets.

    PubMed

    Ismail, Maznah; Mariod, Abdalbasit; Pin, Sia Soh

    2013-01-01

    The effect of preparation methods (raw, half-boiled and hard-boiled) on protein and amino acid contents, as well as the protein quality (amino acid score) of regular, kampung and nutrient enriched Malaysian eggs was investigated. The protein content was determined using a semi-micro Kjeldahl method whereas the amino acid composition was determined using HPLC. The protein content of raw regular, kampung and nutrient enriched eggs were 49.9 ±0.2%, 55.8 ±0.2% and 56.5 ±0.5%, respectively. The protein content of hard-boiled eggs of regular, kampung and nutrient enriched eggs was 56.8 ±0.1%, 54.7 ±0.1%, and 53.7 ±0.5%, while that for half-boiled eggs of regular, kampung and nutrient enriched eggs was 54.7 ±0.6%, 53.4 ±0.4%, and 55.1 ±0.7%, respectively. There were significant differences (p < 0.05) in protein and amino acid contents of half-boiled, hard-boiled as compared with raw samples, and valine was found as the limiting amino acid. It was found that there were significant differences (p < 0.05) of total amino score in regular, kampung and nutrient enriched eggs after heat treatments.Furthermore, hard-boiling (100°C) for 10 minutes and half-boiling (100°C) for 5 minutes affects the total amino score, which in turn alter the protein quality of the egg.

  17. Interaction of foot-and-mouth disease virus non-structural protein 3A with host protein DCTN3 is important for viral virulence in cattle

    USDA-ARS?s Scientific Manuscript database

    Non-structural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids in most FMDVs examined to date. The role of 3A in virus growth and virulence within the natural host is not well understood. Using a yeast two-hybrid approach, we identified cellular ...

  18. Protein location prediction using atomic composition and global features of the amino acid sequence

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cherian, Betsy Sheena, E-mail: betsy.skb@gmail.com; Nair, Achuthsankar S.

    2010-01-22

    Subcellular location of protein is constructive information in determining its function, screening for drug candidates, vaccine design, annotation of gene products and in selecting relevant proteins for further studies. Computational prediction of subcellular localization deals with predicting the location of a protein from its amino acid sequence. For a computational localization prediction method to be more accurate, it should exploit all possible relevant biological features that contribute to the subcellular localization. In this work, we extracted the biological features from the full length protein sequence to incorporate more biological information. A new biological feature, distribution of atomic composition is effectivelymore » used with, multiple physiochemical properties, amino acid composition, three part amino acid composition, and sequence similarity for predicting the subcellular location of the protein. Support Vector Machines are designed for four modules and prediction is made by a weighted voting system. Our system makes prediction with an accuracy of 100, 82.47, 88.81 for self-consistency test, jackknife test and independent data test respectively. Our results provide evidence that the prediction based on the biological features derived from the full length amino acid sequence gives better accuracy than those derived from N-terminal alone. Considering the features as a distribution within the entire sequence will bring out underlying property distribution to a greater detail to enhance the prediction accuracy.« less

  19. Computationally Efficient Multiscale Reactive Molecular Dynamics to Describe Amino Acid Deprotonation in Proteins

    PubMed Central

    2016-01-01

    An important challenge in the simulation of biomolecular systems is a quantitative description of the protonation and deprotonation process of amino acid residues. Despite the seeming simplicity of adding or removing a positively charged hydrogen nucleus, simulating the actual protonation/deprotonation process is inherently difficult. It requires both the explicit treatment of the excess proton, including its charge defect delocalization and Grotthuss shuttling through inhomogeneous moieties (water and amino residues), and extensive sampling of coupled condensed phase motions. In a recent paper (J. Chem. Theory Comput.2014, 10, 2729−273725061442), a multiscale approach was developed to map high-level quantum mechanics/molecular mechanics (QM/MM) data into a multiscale reactive molecular dynamics (MS-RMD) model in order to describe amino acid deprotonation in bulk water. In this article, we extend the fitting approach (called FitRMD) to create MS-RMD models for ionizable amino acids within proteins. The resulting models are shown to faithfully reproduce the free energy profiles of the reference QM/MM Hamiltonian for PT inside an example protein, the ClC-ec1 H+/Cl– antiporter. Moreover, we show that the resulting MS-RMD models are computationally efficient enough to then characterize more complex 2-dimensional free energy surfaces due to slow degrees of freedom such as water hydration of internal protein cavities that can be inherently coupled to the excess proton charge translocation. The FitRMD method is thus shown to be an effective way to map ab initio level accuracy into a much more computationally efficient reactive MD method in order to explicitly simulate and quantitatively describe amino acid protonation/deprotonation in proteins. PMID:26734942

  20. Computationally Efficient Multiscale Reactive Molecular Dynamics to Describe Amino Acid Deprotonation in Proteins.

    PubMed

    Lee, Sangyun; Liang, Ruibin; Voth, Gregory A; Swanson, Jessica M J

    2016-02-09

    An important challenge in the simulation of biomolecular systems is a quantitative description of the protonation and deprotonation process of amino acid residues. Despite the seeming simplicity of adding or removing a positively charged hydrogen nucleus, simulating the actual protonation/deprotonation process is inherently difficult. It requires both the explicit treatment of the excess proton, including its charge defect delocalization and Grotthuss shuttling through inhomogeneous moieties (water and amino residues), and extensive sampling of coupled condensed phase motions. In a recent paper (J. Chem. Theory Comput. 2014, 10, 2729-2737), a multiscale approach was developed to map high-level quantum mechanics/molecular mechanics (QM/MM) data into a multiscale reactive molecular dynamics (MS-RMD) model in order to describe amino acid deprotonation in bulk water. In this article, we extend the fitting approach (called FitRMD) to create MS-RMD models for ionizable amino acids within proteins. The resulting models are shown to faithfully reproduce the free energy profiles of the reference QM/MM Hamiltonian for PT inside an example protein, the ClC-ec1 H(+)/Cl(-) antiporter. Moreover, we show that the resulting MS-RMD models are computationally efficient enough to then characterize more complex 2-dimensional free energy surfaces due to slow degrees of freedom such as water hydration of internal protein cavities that can be inherently coupled to the excess proton charge translocation. The FitRMD method is thus shown to be an effective way to map ab initio level accuracy into a much more computationally efficient reactive MD method in order to explicitly simulate and quantitatively describe amino acid protonation/deprotonation in proteins.

  1. Amino Acid Distribution Rules Predict Protein Fold: Protein Grammar for Beta-Strand Sandwich-Like Structures

    PubMed Central

    Kister, Alexander

    2015-01-01

    We present an alternative approach to protein 3D folding prediction based on determination of rules that specify distribution of “favorable” residues, that are mainly responsible for a given fold formation, and “unfavorable” residues, that are incompatible with that fold, in polypeptide sequences. The process of determining favorable and unfavorable residues is iterative. The starting assumptions are based on the general principles of protein structure formation as well as structural features peculiar to a protein fold under investigation. The initial assumptions are tested one-by-one for a set of all known proteins with a given structure. The assumption is accepted as a “rule of amino acid distribution” for the protein fold if it holds true for all, or near all, structures. If the assumption is not accepted as a rule, it can be modified to better fit the data and then tested again in the next step of the iterative search algorithm, or rejected. We determined the set of amino acid distribution rules for a large group of beta sandwich-like proteins characterized by a specific arrangement of strands in two beta sheets. It was shown that this set of rules is highly sensitive (~90%) and very specific (~99%) for identifying sequences of proteins with specified beta sandwich fold structure. The advantage of the proposed approach is that it does not require that query proteins have a high degree of homology to proteins with known structure. So long as the query protein satisfies residue distribution rules, it can be confidently assigned to its respective protein fold. Another advantage of our approach is that it allows for a better understanding of which residues play an essential role in protein fold formation. It may, therefore, facilitate rational protein engineering design. PMID:25625198

  2. Influence of branched-chain amino acid composition of culture media on the synthesis of plasma proteins by serum-free cultured rat hepatocytes.

    PubMed

    Montoya, A; Gómez-Lechón, M J; Castell, J V

    1989-04-01

    Supplementation of Ham's F12 culture medium with essential amino acids (EAA) up to the rat plasma levels increased the rates of synthesis of albumin and transferrin by cultured rat hepatocytes by 1.3 and 1.7, respectively. Fifty percent of this increase could be attributed to three of the EAA: the branched-chain amino acids (BCAA: Leu Ile and Val). Non-branched-chain essential amino acids (non-BC-EAA) stimulated only 25% of the increase produced by the whole EAA mixture. When each EAA was tested individually, none of them caused an appreciable increase in albumin and transferrin in culture medium. When the concentrations of all EAA were raised to rat postprandial portal levels, albumin and transferrin synthesis rates reached a maximum, increasing by 3.2 and 3.5, respectively. Supplementation with BCAA at postprandial portal concentrations increased albumin and transferrin synthesis rates by 2.2 and 2.0, respectively, and had no noteworthy effect on the synthesis of cellular proteins. Non-BC-EAA at their postprandial portal concentrations increased albumin and transferrin synthesis rates by 1.7 and 1.9, respectively. Supplementation with alanine to reach a nitrogen content equal to that of the modified EAA-enriched medium had no stimulatory effect. Our results show that EAA have a specific effect on the synthesis of plasma proteins by cultured hepatocytes, and that BCAA at physiologic concentrations account for the major part of this stimulatory effect. Consequently, EAA and particularly BCAA concentration should be elevated in serum-free nutrient media to sustain maximum plasma protein synthesis.

  3. Effect of a protein-rich meal on urinary and salivary free amino acid concentrations in human subjects.

    PubMed

    Brand, H S; Jörning, G G; Chamuleau, R A; Abraham-Inpijn, L

    1997-08-08

    The aim of the present study was to investigate whether in healthy volunteers acute changes in plasma free amino acid composition after a protein-rich test meal are reflected in the urinary and salivary concentrations of the corresponding amino acids. The ingestion of a protein-rich meal elicited a significant increase of plasma and urine amino acid concentrations. The postprandial salivary amino acid excretion showed only minor changes. For several amino acids (alanine, arginine, asparagine, glycine, threonine and valine) significant relations were observed between the increase in concentration of these amino acids in venous plasma and urine. In whole saliva, only threonine and valine showed a significant relationship with the corresponding plasma concentration. Our data suggest that the urinary amino acid excretion of several amino acids has the potential for estimating short-term changes in plasma concentrations. Determination of salivary amino acid concentrations seems less appropriate for this purpose.

  4. [Non-enzymatic glycosylation of dietary protein in vitro].

    PubMed

    Bednykh, B S; Evdokimov, I A; Sokolov, A I

    2015-01-01

    Non-enzymatic glycosylation of proteins, based on discovered by Mayarn reaction of carbohydrate aldehyde group with a free amino group of a protein molecule, is well known to experts in biochemistry of food industry. Generated brown solid in some cases give the product marketable qualities--crackling bread--in others conversely, worsen the product. The biological effects of far-advanced products of non-enzymatic protein glycosylation reaction have not been studied enough, although it was reported previously that they are not split by digestive enzymes and couldn't be absorbed by animals. The objective of this work was to compare the depth of glycosylation of different food proteins of animal and vegetable origin. The objects of the study were proteins of animal (casein, lactoglobulin, albumin) and vegetable (soy isolate, proteins of rice flour, buckwheat, oatmeal) origin, glucose and fructose were selected as glycosylation agents, exposure 15 days at 37 degrees C. Lactoglobulin was glycosylated to a lesser extent among the proteins of animal origin while protein of oatmeal was glycosylated in the least degree among vegetable proteins. Conversely, such proteins as casein and soya isolate protein bound rather large amounts of carbohydrates. Fructose binding with protein was generally higher than the binding of glucose. The only exception was a protein of oatmeal. When of glucose and fructose simultaneously presented in the incubation medium, glucose binding usually increased while binding of fructose, in contrast, reduced. According to the total amount of carbohydrate (mcg), which is able to attach a protein (mg) the studied food proteins located in the following order: albumin (38) > soy protein isolate (23) > casein (15,) > whey protein rice flour protein (6) > protein from buckwheat flour (3) > globulin (2) > protein of oatmeal (0.3). The results obtained are to be used to select the optimal combination of proteins and carbohydrates, in which the glycosylation

  5. DNA Asymmetric Strand Bias Affects the Amino Acid Composition of Mitochondrial Proteins

    PubMed Central

    Min, Xiang Jia; Hickey, Donal A.

    2007-01-01

    Abstract Variations in GC content between genomes have been extensively documented. Genomes with comparable GC contents can, however, still differ in the apportionment of the G and C nucleotides between the two DNA strands. This asymmetric strand bias is known as GC skew. Here, we have investigated the impact of differences in nucleotide skew on the amino acid composition of the encoded proteins. We compared orthologous genes between animal mitochondrial genomes that show large differences in GC and AT skews. Specifically, we compared the mitochondrial genomes of mammals, which are characterized by a negative GC skew and a positive AT skew, to those of flatworms, which show the opposite skews for both GC and AT base pairs. We found that the mammalian proteins are highly enriched in amino acids encoded by CA-rich codons (as predicted by their negative GC and positive AT skews), whereas their flatworm orthologs were enriched in amino acids encoded by GT-rich codons (also as predicted from their skews). We found that these differences in mitochondrial strand asymmetry (measured as GC and AT skews) can have very large, predictable effects on the composition of the encoded proteins. PMID:17974594

  6. Abundant storage protein depletion from tuber proteins using ethanol precipitation method: Suitability to proteomics study.

    PubMed

    Lee, Hye Min; Gupta, Ravi; Kim, Sun Hyung; Wang, Yiming; Rakwal, Randeep; Agrawal, Ganesh Kumar; Kim, Sun Tae

    2015-05-01

    High-abundance proteins (HAPs) hamper in-depth proteome study necessitating development of a HAPs depletion method. Here, we report a novel ethanol precipitation method (EPM) for HAPs depletion from total tuber proteins. Ethanol showed a dose-dependent effect on depletion of sporamin from sweet potato and patatin from potato tubers, respectively. The 50% ethanol was an optimal concentration. 2DE analysis of EPM-prepared sweet potato proteins also revealed enrichment of storage proteins (SPs) in ethanol supernatant (ES) resulting in detection of new low-abundance proteins in ethanol pellet (EP), compared to total fraction. The ES fraction showed even higher trypsin inhibitor activity than total proteins, further showing the efficacy of EPM in enrichment of sporamin in ES fraction. Application of this method was demonstrated for comparative proteomics of two sweet potato cultivars (Hwang-geum and Ho-bac) and purification of SP (sporamin) in its native form, as examples. Comparative proteomics identified many cultivar specific protein spots and selected spots were confidently assigned for their protein identity using MALDI-TOF-TOF analysis. Overall, the EPM is simple, reproducible, and economical for depletion of SPs and is suitable for downstream proteomics study. This study opens a door for its potential application to other tuber crops or fruits rich in carbohydrates. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Common amino acid domain among endopolygalacturonases of ascomycete fungi.

    PubMed Central

    Keon, J P; Waksman, G

    1990-01-01

    The endopolygalacturonase (EC 3.2.1.15) enzymes produced in vitro by three ascomycete fungi, Aspergillus niger, Sclerotinia sclerotiorum, and Colletotrichum lindemuthianum were studied by using thin-layer isoelectric focusing and activity stain overlay techniques. The polygalacturonases from A. niger and S. sclerotiorum consisted of numerous isoforms, whereas the endopolygalacturonase from C. lindemuthianum consisted of a single protein species. The most abundant endopolygalacturonase isoform produced by each of these organisms was purified and characterized. Biochemical parameters, including molecular weight, isoelectric point, kinetic parameters, temperature and pH optima, and thermal stability, were determined. Considerable differences in physical and chemical properties were demonstrated among these fungal polygalacturonases. Antibodies raised against individual proteins exhibited little cross-reaction, suggesting that these enzymes differ structurally as well as biochemically. In contrast, the analysis of the N-terminal amino acid sequences of the three proteins showed extensive homology, particularly in a region labeled domain 1 in which 84% of the amino acids were conserved. Images PMID:2403258

  8. Fast computational methods for predicting protein structure from primary amino acid sequence

    DOEpatents

    Agarwal, Pratul Kumar [Knoxville, TN

    2011-07-19

    The present invention provides a method utilizing primary amino acid sequence of a protein, energy minimization, molecular dynamics and protein vibrational modes to predict three-dimensional structure of a protein. The present invention also determines possible intermediates in the protein folding pathway. The present invention has important applications to the design of novel drugs as well as protein engineering. The present invention predicts the three-dimensional structure of a protein independent of size of the protein, overcoming a significant limitation in the prior art.

  9. Climate shapes the protein abundance of dominant soil bacteria.

    PubMed

    Bastida, Felipe; Crowther, Tom W; Prieto, Iván; Routh, Devin; García, Carlos; Jehmlich, Nico

    2018-05-28

    Sensitive models of climate change impacts would require a better integration of multi-omics approaches that connect the abundance and activity of microbial populations. Here, we show that climate is a fundamental driver of the protein abundance of Actinobacteria, Planctomycetes and Proteobacteria, supporting the hypothesis that metabolic activity of some dominant phyla may be closely linked to climate. These results may improve our capacity to construct microbial models that better predict the impact of climate change in ecosystem processes. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Comparison of colorimetric assays with quantitative amino acid analysis for protein quantification of Generalized Modules for Membrane Antigens (GMMA).

    PubMed

    Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A; Saul, Allan; Gerke, Christiane

    2015-01-01

    Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification.

  11. Factors influencing the rate of non-enzymatic activation of carboxylic and amino acids by ATP

    NASA Technical Reports Server (NTRS)

    Mullins, D. W., Jr.; Lacey, J. C., Jr.

    1981-01-01

    The nonenzymatic formation of adenylate anhydrides of carboxylic and amino acids is discussed as a necessary step in the origin of the genetic code and protein biosynthesis. Results of studies are presented which have shown the rate of activation to depend on the pKa of the carboxyl group, the pH of the medium, temperature, the divalent metal ion catalyst, salt concentration, and the nature of the amino acid. In particular, it was found that of the various amino acids investigated, phenylalanine had the greatest affinity for the adenine derivatives adenosine and ATP. Results thus indicate that selective affinities between amino acids and nucleotides were important during prebiotic chemical evolution, and may have played a major role in the origin of protein synthesis and genetic coding.

  12. Structure of the ordered hydration of amino acids in proteins: analysis of crystal structures

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Biedermannová, Lada, E-mail: lada.biedermannova@ibt.cas.cz; Schneider, Bohdan

    2015-10-27

    The hydration of protein crystal structures was studied at the level of individual amino acids. The dependence of the number of water molecules and their preferred spatial localization on various parameters, such as solvent accessibility, secondary structure and side-chain conformation, was determined. Crystallography provides unique information about the arrangement of water molecules near protein surfaces. Using a nonredundant set of 2818 protein crystal structures with a resolution of better than 1.8 Å, the extent and structure of the hydration shell of all 20 standard amino-acid residues were analyzed as function of the residue conformation, secondary structure and solvent accessibility. Themore » results show how hydration depends on the amino-acid conformation and the environment in which it occurs. After conformational clustering of individual residues, the density distribution of water molecules was compiled and the preferred hydration sites were determined as maxima in the pseudo-electron-density representation of water distributions. Many hydration sites interact with both main-chain and side-chain amino-acid atoms, and several occurrences of hydration sites with less canonical contacts, such as carbon–donor hydrogen bonds, OH–π interactions and off-plane interactions with aromatic heteroatoms, are also reported. Information about the location and relative importance of the empirically determined preferred hydration sites in proteins has applications in improving the current methods of hydration-site prediction in molecular replacement, ab initio protein structure prediction and the set-up of molecular-dynamics simulations.« less

  13. Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids.

    PubMed

    Martin, Rey W; Des Soye, Benjamin J; Kwon, Yong-Chan; Kay, Jennifer; Davis, Roderick G; Thomas, Paul M; Majewska, Natalia I; Chen, Cindy X; Marcum, Ryan D; Weiss, Mary Grace; Stoddart, Ashleigh E; Amiram, Miriam; Ranji Charna, Arnaz K; Patel, Jaymin R; Isaacs, Farren J; Kelleher, Neil L; Hong, Seok Hoon; Jewett, Michael C

    2018-03-23

    Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-L-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.

  14. Amino acid nutrition beyond methionine and lysine for milk protein

    USDA-ARS?s Scientific Manuscript database

    Amino acids are involved in many important physiological processes affecting the production, health, and reproduction of high-producing dairy cows. Most research and recommendations for lactating dairy cows has focused on methionine and lysine for increasing milk protein yield. This is because these...

  15. Parenteral amino acids increase albumin and skeletal muscle protein fractional synthetic rates in premature newborn minipigs.

    PubMed

    Hellstern, Gerald; Kaempf-Rotzoll, Daisy; Linderkamp, Otwin; Langhans, Klaus-Dieter; Rating, Dietz

    2002-09-01

    Early administration of parenteral amino acids increases whole body nitrogen retention in premature infants. Tracer kinetic studies suggest an increase in whole body protein synthesis as a possible mechanism for this increase in nitrogen retention. However, the effect of early parenteral amino acids on synthesis of specific proteins remains uncertain. Using premature newborn minipigs as a model for human premature newborns, we investigated the effects of parenterally administered amino acids on albumin and skeletal muscle protein fractional synthetic rates. Fifteen Yucatan minipigs were delivered by cesarean section 6 days before the mean expected delivery date (day 106 of gestation; expected gestation, 111-113 days) and randomized to two groups immediately after birth: 7 piglets received a mixture of amino acids (0.4 g. kg. h ) and glucose (0.8 g. kg. h ) for 5 hours, and 8 piglets (control group) received glucose only. All piglets received a continuous primed infusion of 1-[ C]valine. Arterial plasma free C-valine enrichment was measured by gas chromatography/mass spectrometry, and protein synthetic rates were determined by measuring incorporation of C-valine into albumin and skeletal muscle protein using gas chromatography/combustion/isotope ratio mass spectrometry. Administration of amino acids increased albumin (87.0% +/- 42.1% [mean +/- SD] vs. 37.6% +/- 6.8% per 24 hours; < 0.05) and skeletal muscle fractional synthetic rates (11.60% +/- 6.9% vs. 6.5% +/- 1.5% per 24 hours; < 0.05). We conclude that parenteral amino acids increase albumin and skeletal muscle fractional synthetic rates in premature piglets on the first day of life.

  16. Far Infrared spectroscopy of proteinogenic and other less common amino acids

    NASA Astrophysics Data System (ADS)

    Iglesias-Groth, S.; Cataldo, F.

    2018-05-01

    Far infrared spectroscopy is a powerful tool complementing the potential of mid infrared spectroscopy for the search and identification of organic molecules in space. The far infrared spectra of a total of 29 amino acids are reported in this study. In addition to the spectra of 20 common proteinogenic amino acids, spectra of a selection of 9 non-proteinogenic amino acids are also reported, including the 2-aminoisobutyric acid or α-aminoisobutyric acid which, with glycine, it is one of the most abundant amino acids found in meteorites. The present database of 29 far infrared spectra may serve as reference in the search for amino acids in space environments, given the new apportunities that JWST offers for mid and far IR spectroscopy.

  17. Enhanced Detection of Low-Abundance Human Plasma Proteins by Integrating Polyethylene Glycol Fractionation and Immunoaffinity Depletion

    PubMed Central

    Liu, Haipeng; Yu, Jia; Qiao, Rui; Zhou, Mi; Yang, Yongtao; Zhou, Jian; Xie, Peng

    2016-01-01

    The enormous depth complexity of the human plasma proteome poses a significant challenge for current mass spectrometry-based proteomic technologies in terms of detecting low-level proteins in plasma, which is essential for successful biomarker discovery efforts. Typically, a single-step analytical approach cannot reduce this intrinsic complexity. Current simplex immunodepletion techniques offer limited capacity for detecting low-abundance proteins, and integrated strategies are thus desirable. In this respect, we developed an improved strategy for analyzing the human plasma proteome by integrating polyethylene glycol (PEG) fractionation with immunoaffinity depletion. PEG fractionation of plasma proteins is simple, rapid, efficient, and compatible with a downstream immunodepletion step. Compared with immunodepletion alone, our integrated strategy substantially improved the proteome coverage afforded by PEG fractionation. Coupling this new protocol with liquid chromatography-tandem mass spectrometry, 135 proteins with reported normal concentrations below 100 ng/mL were confidently identified as common low-abundance proteins. A side-by-side comparison indicated that our integrated strategy was increased by average 43.0% in the identification rate of low-abundance proteins, relying on an average 65.8% increase of the corresponding unique peptides. Further investigation demonstrated that this combined strategy could effectively alleviate the signal-suppressive effects of the major high-abundance proteins by affinity depletion, especially with moderate-abundance proteins after incorporating PEG fractionation, thereby greatly enhancing the detection of low-abundance proteins. In sum, the newly developed strategy of incorporating PEG fractionation to immunodepletion methods can potentially aid in the discovery of plasma biomarkers of therapeutic and clinical interest. PMID:27832179

  18. Primordial Earth's Environment Suggested from Equilibrium Conditions among Proteinic Amino Acids

    NASA Astrophysics Data System (ADS)

    Yamaguchi, Yoshimitsu; Nakazawa, K.; Emori, H.

    2006-12-01

    Amino acids are one of the essential substances for terrestrial lives. There are, as is well known, two interesting and important properties on amino acids in terrestrial lives: one is that infinite kinds of amino acids can be synthesized formally but, marvelously, only about 20 amino acids of these are utilized by proteinic materials of a wide variety of terrestrial lives. Another is that the relative molar ratios among the 20 amino acids are almost equal, at least, by the order of magnitude. In our present paper, paying attention to these facts, we will make an attempt to specify physical and chemical environments of the primordial Earth where first vital organic compounds begin to be synthesized. By assuming that two amino acids and appropriate inorganic compounds (CO2, NH3, CH4, etc.) are in chemical equilibrium under the condition of heated water, we can find the activity ratios (or activities) of inorganic compounds. Our results suggest that the heated water must be in a reducing condition and that the oxidizing compounds like O2 or SO2 cannot contribute to the equilibrium reactions.

  19. Visualization and dissemination of multidimensional proteomics data comparing protein abundance during Caenorhabditis elegans development.

    PubMed

    Riffle, Michael; Merrihew, Gennifer E; Jaschob, Daniel; Sharma, Vagisha; Davis, Trisha N; Noble, William S; MacCoss, Michael J

    2015-11-01

    Regulation of protein abundance is a critical aspect of cellular function, organism development, and aging. Alternative splicing may give rise to multiple possible proteoforms of gene products where the abundance of each proteoform is independently regulated. Understanding how the abundances of these distinct gene products change is essential to understanding the underlying mechanisms of many biological processes. Bottom-up proteomics mass spectrometry techniques may be used to estimate protein abundance indirectly by sequencing and quantifying peptides that are later mapped to proteins based on sequence. However, quantifying the abundance of distinct gene products is routinely confounded by peptides that map to multiple possible proteoforms. In this work, we describe a technique that may be used to help mitigate the effects of confounding ambiguous peptides and multiple proteoforms when quantifying proteins. We have applied this technique to visualize the distribution of distinct gene products for the whole proteome across 11 developmental stages of the model organism Caenorhabditis elegans. The result is a large multidimensional dataset for which web-based tools were developed for visualizing how translated gene products change during development and identifying possible proteoforms. The underlying instrument raw files and tandem mass spectra may also be downloaded. The data resource is freely available on the web at http://www.yeastrc.org/wormpes/ . Graphical Abstract ᅟ.

  20. Visualization and Dissemination of Multidimensional Proteomics Data Comparing Protein Abundance During Caenorhabditis elegans Development

    NASA Astrophysics Data System (ADS)

    Riffle, Michael; Merrihew, Gennifer E.; Jaschob, Daniel; Sharma, Vagisha; Davis, Trisha N.; Noble, William S.; MacCoss, Michael J.

    2015-11-01

    Regulation of protein abundance is a critical aspect of cellular function, organism development, and aging. Alternative splicing may give rise to multiple possible proteoforms of gene products where the abundance of each proteoform is independently regulated. Understanding how the abundances of these distinct gene products change is essential to understanding the underlying mechanisms of many biological processes. Bottom-up proteomics mass spectrometry techniques may be used to estimate protein abundance indirectly by sequencing and quantifying peptides that are later mapped to proteins based on sequence. However, quantifying the abundance of distinct gene products is routinely confounded by peptides that map to multiple possible proteoforms. In this work, we describe a technique that may be used to help mitigate the effects of confounding ambiguous peptides and multiple proteoforms when quantifying proteins. We have applied this technique to visualize the distribution of distinct gene products for the whole proteome across 11 developmental stages of the model organism Caenorhabditis elegans. The result is a large multidimensional dataset for which web-based tools were developed for visualizing how translated gene products change during development and identifying possible proteoforms. The underlying instrument raw files and tandem mass spectra may also be downloaded. The data resource is freely available on the web at http://www.yeastrc.org/wormpes/.

  1. Integration of multi-omics data of a genome-reduced bacterium: Prevalence of post-transcriptional regulation and its correlation with protein abundances

    PubMed Central

    Chen, Wei-Hua; van Noort, Vera; Lluch-Senar, Maria; Hennrich, Marco L.; H. Wodke, Judith A.; Yus, Eva; Alibés, Andreu; Roma, Guglielmo; Mende, Daniel R.; Pesavento, Christina; Typas, Athanasios; Gavin, Anne-Claude; Serrano, Luis; Bork, Peer

    2016-01-01

    We developed a comprehensive resource for the genome-reduced bacterium Mycoplasma pneumoniae comprising 1748 consistently generated ‘-omics’ data sets, and used it to quantify the power of antisense non-coding RNAs (ncRNAs), lysine acetylation, and protein phosphorylation in predicting protein abundance (11%, 24% and 8%, respectively). These factors taken together are four times more predictive of the proteome abundance than of mRNA abundance. In bacteria, post-translational modifications (PTMs) and ncRNA transcription were both found to increase with decreasing genomic GC-content and genome size. Thus, the evolutionary forces constraining genome size and GC-content modify the relative contributions of the different regulatory layers to proteome homeostasis, and impact more genomic and genetic features than previously appreciated. Indeed, these scaling principles will enable us to develop more informed approaches when engineering minimal synthetic genomes. PMID:26773059

  2. Protein and amino acid intakes in a rural area of Bangladesh.

    PubMed

    Heck, Julia E; Nieves, Jeri W; Chen, Yu; Parvez, Faruque; Brandt-Rauf, Paul W; Howe, Geoffrey R; Ahsan, Habibul

    2010-06-01

    Few studies have described protein and amino acid intakes in rural Bangladesh, a country with considerable undernutrition. The purpose of this population-based study was to assess and describe protein and amino acid intakes in Araihazar, Bangladesh. The study participants were 11,170 adult men and women who participated in the Health Effects of Arsenic Longitudinal Study (HEALS), which had a 98% participation rate. Dietary exposures were assessed by a food-frequency questionnaire that had been designed and validated for the HEALS study population. The mean body mass index (BMI) was 19.7 among all participants, and 34.9% of women and 44.4% of men had a BMI below 18.5. The average caloric intake was 2142 and 2394 kcal/day among women and men, respectively, and the mean protein intake was 67.5 and 78.2 g/day. The largest sources of protein were from rice and fish. Greater protein intake was related to younger age and several socioeconomic measures, including more years of education, land and television ownership, and employment in business, farming, or as a laborer (for men) or as a homemaker (for women). This study found a high prevalence of underweight among study participants. Nonetheless, most participants had adequate protein intake according to Food and Agriculture Organization standards for body weight.

  3. Inferring topological features of proteins from amino acid residue networks

    NASA Astrophysics Data System (ADS)

    Alves, Nelson Augusto; Martinez, Alexandre Souto

    2007-02-01

    Topological properties of native folds are obtained from statistical analysis of 160 low homology proteins covering the four structural classes. This is done analyzing one, two and three-vertex joint distribution of quantities related to the corresponding network of amino acid residues. Emphasis on the amino acid residue hydrophobicity leads to the definition of their center of mass as vertices in this contact network model with interactions represented by edges. The network analysis helps us to interpret experimental results such as hydrophobic scales and fraction of buried accessible surface area in terms of the network connectivity. Moreover, those networks show assortative mixing by degree. To explore the vertex-type dependent correlations, we build a network of hydrophobic and polar vertices. This procedure presents the wiring diagram of the topological structure of globular proteins leading to the following attachment probabilities between hydrophobic-hydrophobic 0.424(5), hydrophobic-polar 0.419(2) and polar-polar 0.157(3) residues.

  4. Using porphyrin-amino acid pairs to model the electrochemistry of heme proteins: experimental and theoretical investigations.

    PubMed

    Samajdar, Rudra N; Manogaran, Dhivya; Yashonath, S; Bhattacharyya, Aninda J

    2018-04-18

    Quasi reversibility in electrochemical cycling between different oxidation states of iron is an often seen characteristic of iron containing heme proteins that bind dioxygen. Surprisingly, the system becomes fully reversible in the bare iron-porphyrin complex: hemin. This leads to the speculation that the polypeptide bulk (globin) around the iron-porphyrin active site in these heme proteins is probably responsible for the electrochemical quasi reversibility. To understand the effect of such polypeptide bulk on iron-porphyrin, we study the interaction of specific amino acids with the hemin center in solution. We choose three representative amino acids-histidine (a well-known iron coordinator in bio-inorganic systems), tryptophan (a well-known fluoroprobe for proteins), and cysteine (a redox-active organic molecule). The interactions of these amino acids with hemin are studied using electrochemistry, spectroscopy, and density functional theory. The results indicate that among these three, the interaction of histidine with the iron center is strongest. Further, histidine maintains the electrochemical reversibility of iron. On the other hand, tryptophan and cysteine interact weakly with the iron center but disturb the electrochemical reversibility by contributing their own redox active processes to the system. Put together, this study attempts to understand the molecular interactions that can control electrochemical reversibility in heme proteins. The results obtained here from the three representative amino acids can be scaled up to build a heme-amino acid interaction database that may predict the electrochemical properties of any protein with a defined polypeptide sequence.

  5. Fish protein hydrolysates: proximate composition, amino acid composition, antioxidant activities and applications: a review.

    PubMed

    Chalamaiah, M; Dinesh Kumar, B; Hemalatha, R; Jyothirmayi, T

    2012-12-15

    The fish processing industry produces more than 60% by-products as waste, which includes skin, head, viscera, trimmings, liver, frames, bones, and roes. These by-product wastes contain good amount of protein rich material that are normally processed into low market-value products, such as animal feed, fish meal and fertilizer. In view of utilizing these fish industry wastes, and for increasing the value to several underutilised fish species, protein hydrolysates from fish proteins are being prepared by several researchers all over the world. Fish protein hydrolysates are breakdown products of enzymatic conversion of fish proteins into smaller peptides, which normally contain 2-20 amino acids. In recent years, fish protein hydrolysates have attracted much attention of food biotechnologists due to the availability of large quantities of raw material for the process, and presence of high protein content with good amino acid balance and bioactive peptides (antioxidant, antihypertensive, immunomodulatory and antimicrobial peptides). Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Amino acid availability regulates the effect of hyperinsulinemia on skin protein metabolism in pigs

    USDA-ARS?s Scientific Manuscript database

    The effects of amino acid supply and insulin infusion on skin protein kinetics (fractional synthesis rate (FSR), fractional breakdown rate (FBR), and net balance (NB)) in pigs were investigated. Four-month-old pigs were divided into four groups as follows: control, insulin (INS), amino acid (AA), an...

  7. Bacterial Growth, Necromass Turnover, And Endospore Abundance In The Deep Subseafloor Sediments Of The Greenland Shelf Using D:L Amino Acid Model.

    NASA Astrophysics Data System (ADS)

    Mhatre, S. S.; Braun, S.; Jaussi, M.; Røy, H.; Jørgensen, B. B.; Lomstein, B. A.

    2015-12-01

    The subsurface realm is colonized by a large number of microorganisms- about 3 × 1029. Microbial cells in these very stable and oligotrophic settings catabolize at a much slower rate than model organisms in nutrient rich cultures. The aim of this work was to use recently developed D:L-amino acid racemization model for studying the turnover times of microbial biomass and microbial necromass in a ~12,000 years old Greenland shelf marine sediment samples. Sediments were analyzed for total hydrolysable amino acids (THAA), the bacterial endospore marker dipicolinic acid (DPA), and amino acid enantiomers of aspartic acid. The percentage amino acid carbon content (%TAAC) and the percentage amino acid nitrogen content (%TAAN) were used for determining the degradation state of the organic matter. Endospores quantified using DPA quantification method were found to be as abundant as vegetative cells. The microbial necromass turnover times were thousand years, and biomass turnover times were in the range of tens to hundred years. Studies with deeper sediment cores will further improve our understanding of the energetic limits of life in the deep biosphere.

  8. Amino acid sequences of peptides from a chymotryptic digest of a urea-soluble protein fraction (U.S.3) from oxidized wool

    PubMed Central

    Corfield, M. C.; Fletcher, J. C.

    1969-01-01

    1. A chymotryptic digest of the protein fraction U.S.3. from oxidized wool was separated into 51 peptide fractions by chromatography on a column of cation-exchange resin. 2. The less acidic fractions were separated into their component peptides by a combination of cation-exchange-resin chromatography, paper chromatography and paper electrophoresis. 3. The amino acid sequences of 34 of these peptides were elucidated, and those of 14 others partially determined. 4. Overlaps between the tryptic and chymotryptic peptides from fraction U.S.3 have enabled ten extended amino acid sequences to be deduced, the longest containing 20 amino acid residues. 5. The relevance of the results to the structures of the helical and non-helical regions of wool is discussed. PMID:5395876

  9. The Effects of Parent Body Processes on Amino Acids in Carbonaceous Chondrites

    NASA Technical Reports Server (NTRS)

    Glavin, Daniel P.; Callahan, Michael P.; Dworkin, Jason P.; Elsila, Jamie E.

    2010-01-01

    To investigate the effect of parent body processes on the abundance, distribution, and enantiomeric composition of amino acids in carbonaceous chondrites, the water extracts from nine different powdered Cl, CM, and CR carbonaceous chondrites were analyzed for amino acids by ultrahigh performance liquid chromatography-fluorescence detection and time-of-flight mass spectrometry (UPLC-FD/ToF-MS). Four aqueously altered type 1 carbonaceous chondrites including Orgueil (C11), Meteorite Hills (MET) 01070 (CM1), Scott Glacier (SCO) 06043 (CM1), and Grosvenor Mountains (GRO) 95577 (CR1) were analyzed using this technique for the first time. Analyses of these meteorites revealed low levels of two- to five-carbon acyclic amino alkanoic acids with concentrations ranging from -1 to 2,700 parts-per-billion (ppb). The type 1 carbonaceous chondrites have a distinct distribution of the five-carbon (C5) amino acids with much higher relative abundances of the gamma- and delta-amino acids compared to the type 2 and type 3 carbonaceous chondrites, which are dominated by a-amino acids. Much higher amino acid abundances were found in the CM2 chondrites Murchison, Lonewolf Nunataks (LON) 94102, and Lewis Cliffs (LEW) 90500, the CR2 Elephant Moraine (EET) 92042, and the CR3 Queen Alexandra Range (QUE) 99177. For example, a-aminoisobutyric acid ((alpha-AIB) and isovaline were approximately 100 to 1000 times more abundant in the type 2 and 3 chondrites compared to the more aqueously altered type 1 chondrites. Most of the chiral amino acids identified in these meteorites were racemic, indicating an extraterrestrial abiotic origin. However, non-racemic isovaline was observed in the aqueously altered carbonaceous chondrites Murchison, Orgueil, SCO 06043, and GRO 95577 with L-isovaline excesses ranging from approximately 11 to 19%, whereas the most pristine, unaltered carbonaceous chondrites analyzed in this study had no detectable L-isovaline excesses. These results are consistent with the

  10. Oral glutamine and amino acid supplementation inhibit whole-body protein degradation in children with Duchenne muscular dystrophy.

    PubMed

    Mok, Elise; Eléouet-Da Violante, Catherine; Daubrosse, Christel; Gottrand, Frédéric; Rigal, Odile; Fontan, Jean-Eudes; Cuisset, Jean-Marie; Guilhot, Joëlle; Hankard, Régis

    2006-04-01

    Glutamine has been shown to acutely decrease whole-body protein degradation in Duchenne muscular dystrophy (DMD). To improve nutritional support in DMD, we tested whether oral supplementation with glutamine for 10 d decreased whole-body protein degradation significantly more than did an isonitrogenous amino acid control mixture. Twenty-six boys with DMD were included in this randomized, double-blind parallel study; they received an oral supplement of either glutamine (0.5 g . kg(-1) . d(-1)) or an isonitrogenous, nonspecific amino acid mixture (0.8 g . kg(-1) . d(-1)) for 10 d. The subjects in each group were not clinically different at entry. Leucine and glutamine metabolisms were estimated in the postabsorptive state by using a primed continuous intravenous infusion of [1-(13)C]leucine and [2-(15)N]glutamine before and 10 d after supplementation. A significant effect of time was observed on estimates of whole-body protein degradation. A significant (P < 0.05) decrease in the rate of leucine appearance (an index of whole-body protein degradation) was observed after both glutamine and isonitrogenous amino acid supplementation [x +/-SEM: 136 +/- 9 to 124 +/- 6 micromol . kg fat-free mass (FFM)(-1) . h(-1) for glutamine and 136 +/- 6 to 131 +/- 8 micromol . kg FFM(-1) . h(-1) for amino acids]. A significant (P < 0.05) decrease in endogenous glutamine due to protein breakdown was also observed (91 +/- 6 to 83 +/- 4 micromol . kg FFM(-1) . h(-1) for glutamine and 91 +/- 4 to 88 +/- 5 micromol . kg FFM(-1) . h(-1) for amino acids). The decrease in the estimates of whole-body protein degradation did not differ significantly between the 2 supplemental groups. Oral glutamine or amino acid supplementation over 10 d equally inhibits whole-body protein degradation in DMD.

  11. Large differences in proportions of harmful and benign amino acid substitutions between proteins and diseases.

    PubMed

    Schaafsma, Gerard C P; Vihinen, Mauno

    2017-07-01

    Genes and proteins are known to have differences in their sensitivity to alterations. Despite numerous sequencing studies, proportions of harmful and harmless substitutions are not known for proteins and groups of proteins. To address this question, we predicted the outcome for all possible single amino acid substitutions (AASs) in nine representative protein groups by using the PON-P2 method. The effects on 996 proteins were studied and vast differences were noticed. Proteins in the cancer group harbor the largest proportion of harmful variants (42.1%), whereas the non-disease group of proteins not known to have a disease association and not involved in the housekeeping functions had the lowest number of harmful variants (4.2%). Differences in the proportions of the harmful and benign variants are wide within each group, but they still show clear differences between the groups. Frequently appearing protein domains show a wide spectrum of variant frequencies, whereas no major protein structural class-specific differences were noticed. AAS types in the original and variant residues showed distinctive patterns, which are shared by all the protein groups. The observations are relevant for understanding genetic bases of diseases, variation interpretation, and for the development of methods for that purpose. © 2017 Wiley Periodicals, Inc.

  12. Ornithine: at the crossroads of multiple paths to amino acids and polyamines

    Treesearch

    Rajtilak Majumdar; Rakesh Minocha; Subhash Minocha

    2015-01-01

    After the 20 amino acids that make up the proteins in all living organisms, ornithine perhaps occupies the most critical position among the non-protein amino acids. It sits at the crossroads of interconversions of glutamate and arginine on the one hand and the production of proline, polyamines and several alkaloids on the other: all products of tremendous importance to...

  13. An Amino Acid Code for Irregular and Mixed Protein Packing

    PubMed Central

    Joo, Hyun; Chavan, Archana; Fraga, Keith; Tsai, Jerry

    2015-01-01

    To advance our understanding of protein tertiary structure, the development of the knob-socket model is completed in an analysis of the packing in irregular coil and turn secondary structure packing as well as between mixed secondary structure. The knob-socket model simplifies packing based on repeated patterns of 2 motifs: a 3 residue socket for packing within 2° structure and a 4 residue knob-socket for 3° packing. For coil and turn secondary structure, knob-sockets allow identification of a correlation between amino acid composition and tertiary arrangements in space. Coil contributes almost as much as α-helices to tertiary packing. Irregular secondary structure involves 3 residue cliques of consecutive contacting residues or XYZ sockets. In irregular sockets, Gly, Pro, Asp and Ser are favored, while Cys, His, Met and Trp are not. For irregular knobs, the preference order is Arg, Asp, Pro, Asn, Thr, Leu, and Gly, while Cys, His, Met and Trp are not. In mixed packing, the knob amino acid preferences are a function of the socket that they are packing into, whereas the amino acid composition of the sockets does not depend on the secondary structure of the knob. A unique motif of a coil knob with an XYZ β-sheet socket may potentially function to inhibit β-sheet extension. In addition, analysis of the preferred crossing angles for strands within a β-sheet and mixed α-helices/β-sheets identifies canonical packing patterns useful in protein design. Lastly, the knob-socket model abstracts the complexity of protein tertiary structure into an intuitive packing surface topology map. PMID:26370334

  14. In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry

    PubMed Central

    Hatzenpichler, Roland; Scheller, Silvan; Tavormina, Patricia L; Babin, Brett M; Tirrell, David A; Orphan, Victoria J

    2014-01-01

    Here we describe the application of a new click chemistry method for fluorescent tracking of protein synthesis in individual microorganisms within environmental samples. This technique, termed bioorthogonal non-canonical amino acid tagging (BONCAT), is based on the in vivo incorporation of the non-canonical amino acid L-azidohomoalanine (AHA), a surrogate for l-methionine, followed by fluorescent labelling of AHA-containing cellular proteins by azide-alkyne click chemistry. BONCAT was evaluated with a range of phylogenetically and physiologically diverse archaeal and bacterial pure cultures and enrichments, and used to visualize translationally active cells within complex environmental samples including an oral biofilm, freshwater and anoxic sediment. We also developed combined assays that couple BONCAT with ribosomal RNA (rRNA)-targeted fluorescence in situ hybridization (FISH), enabling a direct link between taxonomic identity and translational activity. Using a methanotrophic enrichment culture incubated under different conditions, we demonstrate the potential of BONCAT-FISH to study microbial physiology in situ. A direct comparison of anabolic activity using BONCAT and stable isotope labelling by nano-scale secondary ion mass spectrometry (15NH3 assimilation) for individual cells within a sediment-sourced enrichment culture showed concordance between AHA-positive cells and 15N enrichment. BONCAT-FISH offers a fast, inexpensive and straightforward fluorescence microscopy method for studying the in situ activity of environmental microbes on a single-cell level. PMID:24571640

  15. BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

    PubMed

    Wei, Qing; La, David; Kihara, Daisuke

    2017-01-01

    Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .

  16. A supplemental intravenous amino acid infusion sustains a positive protein balance for 24 hours in critically ill patients.

    PubMed

    Sundström Rehal, Martin; Liebau, Felix; Tjäder, Inga; Norberg, Åke; Rooyackers, Olav; Wernerman, Jan

    2017-12-06

    Providing supplemental amino acids to ICU patients during a 3-h period results in improved whole-body net protein balance, without an increase in amino acid oxidation. The primary objective was to investigate if a 24-h intravenous amino acid infusion in critically ill patients has a sustained effect on whole-body protein balance as was seen after 3 h. Secondary objectives were monitoring of amino acid oxidation rate, urea and free amino acid plasma concentrations. An infusion of [1- 13 C]-phenylalanine was added to ongoing enteral nutrition to quantify the enteral uptake of amino acids. Primed intravenous infusions of [ring- 2 H 5 ]-phenylalanine and [3,3- 2 H 2 ]-tyrosine were used to assess whole-body protein synthesis and breakdown, to calculate net protein balance and to assess amino acid oxidation at baseline and at 3 and 24 hours. An intravenous amino acid infusion was added to nutrition at a rate of 1 g/kg/day and continued for 24 h. Eight patients were studied. The amino acid infusion resulted in improved net protein balance over time, from -1.6 ± 7.9 μmol phe/kg/h at 0 h to 6.0 ± 8.8 at 3 h and 7.5 ± 5.1 at 24 h (p = 0.0016). The sum of free amino acids in plasma increased from 3.1 ± 0.6 mmol/L at 0 h to 3.2 ± 0.3 at 3 h and 3.6 ± 0.5 at 24 h (p = 0.038). Amino acid oxidation and plasma urea were not altered significantly. We demonstrated that the improvement in whole-body net protein balance from a supplemental intravenous amino acid infusion seen after 3 h was sustained after 24 h in critically ill patients. This trial was prospectively registered at Australian New Zealand Clinical Trials Registry. ACTRN, 12615001314516 . Registered on 1 December 2015.

  17. MS-READ: Quantitative measurement of amino acid incorporation.

    PubMed

    Mohler, Kyle; Aerni, Hans-Rudolf; Gassaway, Brandon; Ling, Jiqiang; Ibba, Michael; Rinehart, Jesse

    2017-11-01

    Ribosomal protein synthesis results in the genetically programmed incorporation of amino acids into a growing polypeptide chain. Faithful amino acid incorporation that accurately reflects the genetic code is critical to the structure and function of proteins as well as overall proteome integrity. Errors in protein synthesis are generally detrimental to cellular processes yet emerging evidence suggest that proteome diversity generated through mistranslation may be beneficial under certain conditions. Cumulative translational error rates have been determined at the organismal level, however codon specific error rates and the spectrum of misincorporation errors from system to system remain largely unexplored. In particular, until recently technical challenges have limited the ability to detect and quantify comparatively rare amino acid misincorporation events, which occur orders of magnitude less frequently than canonical amino acid incorporation events. We now describe a technique for the quantitative analysis of amino acid incorporation that provides the sensitivity necessary to detect mistranslation events during translation of a single codon at frequencies as low as 1 in 10,000 for all 20 proteinogenic amino acids, as well as non-proteinogenic and modified amino acids. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Amino acid composition, score and in vitro protein digestibility of foods commonly consumed in northwest Mexico.

    PubMed

    Caire-Juvera, Graciela; Vázquez-Ortiz, Francisco A; Grijalva-Haro, Maria I

    2013-01-01

    A better knowledge of the amino acid composition of foods commonly consumed in different regions is essential to calculate their scores and, therefore, to predict their protein quality. This paper presents the amino acid composition, amino acid score and in vitro protein digestibility of fifteen foods that are commonly consumed in Northwest Mexico. The foods were prepared by the traditional methods and were analyzed by reverse-phase HPLC. The chemical score for each food was determined using the recommendations for children of 1-2 years of age, and the digestibility was evaluated using a multienzyme technique. Lysine was the limiting amino acid in cereal-based products (scores 15 to 54), and methionine and cysteine were limiting in legume products (scores 41 to 47), boiled beef (score = 75) and hamburger (score = 82). The method of preparation had an effect on the content of certain amino acids, some of them increased and others decreased their content. Meat products and regional cheese provided a high amino acid score (scores 67 to 91) and digestibility (80.7 to 87.8%). Bologna, a processed meat product, had a lower digestibility (75.4%). Data on the amino acid composition of foods commonly consumed in Mexico can be used to provide valuable information on food analysis and protein quality, and to contribute to nutrition and health research and health programs. Copyright © AULA MEDICA EDICIONES 2013. Published by AULA MEDICA. All rights reserved.

  19. Gene expression of amino acid transporter in pigeon (Columbia livia) intestine during post-hatch development and its correlation with amino acid in pigeon milk.

    PubMed

    Zhang, X Y; Zhang, N N; Wan, X P; Li, L L; Zou, X T

    2017-05-01

    This study was conducted to evaluate gene expression of the amino acid transporter in post-hatch pigeon small intestine and the association of pigeon milk amino acid with the above transporter's gene expression. A total of 48 pigeon breeding families were randomly allocated to 8 groups of 6 replicates of one parental pigeon pair and 2 squabs. Samples of pigeon milk and duodenum, jejunum, and ileum were collected on d 1, 2, 3, 4, 6, 8, 10, and 14 post hatch. The results showed that levels of crude protein (8.93 to 15.56%) were highest in pigeon milk on an air-dry basis. Amino acid content in pigeon milk remained constant in the first 4 d, declined abruptly at d 6, then increased dramatically from d 8 to 14. There was a significant effect of interaction between age and intestinal segments on those amino acid transporters gene expression. mRNA abundance of ATB0'+, SNAT-2, LAT-4, rBAT, b0'+AT, EAAT-3 and PAT-1 was highest in the ileum; B0AT1, asc-1, and IMINO were predominate in the jejunum; and CAT-1 and y+LAT2 were greatest in the duodenum. Age-related changes of amino acid transporter mRNA was inconsistent. mRNA levels of SNAT-2, rBAT, y+LAT2, b0'+AT, and EAAT-3 ascended with age, whereas that of asc-1, CAT-1, and IMINO diminished significantly. Levels of B0AT1 and PAT-1 mRNA abundance were minimized at d 6. However, few correlations were found between pigeon milk amino acid and the amino acid transporter gene expressions in squab small intestine. Our findings provide a comprehensive elaboration on ontogeny of the amino acid transporter in post-hatch pigeon intestine. © 2016 Poultry Science Association Inc.

  20. Depleting high-abundant and enriching low-abundant proteins in human serum: An evaluation of sample preparation methods using magnetic nanoparticle, chemical depletion and immunoaffinity techniques.

    PubMed

    de Jesus, Jemmyson Romário; da Silva Fernandes, Rafael; de Souza Pessôa, Gustavo; Raimundo, Ivo Milton; Arruda, Marco Aurélio Zezzi

    2017-08-01

    The efficiency of three different depletion methods to remove the most abundant proteins, enriching those human serum proteins with low abundance is checked to make more efficient the search and discovery of biomarkers. These methods utilize magnetic nanoparticles (MNPs), chemical reagents (sequential application of dithiothreitol and acetonitrile, DTT/ACN), and commercial apparatus based on immunoaffinity (ProteoMiner, PM). The comparison between methods shows significant removal of abundant protein, remaining in the supernatant at concentrations of 4.6±0.2, 3.6±0.1, and 3.3±0.2µgµL -1 (n=3) for MNPs, DTT/ACN and PM respectively, from a total protein content of 54µgµL -1 . Using GeLC-MS/MS analysis, MNPs depletion shows good efficiency in removing high molecular weight proteins (>80kDa). Due to the synergic effect between the reagents DTT and ACN, DTT/ACN-based depletion offers good performance in the depletion of thiol-rich proteins, such as albumin and transferrin (DTT action), as well as of high molecular weight proteins (ACN action). Furthermore, PM equalization confirms its efficiency in concentrating low-abundant proteins, decreasing the dynamic range of protein levels in human serum. Direct comparison between the treatments reveals 72 proteins identified when using MNP depletion (43 of them exclusively by this method), but only 20 proteins using DTT/ACN (seven exclusively by this method). Additionally, after PM treatment 30 proteins were identified, seven exclusively by this method. Thus, MNPs and DTT/ACN depletion can be simple, quick, cheap, and robust alternatives for immunochemistry-based protein depletion, providing a potential strategy in the search for disease biomarkers. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Regulation of renal amino acid transporters during metabolic acidosis.

    PubMed

    Moret, Caroline; Dave, Mital H; Schulz, Nicole; Jiang, Jean X; Verrey, Francois; Wagner, Carsten A

    2007-02-01

    The kidney plays a major role in acid-base homeostasis by adapting the excretion of acid equivalents to dietary intake and metabolism. Urinary acid excretion is mediated by the secretion of protons and titratable acids, particularly ammonia. NH(3) is synthesized in proximal tubule cells from glutamine taken up via specific amino acid transporters. We tested whether kidney amino acid transporters are regulated in mice in which metabolic acidosis was induced with NH(4)Cl. Blood gas and urine analysis confirmed metabolic acidosis. Real-time RT-PCR was performed to quantify the mRNAs of 16 amino acid transporters. The mRNA of phosphoenolpyruvate carboxykinase (PEPCK) was quantified as positive control for the regulation and that of GAPDH, as internal standard. In acidosis, the mRNA of kidney system N amino acid transporter SNAT3 (SLC38A3/SN1) showed a strong induction similar to that of PEPCK, whereas all other tested mRNAs encoding glutamine or glutamate transporters were unchanged or reduced in abundance. At the protein level, Western blotting and immunohistochemistry demonstrated an increased abundance of SNAT3 and reduced expression of the basolateral cationic amino acid/neutral amino acid exchanger subunit y(+)-LAT1 (SLC7A7). SNAT3 was localized to the basolateral membrane of the late proximal tubule S3 segment in control animals, whereas its expression was extended to the earlier S2 segment of the proximal tubule during acidosis. Our results suggest that the selective regulation of SNAT3 and y(+)LAT1 expression may serve a major role in the renal adaptation to acid secretion and thus for systemic acid-base balance.

  2. Evolutionary gradient of predicted nuclear localization signals (NLS)-bearing proteins in genomes of family Planctomycetaceae.

    PubMed

    Guo, Min; Yang, Ruifu; Huang, Chen; Liao, Qiwen; Fan, Guangyi; Sun, Chenghang; Lee, Simon Ming-Yuen

    2017-04-04

    The nuclear envelope is considered a key classification marker that distinguishes prokaryotes from eukaryotes. However, this marker does not apply to the family Planctomycetaceae, which has intracellular spaces divided by lipidic intracytoplasmic membranes (ICMs). Nuclear localization signal (NLS), a short stretch of amino acid sequence, destines to transport proteins from cytoplasm into nucleus, and is also associated with the development of nuclear envelope. We attempted to investigate the NLS motifs in Planctomycetaceae genomes to demonstrate the potential molecular transition in the development of intracellular membrane system. In this study, we identified NLS-like motifs that have the same amino acid compositions as experimentally identified NLSs in genomes of 11 representative species of family Planctomycetaceae. A total of 15 NLS types and 170 NLS-bearing proteins were detected in the 11 strains. To determine the molecular transformation, we compared NLS-bearing protein abundances in the 11 representative Planctomycetaceae genomes with them in genomes of 16 taxonomically varied microorganisms: nine bacteria, two archaea and five fungi. In the 27 strains, 29 NLS types and 1101 NLS-bearing proteins were identified, principal component analysis showed a significant transitional gradient from bacteria to Planctomycetaceae to fungi on their NLS-bearing protein abundance profiles. Then, we clustered the 993 non-redundant NLS-bearing proteins into 181 families and annotated their involved metabolic pathways. Afterwards, we aligned the ten types of NLS motifs from the 13 families containing NLS-bearing proteins among bacteria, Planctomycetaceae or fungi, considering their diversity, length and origin. A transition towards increased complexity from non-planctomycete bacteria to Planctomycetaceae to archaea and fungi was detected based on the complexity of the 10 types of NLS-like motifs in the 13 NLS-bearing proteins families. The results of this study reveal that

  3. AMINO ACID MIXTURES EFFECTIVE PARENTERALLY FOR LONG CONTINUED PLASMA PROTEIN PRODUCTION. CASEIN DIGESTS COMPARED

    PubMed Central

    Madden, S. C.; Woods, R. R.; Shull, F. W.; Whipple, G. H.

    1944-01-01

    When blood plasma proteins are depleted by bleeding with return of red cells suspended in saline (plasmapheresis) it is possible to bring dogs to a steady state of hypoproteinemia and a constant level of plasma protein production if the diet nitrogen intake is controlled and limited. Such dogs are outwardly normal but have a lowered resistance to infection and to certain intoxications. The ten growth essential amino acids of Rose plus glycine will maintain nitrogen balance and produce as much new plasma protein as will good diet proteins. This good utilization is demonstrated over periods of several months when the amino acids are given either orally or parenterally. There is no evidence of toxicity in general nor to unnatural forms of these synthetic amino acids in particular. Given parenterally appropriate mixtures of these amino acids are well tolerated even upon rapid injection. The minimal daily requirements for a 10 kilo dog may be given intravenously in 10 minutes without reaction. Subcutaneously a 10 per cent solution may be given rapidly without reaction. Among various mixtures tested Vt approximates a minimum for a 10 kilo dog. It contains in grams (dl-threonine 0.7, dl-valine 1.5, l-(-) leucine 1.5, dl-isoleucine 1.4, dl-lysine hydrochloride 1.5, l(-) tryptophane 0.4, dl-phenylalanine 1.0, dl-methionine 0.6, l(+)-histidine hydrochloride 0.5, l(+)-arginine hydrochloride 0.5, and glycine 1.0. The presence of glycine improves tolerance to rapid intravenous injection, but excess glycine does not improve utilization of the mixture. Over a long period this mixture appears suboptimal in quantity. Doubled it is more than ample. Of two casein digests tested the one prepared by enzymatic hydrolysis provided good nitrogen retention and fairly good plasma protein production but was much less tolerable upon intravenous injection than certain mixtures of pure amino acids. The other one prepared by acid hydrolysis and tryptophane fortification afforded bare nitrogen

  4. LDL receptor-related protein 1 regulates the abundance of diverse cell-signaling proteins in the plasma membrane proteome.

    PubMed

    Gaultier, Alban; Simon, Gabriel; Niessen, Sherry; Dix, Melissa; Takimoto, Shinako; Cravatt, Benjamin F; Gonias, Steven L

    2010-12-03

    LDL receptor-related protein 1 (LRP1) is an endocytic receptor, reported to regulate the abundance of other receptors in the plasma membrane, including uPAR and tissue factor. The goal of this study was to identify novel plasma membrane proteins, involved in cell-signaling, that are regulated by LRP1. Membrane protein ectodomains were prepared from RAW 264.7 cells in which LRP1 was silenced and control cells using protease K. Peptides were identified by LC-MS/MS. By analysis of spectral counts, 31 transmembrane and secreted proteins were regulated in abundance at least 2-fold when LRP1 was silenced. Validation studies confirmed that semaphorin4D (Sema4D), plexin domain-containing protein-1 (Plxdc1), and neuropilin-1 were more abundant in the membranes of LRP1 gene-silenced cells. Regulation of Plxdc1 by LRP1 was confirmed in CHO cells, as a second model system. Plxdc1 coimmunoprecipitated with LRP1 from extracts of RAW 264.7 cells and mouse liver. Although Sema4D did not coimmunoprecipitate with LRP1, the cell-surface level of Sema4D was increased by RAP, which binds to LRP1 and inhibits binding of other ligands. These studies identify Plxdc1, Sema4D, and neuropilin-1 as novel LRP1-regulated cell-signaling proteins. Overall, LRP1 emerges as a generalized regulator of the plasma membrane proteome.

  5. Differential protein abundance of a basolateral MCT1 transporter in the human gastrointestinal tract.

    PubMed

    Al-Mosauwi, Hashemeya; Ryan, Elizabeth; McGrane, Alison; Riveros-Beltran, Stefanie; Walpole, Caragh; Dempsey, Eugene; Courtney, Danielle; Fearon, Naomi; Winter, Desmond; Baird, Alan; Stewart, Gavin

    2016-12-01

    Bacterially derived short chain fatty acids (SCFAs), such as butyrate, are vital in maintaining the symbiotic relationship that exists between humans and their gastrointestinal microbial populations. A key step in this process is the transport of SCFAs across colonic epithelial cells via MCT1 transporters. This study investigated MCT1 protein abundance in various human intestinal tissues. Initial RT-PCR analysis confirmed the expected MCT1 RNA expression pattern of colon > small intestine > stomach. Using surgical resection samples, immunoblot analysis detected higher abundance of a 45 kDa MCT1 protein in colonic tissue compared to ileum tissue (P < 0.001, N = 4, unpaired t-test). Importantly, MCT1 abundance was found to be significantly lower in sigmoid colon compared to ascending colon (P < 0.01, N = 8-11, ANOVA). Finally, immunolocalization studies confirmed MCT1 to be abundant in the basolateral membranes of surface epithelial cells of the ascending, transverse, and descending colon, but significantly less prevalent in the sigmoid colon (P < 0.05, N = 5-21, ANOVA). In conclusion, these data confirm that basolateral MCT1 protein abundance is correlated to levels of bacterially derived SCFAs along the human gastrointestinal tract. These findings highlight the importance of precise tissue location in studies comparing colonic MCT1 abundance between normal and diseased states. © 2016 International Federation for Cell Biology.

  6. Long-term leucine induced stimulation of muscle protein synthesis is amino acid dependent

    USDA-ARS?s Scientific Manuscript database

    Infusing leucine for 1 h increases skeletal muscle protein synthesis in the neonate, but this is not sustained for 2 h unless the corresponding fall in amino acids is prevented. This study aimed to determine whether a continuous leucine infusion can stimulate protein synthesis for a prolonged period...

  7. Umami taste amino acids produced by hydrolyzing extracted protein from tomato seed meal

    USDA-ARS?s Scientific Manuscript database

    Enzymatic hydrolysis was performed for extracting protein to prepare umami taste amino acids from defatted tomato seed meal (DTSM) which is a by-product of tomato processing. Papain was used as an enzyme for the hydrolysis of DTSM. The particle size distribution of DTSM, protein concentration and fr...

  8. A species-specific nucleosomal signature defines a periodic distribution of amino acids in proteins.

    PubMed

    Quintales, Luis; Soriano, Ignacio; Vázquez, Enrique; Segurado, Mónica; Antequera, Francisco

    2015-04-01

    Nucleosomes are the basic structural units of chromatin. Most of the yeast genome is organized in a pattern of positioned nucleosomes that is stably maintained under a wide range of physiological conditions. In this work, we have searched for sequence determinants associated with positioned nucleosomes in four species of fission and budding yeasts. We show that mononucleosomal DNA follows a highly structured base composition pattern, which differs among species despite the high degree of histone conservation. These nucleosomal signatures are present in transcribed and non-transcribed regions across the genome. In the case of open reading frames, they correctly predict the relative distribution of codons on mononucleosomal DNA, and they also determine a periodicity in the average distribution of amino acids along the proteins. These results establish a direct and species-specific connection between the position of each codon around the histone octamer and protein composition.

  9. Depletion of highly abundant proteins in blood plasma by ammonium sulfate precipitation for 2D-PAGE analysis.

    PubMed

    Mahn, Andrea; Ismail, Maritza

    2011-11-15

    Ammonium sulfate precipitation (ASP) was explored as a method for depleting some highly abundant proteins from blood plasma, in order to reduce the dynamic range of protein concentration and to improve the detection of low abundance proteins by 2D-PAGE. 40% ammonium sulfate saturation was chosen since it allowed depleting 39% albumin and 82% α-1-antitrypsin. ASP-depletion showed high reproducibility in 2D-PAGE analysis (4.2% variation in relative abundance of albumin), similar to that offered by commercial affinity-depletion columns. Besides, it allowed detecting 59 spots per gel, very close to the number of spots detected in immuno-affinity-depleted plasma. Thus, ASP at 40% saturation is a reliable depletion method that may help in proteomic analysis of blood plasma. Finally, ASP-depletion seems to be complementary to hydrophobic interaction chromatography (HIC)-depletion, and therefore an ASP-step followed by a HIC-step could probably deplete the most highly abundant plasma proteins, thus improving the detection of low abundance proteins by 2D-PAGE. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Branched Chain Amino Acids: Beyond Nutrition Metabolism.

    PubMed

    Nie, Cunxi; He, Ting; Zhang, Wenju; Zhang, Guolong; Ma, Xi

    2018-03-23

    Branched chain amino acids (BCAAs), including leucine (Leu), isoleucine (Ile), and valine (Val), play critical roles in the regulation of energy homeostasis, nutrition metabolism, gut health, immunity and disease in humans and animals. As the most abundant of essential amino acids (EAAs), BCAAs are not only the substrates for synthesis of nitrogenous compounds, they also serve as signaling molecules regulating metabolism of glucose, lipid, and protein synthesis, intestinal health, and immunity via special signaling network, especially phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signal pathway. Current evidence supports BCAAs and their derivatives as the potential biomarkers of diseases such as insulin resistance (IR), type 2 diabetes mellitus (T2DM), cancer, and cardiovascular diseases (CVDs). These diseases are closely associated with catabolism and balance of BCAAs. Hence, optimizing dietary BCAA levels should have a positive effect on the parameters associated with health and diseases. This review focuses on recent findings of BCAAs in metabolic pathways and regulation, and underlying the relationship of BCAAs to related disease processes.

  11. Branched Chain Amino Acids: Beyond Nutrition Metabolism

    PubMed Central

    2018-01-01

    Branched chain amino acids (BCAAs), including leucine (Leu), isoleucine (Ile), and valine (Val), play critical roles in the regulation of energy homeostasis, nutrition metabolism, gut health, immunity and disease in humans and animals. As the most abundant of essential amino acids (EAAs), BCAAs are not only the substrates for synthesis of nitrogenous compounds, they also serve as signaling molecules regulating metabolism of glucose, lipid, and protein synthesis, intestinal health, and immunity via special signaling network, especially phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signal pathway. Current evidence supports BCAAs and their derivatives as the potential biomarkers of diseases such as insulin resistance (IR), type 2 diabetes mellitus (T2DM), cancer, and cardiovascular diseases (CVDs). These diseases are closely associated with catabolism and balance of BCAAs. Hence, optimizing dietary BCAA levels should have a positive effect on the parameters associated with health and diseases. This review focuses on recent findings of BCAAs in metabolic pathways and regulation, and underlying the relationship of BCAAs to related disease processes. PMID:29570613

  12. A new solar carbon abundance based on non-LTE CN molecular spectra

    NASA Technical Reports Server (NTRS)

    Mount, G. H.; Linsky, J. L.

    1975-01-01

    A detailed non-LTE analysis of solar CN spectra strongly suggest a revised carbon abundance for the sun. We recommend a value of log carbon abundance = 8.35 plus or minus 0.15 which is significantly lower than the presently accepted value of log carbon abundance = 8.55. This revision may have important consequences in astrophysics.

  13. An Assay of Selected Serum Amino Acids in Patients with Type 2 Diabetes Mellitus.

    PubMed

    Drábková, Petra; Šanderová, Jana; Kovařík, Jakub; kanďár, Roman

    2015-01-01

    Amino acids are the building blocks of proteins. In case of insulin resistance, which is typical for type 2 diabetes mellitus (T2DM), proteolysis is increased and protein synthesis is decreased; therefore, we can observe changes in the levels of amino acids in diabetics vs. non-diabetics. The aim of this study was to find differences in the levels of selected amino acids between patients with diabetes (type 2) and a control group. Amino acids were derivatized with naphthalene-2,3-dicarboxaldehyde in the presence of potassium cyanide to form fluorescent 1-cyanobenz(f)isoindole product. Amino acids derivatives were measured using a high-performance liquid chromatography with fluorescence detection. The serum levels of glucose were determined using an automatic biochemistry analyzer, glycated hemoglobin HbA1c was measured by cation exchange chromatography. A total of 19 serum amino acids in T2DM patients and non-diabetics were measured. There were 9 amino acids, which were significantly different in these groups (p<0.05). Significantly decreased levels of arginine, asparagine, glycine, serine, threonine and significantly increased levels of alanine, isoleucine, leucine, valine in diabetics were found. Significant difference in metabolism of amino acids between diabetics and non-diabetics were observed. The altered levels of amino acids in diabetic patients could be a suitable predictor of diabetes.

  14. Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide-protein complexes.

    PubMed

    Kondo, Jiro; Westhof, Eric

    2011-10-01

    Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide-protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson-Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson-Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues.

  15. Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide–protein complexes

    PubMed Central

    Kondo, Jiro; Westhof, Eric

    2011-01-01

    Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide–protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson–Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson–Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues. PMID:21737431

  16. [Comparison of the effects of alpha-keto/ amino acid supplemented low protein diet and diabetes diet in patients with diabetic nephropathy].

    PubMed

    Qiu, Hong-yu; Liu, Fang; Zhao, Li-jun; Huang, Song-min; Zuo, Chuan; Zhong, Hui; Chen, Feng

    2012-05-01

    To investigate if a-keto/amino acid supplemented low protein diet can slow down the progression of diabetic nephrophathy in comparison with non-supplemented diabetes diet. A prospective, randomized, controlled clinical study was conducted. Twenty three cases of type 2 diabetic nephropathy in IV stage were randomly divided into alpha-keto/amino acid supplemented diet group (trial group) and conventional diabetes diet group (control group), The treatment duration was 52 weeks. 24 h urine protein was measured at 0, 12, 20, 36 and 52 weeks. Before and after the 52 weeks treatment, all the patients received the measurement of glomerular filtration rate (GFR), blood glucose, blood lipids, inflammatory markers, as well as nutritional status. After the treatment for 20, 36, 52 weeks, mean 24 h urine protein decreased significantly in trial groups (P < 0.05), and 24 h urine protein in trial group were significantly decreased (P < 0.05) compared with control group in 20 weeks after treatment. Either in trial group or in control group, GFR remained relatively stable during the observation period. Nutrition status, inflammatory markers, and serum calcium, phosphorus levels between the two groups were no significantly difference. The adverse events experienced by the patients in trial group were similar and consistent with the patients underlying renal diseases. Alpha-keto/amino acid can reduce proteinuria more effectively, while improve renal function and nutritional status in diabetic nephropathy patients with well-toleration.

  17. Crystal structure of secretory abundant heat soluble protein 4 from one of the toughest “water bears” micro‐animals Ramazzottius Varieornatus

    PubMed Central

    Fukuda, Yohta

    2018-01-01

    Abstract Though anhydrobiotic tardigrades (micro‐animals also known as water bears) possess many genes of secretory abundant heat soluble (SAHS) proteins unique to Tardigrada, their functions are unknown. A previous crystallographic study revealed that a SAHS protein (RvSAHS1) from one of the toughest tardigrades, Ramazzottius varieornatus, has a β‐barrel architecture similar to fatty acid binding proteins (FABPs) and two putative ligand binding sites (LBS1 and LBS2) where fatty acids can bind. However, some SAHS proteins such as RvSAHS4 have different sets of amino acid residues at LBS1 and LBS2, implying that they prefer other ligands and have different functions. Here RvSAHS4 was crystallized and analyzed under a condition similar to that for RvSAHS1. There was no electron density corresponding to a fatty acid at LBS1 of RvSAHS4, where a putative fatty acid was observed in RvSAHS1. Instead, LBS2 of RvSAHS4, which was composed of uncharged residues, captured a putative polyethylene glycol molecule. These results suggest that RvSAHS4 mainly uses LBS2 for the binding of uncharged molecules. PMID:29493034

  18. New Insight into Quinoa Seed Quality under Salinity: Changes in Proteomic and Amino Acid Profiles, Phenolic Content, and Antioxidant Activity of Protein Extracts

    PubMed Central

    Aloisi, Iris; Parrotta, Luigi; Ruiz, Karina B.; Landi, Claudia; Bini, Luca; Cai, Giampiero; Biondi, Stefania; Del Duca, Stefano

    2016-01-01

    highest TPC and AA under non-saline conditions. Salinity increased TPC in all three landraces, with the strongest increase occurring in R49, and enhanced radical scavenging capacity in R49 and VR. Overall, results show that salinity deeply altered the seed proteome and amino acid profiles and, in general, increased the concentration of bioactive molecules and AA of protein extracts in a genotype-dependent manner. PMID:27242857

  19. Amino acid sequences of the ribosomal proteins HL30 and HmaL5 from the archaebacterium Halobacterium marismortui.

    PubMed

    Hatakeyama, T; Hatakeyama, T

    1990-07-06

    The complete amino acid sequences of the ribosomal proteins HL30 and HmaL5 from the archaebacterium Halobacterium marismortui were determined. Protein HL30 was found to be acetylated at its N-terminal amino acid and shows homology to the eukaryotic ribosomal proteins YL34 from yeast and RL31 from rat. Protein HmaL5 was homologous to the protein L5 from Escherichia coli and Bacillus stearothermophilus as well as to YL16 from yeast. HmaL5 shows more similarities to its eukaryotic counterpart than to eubacterial ones.

  20. An Amino Acid Packing Code for α-helical Structure and Protein Design

    PubMed Central

    Joo, Hyun; Chavan, Archana G.; Phan, Jamie; Day, Ryan; Tsai, Jerry

    2012-01-01

    This work demonstrates that all packing in α-helices can be simplified to repetitive patterns of a single motif: the knob-socket. Using the precision of Voronoi Polyhedra/Deluaney Tessellations to identify contacts, the knob-socket is a 4 residue tetrahedral motif: a knob residue on one α-helix packs into the 3 residue socket on another α-helix. The principle of the knob-socket model relates the packing between levels of protein structure: the intra-helical packing arrangements within secondary structure that permit inter-helix tertiary packing interactions. Within an α-helix, the 3 residue sockets arrange residues into a uniform packing lattice. Inter-helix packing results from a definable pattern of interdigitated knob-socket motifs between 2 α-helices. Furthermore, the knob-socket model classifies 3 types of sockets: 1) free: favoring only intra-helical packing, 2) filled: favoring inter-helical interactions and 3) non: disfavoring α-helical structure. The amino acid propensities in these 3 socket classes essentially represent an amino acid code for structure in α-helical packing. Using this code, a novel yet straightforward approach for the design of α-helical structure was used to validate the knob-socket model. Unique sequences for 3 peptides were created to produce a predicted amount of α-helical structure: mostly helical, some helical, and no-helix. These 3 peptides were synthesized and helical content assessed using CD spectroscopy. The measured α-helicity of each peptide was consistent with the expected predictions. These results and analysis demonstrate that the knob-socket motif functions as the basic unit of packing and presents an intuitive tool to decipher the rules governing packing in protein structure. PMID:22426125

  1. AMINO ACIDURIA IN PRIMATES FOLLOWING IRRADIATION

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hunter, C.G.

    The daily urine amino N excretion of 4 monkeys was followed before and after whole body irradiation with doses in the lower lethal range. In 2 non- survivors, there was little change in the daily quantity excreted until terminally. In 2 survivors given the same food intakes as in the irradiated study and sham irradiated, the daily urine amino N excretion during the first week differed but slightly from the values after irradiation, but in the second week more amino N was excreted after irradiation in one animal and less in the other. It would appear that amino aciduria inmore » primates irradiated with doses in the lower lethal range is inseparable from the natural response of the over-all protein metabolism associated with any injury. (auth)« less

  2. Investigation of non-corrin cobalt(II)-containing sites in protein structures of the Protein Data Bank.

    PubMed

    Abriata, Luciano Andres

    2013-04-01

    Protein X-ray structures with non-corrin cobalt(II)-containing sites, either natural or substituting another native ion, were downloaded from the Protein Data Bank and explored to (i) describe which amino acids are involved in their first ligand shells and (ii) analyze cobalt(II)-donor bond lengths in comparison with previously reported target distances, CSD data and EXAFS data. The set of amino acids involved in Co(II) binding is similar to that observed for catalytic Zn(II) sites, i.e. with a large fraction of carboxylate O atoms from aspartate and glutamate and aromatic N atoms from histidine. The computed Co(II)-donor bond lengths were found to depend strongly on structure resolution, an artifact previously detected for other metal-donor distances. Small corrections are suggested for the target bond lengths to the aromatic N atoms of histidines and the O atoms of water and hydroxide. The available target distance for cysteine (Scys) is confirmed; those for backbone O and other donors remain uncertain and should be handled with caution in refinement and modeling protocols. Finally, a relationship between both Co(II)-O bond lengths in bidentate carboxylates is quantified.

  3. LS-SNP/PDB: annotated non-synonymous SNPs mapped to Protein Data Bank structures.

    PubMed

    Ryan, Michael; Diekhans, Mark; Lien, Stephanie; Liu, Yun; Karchin, Rachel

    2009-06-01

    LS-SNP/PDB is a new WWW resource for genome-wide annotation of human non-synonymous (amino acid changing) SNPs. It serves high-quality protein graphics rendered with UCSF Chimera molecular visualization software. The system is kept up-to-date by an automated, high-throughput build pipeline that systematically maps human nsSNPs onto Protein Data Bank structures and annotates several biologically relevant features. LS-SNP/PDB is available at (http://ls-snp.icm.jhu.edu/ls-snp-pdb) and via links from protein data bank (PDB) biology and chemistry tabs, UCSC Genome Browser Gene Details and SNP Details pages and PharmGKB Gene Variants Downloads/Cross-References pages.

  4. Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mTOR-dependent translation initiation

    USDA-ARS?s Scientific Manuscript database

    In skeletal muscle of adults, sepsis reduces protein synthesis by depressing translation initiation and induces resistance to branched-chain amino acid stimulation. Normal neonates maintain a high basal muscle protein synthesis rate that is sensitive to amino acid stimulation. In the present study...

  5. Amino Acid Composition of an Organic Brown Rice Protein Concentrate and Isolate Compared to Soy and Whey Concentrates and Isolates.

    PubMed

    Kalman, Douglas S

    2014-06-30

    A protein concentrate (Oryzatein-80™) and a protein isolate (Oryzatein-90™) from organic whole-grain brown rice were analyzed for their amino acid composition. Two samples from different batches of Oryzatein-90™ and one sample of Oryzatein-80™ were provided by Axiom Foods (Los Angeles, CA, USA). Preparation and analysis was carried out by Covance Laboratories (Madison, WI, USA). After hydrolysis in 6-N hydrochloric acid for 24 h at approximately 110 °C and further chemical stabilization, samples were analyzed by HPLC after pre-injection derivitization. Total amino acid content of both the isolate and the concentrate was approximately 78% by weight with 36% essential amino acids and 18% branched-chain amino acids. These results are similar to the profiles of raw and cooked brown rice except in the case of glutamic acid which was 3% lower in the isolate and concentrate. The amino acid content and profile of the Oryzatein-90™ isolate was similar to published values for soy protein isolate but the total, essential, and branched-chain amino acid content of whey protein isolate was 20%, 39% and 33% greater, respectively, than that of Oryzatein-90™. These results provide a valuable addition to the nutrient database of protein isolates and concentrates from cereal grains.

  6. Amino Acid Composition of an Organic Brown Rice Protein Concentrate and Isolate Compared to Soy and Whey Concentrates and Isolates

    PubMed Central

    Kalman, Douglas S.

    2014-01-01

    A protein concentrate (Oryzatein-80™) and a protein isolate (Oryzatein-90™) from organic whole-grain brown rice were analyzed for their amino acid composition. Two samples from different batches of Oryzatein-90™ and one sample of Oryzatein-80™ were provided by Axiom Foods (Los Angeles, CA, USA). Preparation and analysis was carried out by Covance Laboratories (Madison, WI, USA). After hydrolysis in 6-N hydrochloric acid for 24 h at approximately 110 °C and further chemical stabilization, samples were analyzed by HPLC after pre-injection derivitization. Total amino acid content of both the isolate and the concentrate was approximately 78% by weight with 36% essential amino acids and 18% branched-chain amino acids. These results are similar to the profiles of raw and cooked brown rice except in the case of glutamic acid which was 3% lower in the isolate and concentrate. The amino acid content and profile of the Oryzatein-90™ isolate was similar to published values for soy protein isolate but the total, essential, and branched-chain amino acid content of whey protein isolate was 20%, 39% and 33% greater, respectively, than that of Oryzatein-90™. These results provide a valuable addition to the nutrient database of protein isolates and concentrates from cereal grains. PMID:28234326

  7. Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product

    NASA Astrophysics Data System (ADS)

    Rustgi, Anil K.; Dyson, Nicholas; Bernards, Rene

    1991-08-01

    THE proteins encoded by the myc gene family are involved in the control of cell proliferation and differentiation, and aberrant expression of myc proteins has been implicated in the genesis of a variety of neoplasms1. In the carboxyl terminus, myc proteins have two domains that encode a basic domain/helix-loop-helix and a leucine zipper motif, respectively. These motifs are involved both in DNA binding and in protein dimerization2-5. In addition, myc protein family members share several regions of highly conserved amino acids in their amino termini that are essential for transformation6,7. We report here that an N-terminal domain present in both the c-myc and N-myc proteins mediates binding to the retinoblastoma gene product, pRb. We show that the human papilloma virus E7 protein competes with c-myc for binding to pRb, indicating that these proteins share overlapping binding sites on pRb. Furthermore, a mutant Rb protein from a human tumour cell line that carried a 35-amino-acid deletion in its C terminus failed to bind to c-myc. Our results suggest that c-myc and pRb cooperate through direct binding to control cell proliferation.

  8. Proteins without unique 3D structures: biotechnological applications of intrinsically unstable/disordered proteins.

    PubMed

    Uversky, Vladimir N

    2015-03-01

    Intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs) are functional proteins or regions that do not have unique 3D structures under functional conditions. Therefore, from the viewpoint of their lack of stable 3D structure, IDPs/IDPRs are inherently unstable. As much as structure and function of normal ordered globular proteins are determined by their amino acid sequences, the lack of unique 3D structure in IDPs/IDPRs and their disorder-based functionality are also encoded in the amino acid sequences. Because of their specific sequence features and distinctive conformational behavior, these intrinsically unstable proteins or regions have several applications in biotechnology. This review introduces some of the most characteristic features of IDPs/IDPRs (such as peculiarities of amino acid sequences of these proteins and regions, their major structural features, and peculiar responses to changes in their environment) and describes how these features can be used in the biotechnology, for example for the proteome-wide analysis of the abundance of extended IDPs, for recombinant protein isolation and purification, as polypeptide nanoparticles for drug delivery, as solubilization tools, and as thermally sensitive carriers of active peptides and proteins. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Rapid protein disappearance rates along the small intestine advantage poultry performance and influence the post-enteral availability of amino acids.

    PubMed

    Truong, Ha H; Chrystal, Peter V; Moss, Amy F; Selle, Peter H; Liu, Sonia Yun

    2017-12-01

    A foundation diet, an intermediate blend and a summit diet were formulated with different levels of soyabean meal, casein and crystalline amino acids to compare 'slow' and 'rapid' protein diets. The diets were offered to male Ross 308 chicks from 7 to 28 d post-hatch and assessed parameters included growth performance, nutrient utilisation, apparent digestibility coefficients and disappearance rates of starch and protein (N) in four small intestinal segments. Digestibility coefficients and disappearance rates of sixteen amino acids in three small intestinal segments and amino acid concentrations in plasma from portal and systemic circulations from the foundation and summit diets were determined. The dietary transition significantly accelerated protein (N) disappearance rates in the distal jejunum and ileum. The transition from foundation to summit diets significantly increased starch digestibility coefficients in the ileum and disappearance rates in all four small intestinal segments. These starch responses were associated with significant enhancements in nutrient utilisation. The dietary transition linearly increased digestibility coefficients and disappearance rates of amino acids in the majority of cases. The summit diet increased plasma concentrations of five amino acids but decreased those of four amino acids relative to the foundation diet to significant extents. Plasma concentrations of free amino acids were higher in the portal than systemic circulations. Rapid protein disappearance rates advantaged poultry performance and influenced post-enteral availability of amino acids. If the underlying mechanisms are to be identified, further research into the impact of protein digestive dynamics on broiler performance is required but appears justified.

  10. Studies on the protein and sulfur amino acid requirements of young bobwhite quail

    USGS Publications Warehouse

    Serafin, J.A.

    1977-01-01

    Four experiments were conducted with purified diets to examine the influence of protein level and to estimate the sulfur amino acid (S.A.A.) requirement of young Bobwhite quail (Colinus virginianus). These studies demonstrated (I) that 26% protein was sufficient for rapid growth when the diet was supplemented with methionine; (2) that diets containing higher levels of protein (29.3% and 31.3%) failed to support satisfactory growth unless they contained supplemental methionine; and (3) that young Bobwhite quail require no more than 1.0% sulfur-containing amino acids for optimal growth and efficiency of feed utilization. A fifth experiment was conducted to examine the protein and S.A.A. requirements of young Bobwhite quail using practical rations and to compare results with those obtained with purified diets. Diets containing 24%, 26% and 28% protein were supplied with and without supplemental methionine in a five week study. Results showed significant growth responses to protein and supplemental methionine. Responses showed that Bobwhite quail require no more than 26% protein for maximum growth and efficiency of feed utilization when the S.A.A. level of the diet was approximately 1.0%. The results were in close agreement with those obtained with purified diets. These findings define more precisely than had been known the quantitative requirements of young Bobwhite quail for protein and for the S.A.A. necessary for optimal growth.

  11. Effects of Whey Protein Hydrolysate Ingestion on Postprandial Aminoacidemia Compared with a Free Amino Acid Mixture in Young Men

    PubMed Central

    Nakayama, Kyosuke; Sanbongi, Chiaki; Ikegami, Shuji

    2018-01-01

    To stimulate muscle protein synthesis, it is important to increase the plasma levels of essential amino acids (EAA), especially leucine, by ingesting proteins. Protein hydrolysate ingestion can induce postprandial hyperaminoacidemia; however, it is unclear whether protein hydrolysate is associated with higher levels of aminoacidemia compared with a free amino acid mixture when both are ingested orally. We assessed the effects of whey protein hydrolysate (WPH) ingestion on postprandial aminoacidemia, especially plasma leucine levels, compared to ingestion of a free amino acid mixture. This study was an open-label, randomized, 4 × 4 Latin square design. After 12–15 h of fasting, 11 healthy young men ingested the WPH (3.3, 5.0, or 7.5 g of protein) or the EAA mixture (2.5 g). Blood samples were collected before ingestion and at time points from 10 to 120 min after ingestion, and amino acids, insulin, glucose and insulin-like growth factor-1 (IGF-1) concentrations in plasma were measured. Even though the EAA mixture and 5.0 g of the WPH contained similar amounts of EAA and leucine, the WPH was associated with significantly higher plasma EAA and leucine levels. These results suggest that the WPH can induce a higher level of aminoacidemia compared with a free amino acid mixture when both are ingested orally. PMID:29671767

  12. Effects of Whey Protein Hydrolysate Ingestion on Postprandial Aminoacidemia Compared with a Free Amino Acid Mixture in Young Men.

    PubMed

    Nakayama, Kyosuke; Sanbongi, Chiaki; Ikegami, Shuji

    2018-04-19

    To stimulate muscle protein synthesis, it is important to increase the plasma levels of essential amino acids (EAA), especially leucine, by ingesting proteins. Protein hydrolysate ingestion can induce postprandial hyperaminoacidemia; however, it is unclear whether protein hydrolysate is associated with higher levels of aminoacidemia compared with a free amino acid mixture when both are ingested orally. We assessed the effects of whey protein hydrolysate (WPH) ingestion on postprandial aminoacidemia, especially plasma leucine levels, compared to ingestion of a free amino acid mixture. This study was an open-label, randomized, 4 × 4 Latin square design. After 12⁻15 h of fasting, 11 healthy young men ingested the WPH (3.3, 5.0, or 7.5 g of protein) or the EAA mixture (2.5 g). Blood samples were collected before ingestion and at time points from 10 to 120 min after ingestion, and amino acids, insulin, glucose and insulin-like growth factor-1 (IGF-1) concentrations in plasma were measured. Even though the EAA mixture and 5.0 g of the WPH contained similar amounts of EAA and leucine, the WPH was associated with significantly higher plasma EAA and leucine levels. These results suggest that the WPH can induce a higher level of aminoacidemia compared with a free amino acid mixture when both are ingested orally.

  13. Proteolysis of chloroplast proteins is responsible for accumulation of free amino acids in dark-treated tea (Camellia sinensis) leaves.

    PubMed

    Chen, Yiyong; Fu, Xiumin; Mei, Xin; Zhou, Ying; Cheng, Sihua; Zeng, Lanting; Dong, Fang; Yang, Ziyin

    2017-03-22

    Shade management (dark treatment) on tea (Camellia sinensis) plants is a common approach to improve free amino acids in raw materials of tea leaves. However, the reason for amino acid accumulation in dark-treated tea leaves is still unknown. In the present study, dark treatment significantly increased content of free amino acids and reduced content of soluble proteins in tea leaves. Quantitative proteomics analysis showed that most enzymes involved in biosyntheses of amino acids were down-accumulated by dark treatment. Chloroplast numbers reduced in dark-treated leaves and the content of soluble proteins reduced in the chloroplasts isolated from dark-treated leaves compared to control. These suggest that proteolysis of chloroplast proteins contributed to amino acid accumulation in dark-treated leaves. Two chloroplasts proteases, ATP-dependent Clp protease proteolytic subunit 3 and protease Do-like 2, were up-accumulated in dark-treated leaves. This study firstly elucidated the mechanism of accumulation of amino acids in dark-treated tea leaves. Effect of dark on crop growth has been widely studied, while less attention has been paid to effect of dark on quality-related metabolites in crops. Shade management (dark treatment) on tea plants is a common approach to improve free amino acids in tea leaves. However, the reason for accumulation of free amino acids in dark-treated tea leaves is still unknown. In the present study, an iTRAQ-based quantitative proteomic analysis was performed and the results revealed the accumulation of free amino acids in dark-treated tea leaves was not due to activation of biosyntheses of amino acids, but resulted from proteolysis of chloroplast proteins. The information will advance our understanding of formation of quality or function-related metabolites in agricultural crops exposed to dark stress/shade management. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces

    PubMed Central

    Zhu, Yingying; Lin, Xisha; Li, He; Li, Yingqiu; Shi, Xuebin; Zhao, Fan; Xu, Xinglian; Li, Chunbao; Zhou, Guanghong

    2016-01-01

    Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota. PMID:27042829

  15. Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces.

    PubMed

    Zhu, Yingying; Lin, Xisha; Li, He; Li, Yingqiu; Shi, Xuebin; Zhao, Fan; Xu, Xinglian; Li, Chunbao; Zhou, Guanghong

    2016-01-01

    Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota.

  16. Upregulation of capacity for glutathione synthesis in response to amino acid deprivation: regulation of glutamate-cysteine ligase subunits

    PubMed Central

    Sikalidis, Angelos K.; Mazor, Kevin M.; Lee, Jeong-In; Roman, Heather B.; Hirschberger, Lawrence L.; Stipanuk, Martha H.

    2014-01-01

    Using HepG2/C3A cells and MEFs, we investigated whether induction of GSH synthesis in response to sulfur amino acid deficiency is mediated by the decrease in cysteine levels or whether it requires a decrease in GSH levels per se. Both the glutamate-cysteine ligase catalytic (GCLC) and modifier (GCLM) subunit mRNA levels were upregulated in response to a lack of cysteine or other essential amino acids, independent of GSH levels. This upregulation did not occur in MEFs lacking GCN2 (general control non-derepressible 2, also known as eIF2α kinase 4) or in cells expressing mutant eIF2α lacking the eIF2α kinase Ser51 phosphorylation site, indicating that expression of both GCLC and GCLM was mediated by the GCN2/ATF4 stress response pathway. Only the increase in GCLM mRNA level, however, was accompanied by a parallel increase in protein expression, suggesting that the enhanced capacity for GSH synthesis depended largely on increased association of GCLC with its regulatory subunit. Upregulation of both GCLC and GLCM mRNA levels in response to cysteine deprivation was dependent on new protein synthesis, which is consistent with expression of GCLC and GCLM being mediated by proteins whose synthesis depends on activation of the GCN2/ATF4 pathway. Our data suggest that the regulation of GCLC expression may be mediated by changes in the abundance of transcriptional regulators, whereas the regulation of GCLM expression may be mediated by changes in the abundance of mRNA stabilizing or destabilizing proteins. Upregulation of GCLM levels in response to low cysteine levels may serve to protect the cell in the face of a future stress requiring GSH as an antioxidant or conjugating/detoxifying agent. PMID:24557597

  17. Intact Protein Analysis at 21 Tesla and X-Ray Crystallography Define Structural Differences in Single Amino Acid Variants of Human Mitochondrial Branched-Chain Amino Acid Aminotransferase 2 (BCAT2)

    NASA Astrophysics Data System (ADS)

    Anderson, Lissa C.; Håkansson, Maria; Walse, Björn; Nilsson, Carol L.

    2017-09-01

    Structural technologies are an essential component in the design of precision therapeutics. Precision medicine entails the development of therapeutics directed toward a designated target protein, with the goal to deliver the right drug to the right patient at the right time. In the field of oncology, protein structural variants are often associated with oncogenic potential. In a previous proteogenomic screen of patient-derived glioblastoma (GBM) tumor materials, we identified a sequence variant of human mitochondrial branched-chain amino acid aminotransferase 2 as a putative factor of resistance of GBM to standard-of-care-treatments. The enzyme generates glutamate, which is neurotoxic. To elucidate structural coordinates that may confer altered substrate binding or activity of the variant BCAT2 T186R, a 45 kDa protein, we applied combined ETD and CID top-down mass spectrometry in a LC-FT-ICR MS at 21 T, and X-Ray crystallography in the study of both the variant and non-variant intact proteins. The combined ETD/CID fragmentation pattern allowed for not only extensive sequence coverage but also confident localization of the amino acid variant to its position in the sequence. The crystallographic experiments confirmed the hypothesis generated by in silico structural homology modeling, that the Lys59 side-chain of BCAT2 may repulse the Arg186 in the variant protein (PDB code: 5MPR), leading to destabilization of the protein dimer and altered enzyme kinetics. Taken together, the MS and novel 3D structural data give us reason to further pursue BCAT2 T186R as a precision drug target in GBM. [Figure not available: see fulltext.

  18. Effect of a keto acid-amino acid supplement on the metabolism and renal elimination of branched-chain amino acids in patients with chronic renal insufficiency on a low protein diet.

    PubMed

    Teplan, V; Schück, O; Horácková, M; Skibová, J; Holecek, M

    2000-10-27

    The aim of our study was to evaluate the effect of a low-protein diet supplemented with keto acids-amino acids on renal function and urinary excretion of branched-chain amino acids (BCAA) in patients with chronic renal insufficiency (CRI). In a prospective investigation 28 patients with CRI (16 male, 12 female, aged 28-66 yrs, CCr 18.6 +/- 10.2 ml/min) on a low-protein diet (0.6 g of protein /kg BW/day and energy intake 140 kJ/kg BW/day) for a period of one month were included. Subsequently, this low protein diet was supplemented with keto acids-amino acids at a dose of 0.1 g/kg BW/day orally for a period of 3 months. Examinations performed at baseline and at the end of the follow-up period revealed significant increase in the serum levels of BCAA leucine (p < 0.02), isoleucine (p < 0.03), and valine (p < 0.02) while their renal fractional excretion declined (p < 0.02, p < 0.01 resp.). Keto acid-amino acid administration had no effect on renal function and on the clearance of inulin, para-aminohippuric acid. Endogenous creatinine and urea clearance remained unaltered. A significant correlation between fractional excretion of sodium and leucine (p < 0.05) and a hyperbolic relationship between inulin clearance and fractional excretion of BCAA (p < 0.01) were seen. Moreover, a significant decrease in proteinuria (p < 0.02), plasma urea concentration and renal urea excretion and a rise in albumin level (p < 0.03) were noted. We conclude that in patients with CRI on a low protein diet the supplementation of keto acids-amino acids does not affect renal hemodynamics, but is associated--despite increases in plasma concentrations--with a reduction of renal amino acid and protein excretion suggesting induction of alterations in the tubular transport mechanisms.

  19. Prediction protein structural classes with pseudo-amino acid composition: approximate entropy and hydrophobicity pattern.

    PubMed

    Zhang, Tong-Liang; Ding, Yong-Sheng; Chou, Kuo-Chen

    2008-01-07

    Compared with the conventional amino acid (AA) composition, the pseudo-amino acid (PseAA) composition as originally introduced for protein subcellular location prediction can incorporate much more information of a protein sequence, so as to remarkably enhance the power of using a discrete model to predict various attributes of a protein. In this study, based on the concept of PseAA composition, the approximate entropy and hydrophobicity pattern of a protein sequence are used to characterize the PseAA components. Also, the immune genetic algorithm (IGA) is applied to search the optimal weight factors in generating the PseAA composition. Thus, for a given protein sequence sample, a 27-D (dimensional) PseAA composition is generated as its descriptor. The fuzzy K nearest neighbors (FKNN) classifier is adopted as the prediction engine. The results thus obtained in predicting protein structural classification are quite encouraging, indicating that the current approach may also be used to improve the prediction quality of other protein attributes, or at least can play a complimentary role to the existing methods in the relevant areas. Our algorithm is written in Matlab that is available by contacting the corresponding author.

  20. Influence of the yeast strain on the changes of the amino acids, peptides and proteins during sparkling wine production by the traditional method.

    PubMed

    Martínez-Rodríguez, A J; Carrascosa, A V; Martín-Alvarez, P J; Moreno-Arribas, V; Polo, M C

    2002-12-01

    The influence of five yeast strains on the nitrogen fractions, amino acids, peptides and proteins, during 12 months of aging of sparkling wines produced by the traditional or Champenoise method, was studied. High-performance liquid chromatography (HPLC) techniques were used for analysis of the amino acid and peptide fractions. Proteins plus polypeptides were determined by the colorimetric Bradford method. Four main stages were detected in the aging of wines with yeast. In the first stage, a second fermentation took place; amino acids and proteins plus polypeptides diminished, and peptides were liberated. In the second stage, there was a release of amino acids and proteins, and peptides were degraded. In the third stage, the release of proteins and peptides predominated. In the fourth stage, the amino acid concentration diminished. The yeast strain used influenced the content of free amino acids and peptides and the aging time in all the nitrogen fractions.

  1. [Dynamics of amino acid and protein metabolism in laying hens after the administration of 15N-labeled wheat protein. 11. Incorporation of 15N in the tissues and the amino acids of the muscles].

    PubMed

    Gruhn, K; Zander, R

    1989-03-01

    Over a period of 4 days 12 colostomized laying hens daily received 36 g 15N labelled wheat with 15N excess (15N') of 14.37 atom-% together with a conventional feed mixture for laying hens. The labelling of the lysine N in the wheat was 13.58 atom-%, that of histidine N 14.38 and that of arginine 15N' 13.63 atom-% 15N'. Three hens each were butchered 12, 36, 60 and 108 h after the last 15N' feeding. The first three hens did not receive any feed before being butchered. The following three hens each received the unlabelled feed ration for another 1, 2 or 4 days resp. after the main period until they were butchered. The total of skeleton muscles, the heart and the stomach muscle (without inner skin) of each hen were combined into one sample, cut thinly, drenched with fluid nitrogen and pulverized. N, 15N' and the basic and non-basic amino acids as well as their 15N' were determined in the individual samples. In contrast to the organs, the proteins in the muscle tissue have a long half life so that a slight decrease of atom-% 15N' in the muscles could only be detected after 108 h. The 14N and 15N' quota of the non-basic amino acids in the total nitrogen of the muscles is 50%. The 14N quota of the basic amino acids is 30% and the 15N' quota only 22.5% in the total muscle N. The heavy nitrogen of the free lysine in the TCA soluble N fraction is hardly detectable 36 h and 60 h after the last 15N' supply and not at all after 108 h. In contrast to this, the other two free basic amino acids remain significantly higher labelled in dependence on the last butchering time.

  2. PLASMA PROTEIN AND HEMOGLOBIN PRODUCTION

    PubMed Central

    Robscheit-Robbins, F. S.; Miller, L. L.; Whipple, G. H.

    1947-01-01

    Given healthy dogs fed abundant iron and protein-free or low protein diets with sustained anemia and hypoproteinemia, we can study the capacity of these animals to produce simultaneously new hemoglobin and plasma protein. Reserve stores of blood protein-building materials are measurably depleted and levels of 6 to 8 gm. per cent for hemoglobin and 4 to 5 gm. per cent for plasma protein can be maintained for weeks or months depending upon the intake of food proteins or amino acid mixtures. These dogs are very susceptible to infection and various poisons. Dogs tire of these diets and loss of appetite terminates many experiments. Under these conditions (double depletion) standard growth mixtures of essential amino acids are tested to show the response in blood protein output and urinary nitrogen balance. As a part of each tabulated experiment one of the essential amino acids is deleted from the complete growth mixture to compare such response with that of the whole mixture. Methionine, threonine, phenylalanine, and tryptophane when singly eliminated from the complete amino acid mixture do effect a sharp rise in urinary nitrogen. This loss of urinary nitrogen is corrected when the individual amino acid is replaced in the mixture. Histidine, lysine, and valine have a moderate influence upon urinary nitrogen balance toward nitrogen conservation. Leucine, isoleucine, and arginine have minimal or no effect upon urinary nitrogen balance when these individual amino acids are deleted from the complete growth mixture of amino acids during 3 to 4 week periods. Tryptophane and to a less extent phenylalanine and threonine when returned to the amino acid mixture are associated with a conspicuous preponderance of plasma protein output over the hemoglobin output (Table 4). Arginine, lysine, and histidine when returned to the amino acid mixture are associated with a large preponderance of hemoglobin output. Various amino acid mixtures under these conditions may give a positive

  3. Quantitative expression of protein heterogeneity: Response of amino acid side chains to their local environment.

    PubMed

    Bandyopadhyay, Debashree; Mehler, Ernest L

    2008-08-01

    A general method has been developed to characterize the hydrophobicity or hydrophilicity of the microenvironment (MENV), in which a given amino acid side chain is immersed, by calculating a quantitative property descriptor (QPD) based on the relative (to water) hydrophobicity of the MENV. Values of the QPD were calculated for a test set of 733 proteins to analyze the modulating effects on amino acid residue properties by the MENV in which they are imbedded. The QPD values and solvent accessibility were used to derive a partitioning of residues based on the MENV hydrophobicities. From this partitioning, a new hydrophobicity scale was developed, entirely in the context of protein structure, where amino acid residues are immersed in one or more "MENVpockets." Thus, the partitioning is based on the residues "sampling" a large number of "solvents" (MENVs) that represent a very large range of hydrophobicity values. It was found that the hydrophobicity of around 80% of amino acid side chains and their MENV are complementary to each other, but for about 20%, the MENV and their imbedded residue can be considered as mismatched. Many of these mismatches could be rationalized in terms of the structural stability of the protein and/or the involvement of the imbedded residue in function. The analysis also indicated a remarkable conservation of local environments around highly conserved active site residues that have similar functions across protein families, but where members have relatively low sequence homology. Thus, quantitative evaluation of this QPD is suggested, here, as a tool for structure-function prediction, analysis, and parameter development for the calculation of properties in proteins. (c) 2008 Wiley-Liss, Inc.

  4. Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer-derived exosomes.

    PubMed

    Clark, David J; Fondrie, William E; Yang, Austin; Mao, Li

    2016-02-05

    Exosomes are 30-100 nm sized membrane vesicles released by cells into the extracellular space that mediate intercellular communication via transfer of proteins and other biological molecules. To better understand the role of these microvesicles in lung carcinogenesis, we employed a Triple SILAC quantitative proteomic strategy to examine the differential protein abundance between exosomes derived from an immortalized normal bronchial epithelial cell line and two non-small cell lung cancer (NSCLC) cell lines harboring distinct activating mutations in the cell signaling molecules: Kirsten rat sarcoma viral oncogene homolog (KRAS) or epidermal growth factor receptor (EGFR). In total, we were able to quantify 721 exosomal proteins derived from the three cell lines. Proteins associated with signal transduction, including EGFR, GRB2 and SRC, were enriched in NSCLC exosomes, and could actively regulate cell proliferation in recipient cells. This study's investigation of the NSCLC exosomal proteome has identified enriched protein cargo that can contribute to lung cancer progression, which may have potential clinical implications in biomarker development for patients with NSCLC. The high mortality associated with lung cancer is a result of late-stage diagnosis of the disease. Current screening techniques used for early detection of lung cancer lack the specificity for accurate diagnosis. Exosomes are nano-sized extracellular vesicles, and the increased abundance of select protein cargo in exosomes derived from cancer cells may be used for diagnostic purposes. In this paper, we applied quantitative proteomic analysis to elucidate abundance differences in exosomal protein cargo between two NSCLC cell lines with distinctive oncogene mutations and an immortalized normal bronchial epithelial cell line. This study revealed proteins associated with cell adhesion, the extracellular matrix, and a variety of signaling molecules were enriched in NSCLC exosomes. The present data reveals

  5. Boron Stress Activates the General Amino Acid Control Mechanism and Inhibits Protein Synthesis

    PubMed Central

    Uluisik, Irem; Kaya, Alaattin; Fomenko, Dmitri E.; Karakaya, Huseyin C.; Carlson, Bradley A.; Gladyshev, Vadim N.; Koc, Ahmet

    2011-01-01

    Boron is an essential micronutrient for plants, and it is beneficial for animals. However, at high concentrations boron is toxic to cells although the mechanism of this toxicity is not known. Atr1 has recently been identified as a boron efflux pump whose expression is upregulated in response to boron treatment. Here, we found that the expression of ATR1 is associated with expression of genes involved in amino acid biosynthesis. These mechanisms are strictly controlled by the transcription factor Gcn4 in response to boron treatment. Further analyses have shown that boron impaired protein synthesis by promoting phosphorylation of eIF2α in a Gcn2 kinase dependent manner. The uncharged tRNA binding domain (HisRS) of Gcn2 is necessary for the phosphorylation of eIF2α in the presence of boron. We postulate that boron exerts its toxic effect through activation of the general amino acid control system and inhibition of protein synthesis. Since the general amino acid control pathway is conserved among eukaryotes, this mechanism of boron toxicity may be of general importance. PMID:22114689

  6. The effects of reduced dietary protein level on amino acid transporters and mTOR signaling pathway in pigs.

    PubMed

    Wang, Dan; Wan, Xuebin; Peng, Jian; Xiong, Qi; Niu, Hongdan; Li, Huanan; Chai, Jin; Jiang, Siwen

    2017-04-01

    Amino acid transporter plays an important role in regulating mTOR signaling pathway. This study investigated the effects of reduced dietary protein levels on amino acid transporters and mTOR signaling pathway. A total of 54 weaning pigs were randomly allocated into a 3 × 3 factorial design, followed by slaughtering the pigs separately after 10-, 25- and 45-day feeding, with 18 pigs from each feeding period divided into three subgroups for treatment with three different protein-level diets: 20% crude protein (CP) diet (normal recommended, high protein, HP), 17% CP diet (medium protein, MP) and 14% CP diet (low protein, LP). The results indicated that reduced dietary protein level decreased the weight of longissimus dorsi. Additionally, quantitative PCR chip analysis showed that mRNA expression of amino acid transporters SLC38A2, SLC1A7, SLC7A1, SLC7A5, SLC16A10 and SLC3A2 in the LP group were significantly (P < 0.05) higher than those in the MP or HP group, and the phosphorylation of mTOR and S6K1 decreased in the LP group after 25-day feeding. Furthermore, the vitro experimental results further confirmed that the mRNA levels for SLC7A1, SLC7A5, SLC3A2, SLC38A2 and SLC36A1 were increased and the phosphorylation of mTOR and S6K1 was decreased when the concentration of amino acids in C2C12 myoblasts was reduced. All these results indicated that the LP diet induced a high expression of amino acid transporters and the inhibition of the mTOR activity, which resulting in restriction on protein synthesis and longissimus dorsi growth. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Highly abundant defense proteins in human sweat as revealed by targeted proteomics and label-free quantification mass spectrometry.

    PubMed

    Csősz, É; Emri, G; Kalló, G; Tsaprailis, G; Tőzsér, J

    2015-10-01

    The healthy human skin with its effective antimicrobial defense system forms an efficient barrier against invading pathogens. There is evidence suggesting that the composition of this chemical barrier varies between diseases, making the easily collected sweat an ideal candidate for biomarker discoveries. Our aim was to provide information about the normal composition of the sweat, and to study the chemical barrier found at the surface of skin. Sweat samples from healthy individuals were collected during sauna bathing, and the global protein panel was analysed by label-free mass spectrometry. SRM-based targeted proteomic methods were designed and stable isotope labelled reference peptides were used for method validation. Ninety-five sweat proteins were identified, 20 of them were novel proteins. It was shown that dermcidin is the most abundant sweat protein, and along with apolipoprotein D, clusterin, prolactin-inducible protein and serum albumin, they make up 91% of secreted sweat proteins. The roles of these highly abundant proteins were reviewed; all of which have protective functions, highlighting the importance of sweat glands in composing the first line of innate immune defense system, and maintaining the epidermal barrier integrity. Our findings with regard to the proteins forming the chemical barrier of the skin as determined by label-free quantification and targeted proteomics methods are in accordance with previous studies, and can be further used as a starting point for non-invasive sweat biomarker research. © 2015 European Academy of Dermatology and Venereology.

  8. Strategies for Characterization of Low-Abundant Intact or Truncated Low-Molecular-Weight Proteins From Human Plasma.

    PubMed

    Cai, Tanxi; Yang, Fuquan

    2017-01-01

    Low-molecular-weight region (LMW, MW≤30kDa) of human serum/plasma proteins, including small intact proteins, truncated fragments of larger proteins, along with some other small components, has been associated with the ongoing physiological and pathological events, and thereby represent a treasure trove of diagnostic molecules. Great progress in the mining of novel biomarkers from this diagnostic treasure trove for disease diagnosis and health monitoring has been achieved based on serum samples from healthy individuals and patients and powerful new approaches in biochemistry and systems biology. However, cumulative evidence indicates that many potential LMW protein biomarkers might still have escaped from detection due to their low abundance, the dynamic complexity of serum/plasma, and the limited efficiency of characterization approaches. Here, we provide an overview of the current state of knowledge with respect to strategies for the characterization of low-abundant LMW proteins (small intact or truncated proteins) from human serum/plasma, involving prefractionation or enrichment methods to reduce dynamic range and mass spectrometry-based characterization of low-abundant LMW proteins. © 2017 Elsevier Inc. All rights reserved.

  9. Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

    NASA Astrophysics Data System (ADS)

    He, Yi-Ming; Ma, Bin-Guang

    2016-05-01

    Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions.

  10. A 100-Year Review: Protein and amino acid nutrition in dairy cows.

    PubMed

    Schwab, Charles G; Broderick, Glen A

    2017-12-01

    Considerable progress has been made in understanding the protein and amino acid (AA) nutrition of dairy cows. The chemistry of feed crude protein (CP) appears to be well understood, as is the mechanism of ruminal protein degradation by rumen bacteria and protozoa. It has been shown that ammonia released from AA degradation in the rumen is used for bacterial protein formation and that urea can be a useful N supplement when lower protein diets are fed. It is now well documented that adequate rumen ammonia levels must be maintained for maximal synthesis of microbial protein and that a deficiency of rumen-degradable protein can decrease microbial protein synthesis, fiber digestibility, and feed intake. Rumen-synthesized microbial protein accounts for most of the CP flowing to the small intestine and is considered a high-quality protein for dairy cows because of apparent high digestibility and good AA composition. Much attention has been given to evaluating different methods to quantify ruminal protein degradation and escape and for measuring ruminal outflows of microbial protein and rumen-undegraded feed protein. The methods and accompanying results are used to determine the nutritional value of protein supplements and to develop nutritional models and evaluate their predictive ability. Lysine, methionine, and histidine have been identified most often as the most-limiting amino acids, with rumen-protected forms of lysine and methionine available for ration supplementation. Guidelines for protein feeding have evolved from simple feeding standards for dietary CP to more complex nutrition models that are designed to predict supplies and requirements for rumen ammonia and peptides and intestinally absorbable AA. The industry awaits more robust and mechanistic models for predicting supplies and requirements of rumen-available N and absorbed AA. Such models will be useful in allowing for feeding lower protein diets and increased efficiency of microbial protein synthesis

  11. Relative Amino Acid Composition Signatures of Organisms and Environments

    PubMed Central

    Moura, Alexandra; Savageau, Michael A.; Alves, Rui

    2013-01-01

    Background Identifying organism-environment interactions at the molecular level is crucial to understanding how organisms adapt to and change the chemical and molecular landscape of their habitats. In this work we investigated whether relative amino acid compositions could be used as a molecular signature of an environment and whether such a signature could also be observed at the level of the cellular amino acid composition of the microorganisms that inhabit that environment. Methodologies/Principal Findings To address these questions we collected and analyzed environmental amino acid determinations from the literature, and estimated from complete genomic sequences the global relative amino acid abundances of organisms that are cognate to the different types of environment. Environmental relative amino acid abundances clustered into broad groups (ocean waters, host-associated environments, grass land environments, sandy soils and sediments, and forest soils), indicating the presence of amino acid signatures specific for each environment. These signatures correlate to those found in organisms. Nevertheless, relative amino acid abundance of organisms was more influenced by GC content than habitat or phylogeny. Conclusions Our results suggest that relative amino acid composition can be used as a signature of an environment. In addition, we observed that the relative amino acid composition of organisms is not highly determined by environment, reinforcing previous studies that find GC content to be the major factor correlating to amino acid composition in living organisms. PMID:24204807

  12. Relative amino acid composition signatures of organisms and environments.

    PubMed

    Moura, Alexandra; Savageau, Michael A; Alves, Rui

    2013-01-01

    Identifying organism-environment interactions at the molecular level is crucial to understanding how organisms adapt to and change the chemical and molecular landscape of their habitats. In this work we investigated whether relative amino acid compositions could be used as a molecular signature of an environment and whether such a signature could also be observed at the level of the cellular amino acid composition of the microorganisms that inhabit that environment. To address these questions we collected and analyzed environmental amino acid determinations from the literature, and estimated from complete genomic sequences the global relative amino acid abundances of organisms that are cognate to the different types of environment. Environmental relative amino acid abundances clustered into broad groups (ocean waters, host-associated environments, grass land environments, sandy soils and sediments, and forest soils), indicating the presence of amino acid signatures specific for each environment. These signatures correlate to those found in organisms. Nevertheless, relative amino acid abundance of organisms was more influenced by GC content than habitat or phylogeny. Our results suggest that relative amino acid composition can be used as a signature of an environment. In addition, we observed that the relative amino acid composition of organisms is not highly determined by environment, reinforcing previous studies that find GC content to be the major factor correlating to amino acid composition in living organisms.

  13. Amino acid sequence of the Amur tiger prion protein.

    PubMed

    Wu, Changde; Pang, Wanyong; Zhao, Deming

    2006-10-01

    Prion diseases are fatal neurodegenerative disorders in human and animal associated with conformational conversion of a cellular prion protein (PrP(C)) into the pathologic isoform (PrP(Sc)). Various data indicate that the polymorphisms within the open reading frame (ORF) of PrP are associated with the susceptibility and control the species barrier in prion diseases. In the present study, partial Prnp from 25 Amur tigers (tPrnp) were cloned and screened for polymorphisms. Four single nucleotide polymorphisms (T423C, A501G, C511A, A610G) were found; the C511A and A610G nucleotide substitutions resulted in the amino acid changes Lysine171Glutamine and Alanine204Threoine, respectively. The tPrnp amino acid sequence is similar to house cat (Felis catus ) and sheep, but differs significantly from other two cat Prnp sequences that were previously deposited in GenBank.

  14. Amino acid sequences of ribosomal proteins S11 from Bacillus stearothermophilus and S19 from Halobacterium marismortui. Comparison of the ribosomal protein S11 family.

    PubMed

    Kimura, M; Kimura, J; Hatakeyama, T

    1988-11-21

    The complete amino acid sequences of ribosomal proteins S11 from the Gram-positive eubacterium Bacillus stearothermophilus and of S19 from the archaebacterium Halobacterium marismortui have been determined. A search for homologous sequences of these proteins revealed that they belong to the ribosomal protein S11 family. Homologous proteins have previously been sequenced from Escherichia coli as well as from chloroplast, yeast and mammalian ribosomes. A pairwise comparison of the amino acid sequences showed that Bacillus protein S11 shares 68% identical residues with S11 from Escherichia coli and a slightly lower homology (52%) with the homologous chloroplast protein. The halophilic protein S19 is more related to the eukaryotic (45-49%) than to the eubacterial counterparts (35%).

  15. Structural and dynamic changes associated with beneficial engineered single-amino-acid deletion mutations in enhanced green fluorescent protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Arpino, James A. J.; Rizkallah, Pierre J., E-mail: rizkallahp@cardiff.ac.uk; Jones, D. Dafydd, E-mail: rizkallahp@cardiff.ac.uk

    2014-08-01

    The beneficial engineered single-amino-acid deletion variants EGFP{sup D190Δ} and EGFP{sup A227Δ} have been studied. Single-amino-acid deletions are a common part of the natural evolutionary landscape but are rarely sampled during protein engineering owing to limited and prejudiced molecular understanding of mutations that shorten the protein backbone. Single-amino-acid deletion variants of enhanced green fluorescent protein (EGFP) have been identified by directed evolution with the beneficial effect of imparting increased cellular fluorescence. Biophysical characterization revealed that increased functional protein production and not changes to the fluorescence parameters was the mechanism that was likely to be responsible. The structure EGFP{sup D190Δ} containing amore » deletion within a loop revealed propagated changes only after the deleted residue. The structure of EGFP{sup A227Δ} revealed that a ‘flipping’ mechanism was used to adjust for residue deletion at the end of a β-strand, with amino acids C-terminal to the deletion site repositioning to take the place of the deleted amino acid. In both variants new networks of short-range and long-range interactions are generated while maintaining the integrity of the hydrophobic core. Both deletion variants also displayed significant local and long-range changes in dynamics, as evident by changes in B factors compared with EGFP. Rather than being detrimental, deletion mutations can introduce beneficial structural effects through altering core protein properties, folding and dynamics, as well as function.« less

  16. Virulence-Affecting Amino Acid Changes in the PA Protein of H7N9 Influenza A Viruses

    PubMed Central

    Yamayoshi, Seiya; Yamada, Shinya; Fukuyama, Satoshi; Murakami, Shin; Zhao, Dongming; Uraki, Ryuta; Watanabe, Tokiko; Tomita, Yuriko; Macken, Catherine; Neumann, Gabriele

    2014-01-01

    ABSTRACT Novel avian-origin influenza A(H7N9) viruses were first reported to infect humans in March 2013. To date, 143 human cases, including 45 deaths, have been recorded. By using sequence comparisons and phylogenetic and ancestral inference analyses, we identified several distinct amino acids in the A(H7N9) polymerase PA protein, some of which may be mammalian adapting. Mutant viruses possessing some of these amino acid changes, singly or in combination, were assessed for their polymerase activities and growth kinetics in mammalian and avian cells and for their virulence in mice. We identified several mutants that were slightly more virulent in mice than the wild-type A(H7N9) virus, A/Anhui/1/2013. These mutants also exhibited increased polymerase activity in human cells but not in avian cells. Our findings indicate that the PA protein of A(H7N9) viruses has several amino acid substitutions that are attenuating in mammals. IMPORTANCE Novel avian-origin influenza A(H7N9) viruses emerged in the spring of 2013. By using computational analyses of A(H7N9) viral sequences, we identified several amino acid changes in the polymerase PA protein, which we then assessed for their effects on viral replication in cultured cells and mice. We found that the PA proteins of A(H7N9) viruses possess several amino acid substitutions that cause attenuation in mammals. PMID:24371069

  17. Amino acids

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/002222.htm Amino acids To use the sharing features on this page, please enable JavaScript. Amino acids are organic compounds that combine to form proteins . ...

  18. Comparison of Depletion Strategies for the Enrichment of Low-Abundance Proteins in Urine.

    PubMed

    Filip, Szymon; Vougas, Konstantinos; Zoidakis, Jerome; Latosinska, Agnieszka; Mullen, William; Spasovski, Goce; Mischak, Harald; Vlahou, Antonia; Jankowski, Joachim

    2015-01-01

    Proteome analysis of complex biological samples for biomarker identification remains challenging, among others due to the extended range of protein concentrations. High-abundance proteins like albumin or IgG of plasma and urine, may interfere with the detection of potential disease biomarkers. Currently, several options are available for the depletion of abundant proteins in plasma. However, the applicability of these methods in urine has not been thoroughly investigated. In this study, we compared different, commercially available immunodepletion and ion-exchange based approaches on urine samples from both healthy subjects and CKD patients, for their reproducibility and efficiency in protein depletion. A starting urine volume of 500 μL was used to simulate conditions of a multi-institutional biomarker discovery study. All depletion approaches showed satisfactory reproducibility (n=5) in protein identification as well as protein abundance. Comparison of the depletion efficiency between the unfractionated and fractionated samples and the different depletion strategies, showed efficient depletion in all cases, with the exception of the ion-exchange kit. The depletion efficiency was found slightly higher in normal than in CKD samples and normal samples yielded more protein identifications than CKD samples when using both initial as well as corresponding depleted fractions. Along these lines, decrease in the amount of albumin and other targets as applicable, following depletion, was observed. Nevertheless, these depletion strategies did not yield a higher number of identifications in neither the urine from normal nor CKD patients. Collectively, when analyzing urine in the context of CKD biomarker identification, no added value of depletion strategies can be observed and analysis of unfractionated starting urine appears to be preferable.

  19. Comparison of Depletion Strategies for the Enrichment of Low-Abundance Proteins in Urine

    PubMed Central

    Filip, Szymon; Vougas, Konstantinos; Zoidakis, Jerome; Latosinska, Agnieszka; Mullen, William; Spasovski, Goce; Mischak, Harald; Vlahou, Antonia; Jankowski, Joachim

    2015-01-01

    Proteome analysis of complex biological samples for biomarker identification remains challenging, among others due to the extended range of protein concentrations. High-abundance proteins like albumin or IgG of plasma and urine, may interfere with the detection of potential disease biomarkers. Currently, several options are available for the depletion of abundant proteins in plasma. However, the applicability of these methods in urine has not been thoroughly investigated. In this study, we compared different, commercially available immunodepletion and ion-exchange based approaches on urine samples from both healthy subjects and CKD patients, for their reproducibility and efficiency in protein depletion. A starting urine volume of 500 μL was used to simulate conditions of a multi-institutional biomarker discovery study. All depletion approaches showed satisfactory reproducibility (n=5) in protein identification as well as protein abundance. Comparison of the depletion efficiency between the unfractionated and fractionated samples and the different depletion strategies, showed efficient depletion in all cases, with the exception of the ion-exchange kit. The depletion efficiency was found slightly higher in normal than in CKD samples and normal samples yielded more protein identifications than CKD samples when using both initial as well as corresponding depleted fractions. Along these lines, decrease in the amount of albumin and other targets as applicable, following depletion, was observed. Nevertheless, these depletion strategies did not yield a higher number of identifications in neither the urine from normal nor CKD patients. Collectively, when analyzing urine in the context of CKD biomarker identification, no added value of depletion strategies can be observed and analysis of unfractionated starting urine appears to be preferable. PMID:26208298

  20. Molecular characterization of amino acid deletion in VP1 (1D) protein and novel amino acid substitutions in 3D polymerase protein of foot and mouth disease virus subtype A/Iran87.

    PubMed

    Esmaelizad, Majid; Jelokhani-Niaraki, Saber; Hashemnejad, Khadije; Kamalzadeh, Morteza; Lotfi, Mohsen

    2011-12-01

    The nucleotide sequence of the VP1 (1D) and partial 3D polymerase (3D(pol)) coding regions of the foot and mouth disease virus (FMDV) vaccine strain A/Iran87, a highly passaged isolate (~150 passages), was determined and aligned with previously published FMDV serotype A sequences. Overall analysis of the amino acid substitutions revealed that the partial 3D(pol) coding region contained four amino acid alterations. Amino acid sequence comparison of the VP1 coding region of the field isolates revealed deletions in the highly passaged Iranian isolate (A/Iran87). The prominent G-H loop of the FMDV VP1 protein contains the conserved arginine-glycine-aspartic acid (RGD) tripeptide, which is a well-known ligand for a specific cell surface integrin. Despite losing the RGD sequence of the VP1 protein and an Asp(26)→Glu substitution in a beta sheet located within a small groove of the 3D(pol) protein, the virus grew in BHK 21 suspension cell cultures. Since this strain has been used as a vaccine strain, it may be inferred that the RGD deletion has no critical role in virus attachment to the cell during the initiation of infection. It is probable that this FMDV subtype can utilize other pathways for cell attachment.

  1. Radiation-induced reductive modifications of sulfur-containing amino acids within peptides and proteins.

    PubMed

    Chatgilialoglu, Chryssostomos; Ferreri, Carla; Torreggiani, Armida; Salzano, Anna Maria; Renzone, Giovanni; Scaloni, Andrea

    2011-10-19

    The complex scenario of radical stress reactions affecting peptides/proteins can be better elucidated through the design of biomimetic studies simulating the consequences of the different free radicals attacking amino acids. In this context, ionizing radiations allowed to examine the specific damages caused by H-atoms and electrons coupled with protons, thus establishing the molecular basis of reductive radical stress. This is an innovative concept that complements the well-known oxidative stress also in view of a complete understanding of the global consequences of radical species reactivities on living systems. This review summarizes the knowledge of the chemical changes present in sulfur-containing amino acids occurring in polypeptides under reductive radical conditions, in particular the transformation of Met and Cys residues into α-amino butyric acid and alanine, respectively. Reductive radical stress causing a desulfurization process, is therefore coupled with the formation of S-centered radicals, which in turn can diffuse apart and become responsible of the damage transfer from proteins to lipids. These reductive modifications assayed in different peptide/protein sequences constitute an integration of the molecular inventories that up to now take into account only oxidative transformations. They can be useful to achieve an integrated vision of the free radical reactivities in a multifunctional system and, overall, for wider applications in the redox proteomics field. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. New concepts of microbial treatment processes for the nitrogen removal: effect of protein and amino acids degradation.

    PubMed

    González-Martínez, Alejandro; Calderón, Kadiya; González-López, Jesús

    2016-05-01

    High concentrations of proteins and amino acids can be found in wastewater and wastewater stream produced in anaerobic digesters, having shown that amino acids could persist over different managements for nitrogen removal affecting the nitrogen removal processes. Nitrogen removal is completely necessary because of their implications and the significant adverse environmental impact of ammonium such as eutrophication and toxicity to aquatic life on the receiving bodies. In the last decade, the treatment of effluents with high ammonium concentration through anammox-based bioprocesses has been enhanced because these biotechnologies are cheaper and more environmentally friendly than conventional technologies. However, it has been shown that the presence of important amounts of proteins and amino acids in the effluents seriously affects the microbial autotrophic consortia leading to important losses in terms of ammonium oxidation efficiency. Particularly the presence of sulfur amino acids such as methionine and cysteine has been reported to drastically decrease the autotrophic denitrification processes as well as affect the microbial community structure promoting the decline of ammonium oxidizing bacteria in favor of other phylotypes. In this context we discuss that new biotechnological processes that improve the degradation of protein and amino acids must be considered as a priority to increase the performance of the autotrophic denitrification biotechnologies.

  3. White clover fractions as protein source for monogastrics: dry matter digestibility and protein digestibility-corrected amino acid scores.

    PubMed

    Stødkilde, Lene; Damborg, Vinni K; Jørgensen, Henry; Laerke, Helle N; Jensen, Søren K

    2018-05-01

    The present study aimed to evaluate the use of white clover as an alternative protein source for monogastrics. White clover plant and leaves were processed using a screw-press resulting in a solid pulp and a juice from which protein was acid-precipitated. The chemical composition of all fractions was determined and digestibility of dry matter (DM) and protein was assessed in an experiment with growing rats. Protein concentrates were produced with crude protein (CP) contents of 451 g kg -1 and 530 g kg -1 DM for white clover plant and leaves, respectively, and a pulp with CP contents of 313 and 374 g kg -1 DM from plant and leaves, respectively. The amino acid composition ranged from 4.72 to 6.49 g per 16 g of nitrogen (N) for lysine, 1.82-2.6 g per 16 g N for methionine and cysteine, and 3.66-5.24 g per 16 g N for threonine. True faecal digestibility of protein varied from 0.81 to 0.88, whereas DM digestibility was in the range 0.72-0.80. Methionine and cysteine were found to be limiting in all fractions, regardless of the reference group used. A high digestibility of white clover protein was found irrespective of the physical fractionation. Together with a well-balanced amino acid composition, this makes white clover a promising protein source for monogastrics. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  4. Identification of proteins of altered abundance in oil palm infected with Ganoderma boninense.

    PubMed

    Al-Obaidi, Jameel R; Mohd-Yusuf, Yusmin; Razali, Nurhanani; Jayapalan, Jaime Jacqueline; Tey, Chin-Chong; Md-Noh, Normahnani; Junit, Sarni Mat; Othman, Rofina Yasmin; Hashim, Onn Haji

    2014-03-24

    Basal stem rot is a common disease that affects oil palm, causing loss of yield and finally killing the trees. The disease, caused by fungus Ganoderma boninense, devastates thousands of hectares of oil palm plantings in Southeast Asia every year. In the present study, root proteins of healthy oil palm seedlings, and those infected with G. boninense, were analyzed by 2-dimensional gel electrophoresis (2-DE). When the 2-DE profiles were analyzed for proteins, which exhibit consistent significant change of abundance upon infection with G. boninense, 21 passed our screening criteria. Subsequent analyses by mass spectrometry and database search identified caffeoyl-CoA O-methyltransferase, caffeic acid O-methyltransferase, enolase, fructokinase, cysteine synthase, malate dehydrogenase, and ATP synthase as among proteins of which abundances were markedly altered.

  5. Identification of Proteins of Altered Abundance in Oil Palm Infected with Ganoderma boninense

    PubMed Central

    Al-Obaidi, Jameel R.; Mohd-Yusuf, Yusmin; Razali, Nurhanani; Jayapalan, Jaime Jacqueline; Tey, Chin-Chong; Md-Noh, Normahnani; Junit, Sarni Mat; Othman, Rofina Yasmin; Hashim, Onn Haji

    2014-01-01

    Basal stem rot is a common disease that affects oil palm, causing loss of yield and finally killing the trees. The disease, caused by fungus Ganoderma boninense, devastates thousands of hectares of oil palm plantings in Southeast Asia every year. In the present study, root proteins of healthy oil palm seedlings, and those infected with G. boninense, were analyzed by 2-dimensional gel electrophoresis (2-DE). When the 2-DE profiles were analyzed for proteins, which exhibit consistent significant change of abundance upon infection with G. boninense, 21 passed our screening criteria. Subsequent analyses by mass spectrometry and database search identified caffeoyl-CoA O-methyltransferase, caffeic acid O-methyltransferase, enolase, fructokinase, cysteine synthase, malate dehydrogenase, and ATP synthase as among proteins of which abundances were markedly altered. PMID:24663087

  6. Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

    PubMed Central

    He, Yi-Ming; Ma, Bin-Guang

    2016-01-01

    Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions. PMID:27220911

  7. Effect of Bacillus subtilis and Bacillus licheniformis supplementation in diets with low- and high-protein content on ileal crude protein and amino acid digestibility and intestinal microbiota composition of growing pigs.

    PubMed

    Kaewtapee, Chanwit; Burbach, Katharina; Tomforde, Georgina; Hartinger, Thomas; Camarinha-Silva, Amélia; Heinritz, Sonja; Seifert, Jana; Wiltafsky, Markus; Mosenthin, Rainer; Rosenfelder-Kuon, Pia

    2017-01-01

    Bacillus spp. seem to be an alternative to antimicrobial growth promoters for improving animals' health and performance. However, there is little information on the effect of Bacillus spp. in combination with different dietary crude protein (CP) levels on the ileal digestibility and microbiota composition. Therefore, the objective of this study was to determine the effect of Bacillus spp. supplementation to low- (LP) and high-protein diets (HP) on ileal CP and amino acid (AA) digestibility and intestinal microbiota composition. Eight ileally cannulated pigs with an initial body weight of 28.5 kg were randomly allocated to a row-column design with 8 pigs and 3 periods of 16 d each. The assay diets were based on wheat-barley-soybean meal with two protein levels: LP (14% CP, as-fed) and HP diet (18% CP, as-fed). The LP and HP diets were supplemented with or without Bacillus spp. at a level of 0.04% (as-fed). The apparent ileal digestibility (AID) and standardized ileal digestibility (SID) of CP and AA was determined. Bacterial community composition from ileal digesta was analyzed by Illumina amplicon sequencing and quantitative real-time PCR. Data were analyzed as a 2 × 2 factorial design using the GLIMMIX procedures of SAS. The supplementation with Bacillus spp. did not affect both AID and SID of CP and AA in growing pigs. Moreover, there was no difference in AID of CP and AA between HP and LP diets, but SID of cystine, glutamic acid, glycine, and proline was lower ( P  < 0.05) in pigs fed the HP diets. The HP diets increased abundance of Bifidobacterium spp. and Lactobacillus spp., ( P  < 0.05) and by amplicon sequencing the latter was identified as predominant genus in microbiota from HP with Bacillus spp., whereas dietary supplementation of Bacillus spp. increased ( P  < 0.05) abundance of Roseburia spp.. The HP diet increased abundance of Lactobacillus spp. and Bifidobacterium spp.. The supplementation of Bacillus spp. resulted in a higher

  8. LC-MS display of the total modified amino acids in cataract lens proteins and in lens proteins glycated by ascorbic acid in vitro.

    PubMed

    Cheng, Rongzhu; Feng, Qi; Ortwerth, Beryl J

    2006-05-01

    We previously reported chromatographic evidence supporting the similarity of yellow chromophores isolated from aged human lens proteins, early brunescent cataract lens proteins and calf lens proteins ascorbylated in vitro [Cheng, R. et al. Biochimica et Biophysica Acta 1537, 14-26, 2001]. In this paper, new evidence supporting the chemical identity of the modified amino acids in these protein populations were collected by using a newly developed two-dimensional LC-MS mapping technique supported by tandem mass analysis of the major species. The pooled water-insoluble proteins from aged normal human lenses, early stage brunescent cataract lenses and calf lens proteins reacted with or without 20 mM ascorbic acid in air for 4 weeks were digested with a battery of proteolytic enzymes under argon to release the modified amino acids. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column and four major A330nm-absorbing peaks were collected. Peaks 1, 2 and 3, which contained most of the modified amino acids were concentrated and subjected to RP-HPLC/ESI-MS, and the mass elution maps were determined. The samples were again analyzed and those peaks with a 10(4) - 10(6) response factor were subjected to MS/MS analysis to identify the daughter ions of each modification. Mass spectrometric maps of peaks 1, 2 and 3 from cataract lenses showed 58, 40 and 55 mass values, respectively, ranging from 150 to 600 Da. Similar analyses of the peaks from digests of the ascorbylated calf lens proteins gave 81, 70 and 67 mass values, respectively, of which 100 were identical to the peaks in the cataract lens proteins. A total of 40 of the major species from each digest were analyzed by LC-MS/MS and 36 were shown to be identical. Calf lens proteins incubated without ascorbic acid showed several similar mass values, but the response factors were 100 to 1000-fold less for every modification. Based upon these data, we conclude

  9. Insights on Structure and Function of a Late Embryogenesis Abundant Protein from Amaranthus cruentus: An Intrinsically Disordered Protein Involved in Protection against Desiccation, Oxidant Conditions, and Osmotic Stress

    PubMed Central

    Saucedo, Alma L.; Hernández-Domínguez, Eric E.; de Luna-Valdez, Luis A.; Guevara-García, Angel A.; Escobedo-Moratilla, Abraham; Bojorquéz-Velázquez, Esaú; del Río-Portilla, Federico; Fernández-Velasco, Daniel A.; Barba de la Rosa, Ana P.

    2017-01-01

    Late embryogenesis abundant (LEA) proteins are part of a large protein family that protect other proteins from aggregation due to desiccation or osmotic stresses. Recently, the Amaranthus cruentus seed proteome was characterized by 2D-PAGE and one highly accumulated protein spot was identified as a LEA protein and was named AcLEA. In this work, AcLEA cDNA was cloned into an expression vector and the recombinant protein was purified and characterized. AcLEA encodes a 172 amino acid polypeptide with a predicted molecular mass of 18.34 kDa and estimated pI of 8.58. Phylogenetic analysis revealed that AcLEA is evolutionarily close to the LEA3 group. Structural characteristics were revealed by nuclear magnetic resonance and circular dichroism methods. We have shown that recombinant AcLEA is an intrinsically disordered protein in solution even at high salinity and osmotic pressures, but it has a strong tendency to take a secondary structure, mainly folded as α-helix, when an inductive additive is present. Recombinant AcLEA function was evaluated using Escherichia coli as in vivo model showing the important protection role against desiccation, oxidant conditions, and osmotic stress. AcLEA recombinant protein was localized in cytoplasm of Nicotiana benthamiana protoplasts and orthologs were detected in seeds of wild and domesticated amaranth species. Interestingly AcLEA was detected in leaves, stems, and roots but only in plants subjected to salt stress. This fact could indicate the important role of AcLEA protection during plant stress in all amaranth species studied. PMID:28439280

  10. Natural proteins: Sources, isolation, characterization and applications

    PubMed Central

    Nehete, Jitendra Y.; Bhambar, Rajendra S.; Narkhede, Minal R.; Gawali, Sonali R.

    2013-01-01

    Worldwide, plant protein contributes substantially as a food resource because it contains essential amino acids for meeting human physiological requirements. However, many versatile plant proteins are used as medicinal agents as they are produced by using molecular tools of biotechnology. Proteins can be obtained from plants, animals and microorganism cells. The abundant economical proteins can be obtained from plant seeds. These natural proteins are obtained by isolation procedures depending on the physicochemical properties of proteins. Isolation and purification of single protein from cells containing mixtures of unrelated proteins is achievable due to the physical and chemical attributes of proteins. The following characteristics are unique to each protein: Amino acid composition, sequence, subunit structures, size, shape, net charge, isoelectric point, solubility, heat stability and hydrophobicity. Based on these properties, various methods of isolation exist, like salting out and isoionic precipitation. Purification of proteins is quiet challenging and, therefore, several approaches like sodium dodecyl sulfate gel electrophoresis and chromatography are available. Characterization of proteins can be performed by mass spectrometry/liquid chromatography-mass spectrometry (LC-MS). The amino acid sequence of a protein can be detected by using tandem mass spectrometry. In this article, a review has been made on the sources, isolation, purification and characterization of natural proteins. PMID:24347918

  11. Association between insulin resistance and plasma amino acid profile in non-diabetic Japanese subjects.

    PubMed

    Yamada, Chizumi; Kondo, Masumi; Kishimoto, Noriaki; Shibata, Takeo; Nagai, Yoko; Imanishi, Tadashi; Oroguchi, Takashige; Ishii, Naoaki; Nishizaki, Yasuhiro

    2015-07-01

    Elevation of the branched-chain amino acids (BCAAs), valine, leucine and isoleucine; and the aromatic amino acids, tyrosine and phenylalanine, has been observed in obesity-related insulin resistance. However, there have been few studies on Asians, who are generally less obese and less insulin-resistant than Caucasian or African-Americans. In the present study, we investigated the relationship between homeostasis model assessment of insulin resistance (HOMA-IR) and plasma amino acid concentration in non-diabetic Japanese participants. A total of 94 healthy men and women were enrolled, and plasma amino acid concentration was measured by liquid chromatography/mass spectrometry after overnight fasting. The associations between HOMA-IR and 20 amino acid concentrations, and anthropometric and clinical parameters of lifestyle-related diseases were evaluated. The mean age and body mass index were 40.1 ± 9.6 years and 22.7 ± 3.9, respectively. Significantly positive correlations were observed between HOMA-IR and valine, isoleucine, leucine, tyrosine, phenylalanine and total BCAA concentration. Compared with the HOMA-IR ≤ 1.6 group, the HOMA-IR > 1.6 group showed significantly exacerbated anthropometric and clinical parameters, and significantly elevated levels of valine, isoleucine, leucine, tyrosine, phenylalanine and BCAA. The present study shows that the insulin resistance-related change in amino acid profile is also observed in non-diabetic Japanese subjects. These amino acids include BCAAs (valine, isoleucine and leucine) and aromatic amino acids (tyrosine and phenylalanine), in agreement with previous studies carried out using different ethnic groups with different degrees of obesity and insulin resistance.

  12. Abundance of intrinsic structural disorder in the histone H1 subtypes.

    PubMed

    Kowalski, Andrzej

    2015-12-01

    The intrinsically disordered proteins consist of partially structured regions linked to the unstructured stretches, which consequently form the transient and dynamic conformational ensembles. They undergo disorder to order transition upon binding their partners. Intrinsic disorder is attributed to histones H1, perceived as assemblers of chromatin structure and the regulators of DNA and proteins activity. In this work, the comparison of intrinsic disorder abundance in the histone H1 subtypes was performed both by the analysis of their amino acid composition and by the prediction of disordered stretches, as well as by identifying molecular recognition features (MoRFs) and ANCHOR protein binding regions (APBR) that are responsible for recognition and binding. Both human and model organisms-animals, plants, fungi and protists-have H1 histone subtypes with the properties typical of disordered state. They possess a significantly higher content of hydrophilic and charged amino acid residues, arranged in the long regions, covering over half of the whole amino acid residues in chain. Almost complete disorder corresponds to histone H1 terminal domains, including MoRFs and ANCHOR. Those motifs were also identified in a more ordered histone H1 globular domain. Compared to the control (globular and fibrous) proteins, H1 histones demonstrate the increased folding rate and a higher proportion of low-complexity segments. The results of this work indicate that intrinsic disorder is an inherent structural property of histone H1 subtypes and it is essential for establishing a protein conformation which defines functional outcomes affecting on DNA- and/or partner protein-dependent cell processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Quantitative protein expression and cell surface characteristics of Escherichia coli MG1655 biofilms.

    PubMed

    Mukherjee, Joy; Ow, Saw Yen; Noirel, Josselin; Biggs, Catherine A

    2011-02-01

    Cell surface physicochemical characterization techniques were combined with quantitative changes in protein expression, to investigate the biological and biophysical changes of Escherichia coli MG1655 cells when grown as a biofilm (BIO). The overall surface charge of BIO cells was found to be less negative, highlighting the need for a lower electrophoretic mobility for attachment to occur. Comparison of the chemical functional groups on the cell surface showed similar profiles, with the absorbance intensity higher for proteins and carbohydrates in the BIO cells. Quantitative proteomic analysis demonstrated that 3 proteins were significantly increased, and 9 proteins significantly decreased in abundance, in cells grown as a BIO compared to their planktonic counterparts, with 7 of these total 12 proteins unique to this study. Proteins showing significant increased or decreased abundance include proteins involved in acid resistance, DNA protection and binding and ABC transporters. Further predictive analysis of the metabolic pathways showed an increased abundance of the amino acid metabolism and tricarboxylic acid (TCA) cycle, with a decrease in expression within the pentose phosphate and glycolysis pathways. It is therefore hypothesized that cells grown as a BIO are still energetically viable potentially using amino acids as an indirect carbon backbone source into the TCA cycle. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Amino acid sequence analysis of the annexin super-gene family of proteins.

    PubMed

    Barton, G J; Newman, R H; Freemont, P S; Crumpton, M J

    1991-06-15

    The annexins are a widespread family of calcium-dependent membrane-binding proteins. No common function has been identified for the family and, until recently, no crystallographic data existed for an annexin. In this paper we draw together 22 available annexin sequences consisting of 88 similar repeat units, and apply the techniques of multiple sequence alignment, pattern matching, secondary structure prediction and conservation analysis to the characterisation of the molecules. The analysis clearly shows that the repeats cluster into four distinct families and that greatest variation occurs within the repeat 3 units. Multiple alignment of the 88 repeats shows amino acids with conserved physicochemical properties at 22 positions, with only Gly at position 23 being absolutely conserved in all repeats. Secondary structure prediction techniques identify five conserved helices in each repeat unit and patterns of conserved hydrophobic amino acids are consistent with one face of a helix packing against the protein core in predicted helices a, c, d, e. Helix b is generally hydrophobic in all repeats, but contains a striking pattern of repeat-specific residue conservation at position 31, with Arg in repeats 4 and Glu in repeats 2, but unconserved amino acids in repeats 1 and 3. This suggests repeats 2 and 4 may interact via a buried saltbridge. The loop between predicted helices a and b of repeat 3 shows features distinct from the equivalent loop in repeats 1, 2 and 4, suggesting an important structural and/or functional role for this region. No compelling evidence emerges from this study for uteroglobin and the annexins sharing similar tertiary structures, or for uteroglobin representing a derivative of a primordial one-repeat structure that underwent duplication to give the present day annexins. The analyses performed in this paper are re-evaluated in the Appendix, in the light of the recently published X-ray structure for human annexin V. The structure confirms most of

  15. Designing non-native iron-binding site on a protein cage for biological synthesis of nanoparticles.

    PubMed

    Peng, Tao; Paramelle, David; Sana, Barindra; Lee, Chiu Fan; Lim, Sierin

    2014-08-13

    In biomineralization processes, a supramolecular organic structure is often used as a template for inorganic nanomaterial synthesis. The E2 protein cage derived from Geobacillus stearothermophilus pyruvate dehydrogenase and formed by the self-assembly of 60 subunits, has been functionalized with non-native iron-mineralization capability by incorporating two types of iron-binding peptides. The non-native peptides introduced at the interior surface do not affect the self-assembly of E2 protein subunits. In contrast to the wild-type, the engineered E2 protein cages can serve as size- and shape-constrained reactors for the synthesis of iron nanoparticles. Electrostatic interactions between anionic amino acids and cationic iron molecules drive the formation of iron oxide nanoparticles within the engineered E2 protein cages. The work expands the investigations on nanomaterial biosynthesis using engineered host-guest encapsulation properties of protein cages. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Association of Takayasu arteritis with HLA-B 67:01 and two amino acids in HLA-B protein.

    PubMed

    Terao, Chikashi; Yoshifuji, Hajime; Ohmura, Koichiro; Murakami, Kosaku; Kawabata, Daisuke; Yurugi, Kimiko; Tazaki, Junichi; Kinoshita, Hideyuki; Kimura, Akinori; Akizuki, Masashi; Kawaguchi, Yasushi; Yamanaka, Hisashi; Miura, Yasuo; Maekawa, Taira; Saji, Hiroo; Mimori, Tsuneyo; Matsuda, Fumihiko

    2013-10-01

    Takayasu arteritis (TAK) is a rare autoimmune arteritis that affects large arteries. Although the association between TAK and HLA-B 52:01 is established, the other susceptibility HLA-B alleles are not fully known. We performed genetic association studies to determine independent HLA-B susceptibility alleles other than HLA-B 52:01 and to identify important amino acids of HLA-B protein in TAK susceptibility. One hundred patients with TAK and 1000 unrelated healthy controls were genotyped for HLA-B alleles in the first set, followed by a replication set containing 73 patients with TAK and 1000 controls to compare the frequencies of HLA-B alleles. Step-up logistic regression analysis was performed to identify susceptibility amino acids of HLA-B protein. Strong associations of susceptibility to TAK with HLA-B 52:01 and HLA-B 67:01 were observed (P = 1.0 × 10(-16) and 9.5 × 10(-6), respectively). An independent susceptibility effect of HLA-B 67:01 from HLA-B 52:01 was also detected (P = 1.8 × 10(-7)). Amino acid residues of histidine at position 171 and phenylalanine at position 67, both of which are located in antigen binding grooves of the HLA-B protein, were associated with TAK susceptibility (P ≤ 3.8 × 10(-5)) with a significant difference from other amino acid variations (ΔAIC ≥ 9.65). HLA-B 67:01 is associated with TAK independently from HLA-B 52:01. Two amino acids in HLA-B protein are strongly associated with TAK susceptibility.

  17. Dietary Protein and Amino Acid Supplementation in Inflammatory Bowel Disease Course: What Impact on the Colonic Mucosa?

    PubMed Central

    Vidal-Lletjós, Sandra; Beaumont, Martin; Tomé, Daniel; Benamouzig, Robert; Blachier, François; Lan, Annaïg

    2017-01-01

    Inflammatory bowel diseases (IBD), after disease onset, typically progress in two cyclically repeated phases, namely inflammatory flare and remission, with possible nutritional status impairment. Some evidence, either from epidemiological, clinical, and experimental studies indicate that the quantity and the quality of dietary protein consumption and amino acid supplementation may differently influence the IBD course according to the disease phases. For instance, although the dietary protein needs for mucosal healing after an inflammatory episode remain undetermined, there is evidence that amino acids derived from dietary proteins display beneficial effects on this process, serving as building blocks for macromolecule synthesis in the wounded mucosal area, energy substrates, and/or precursors of bioactive metabolites. However, an excessive amount of dietary proteins may result in an increased intestinal production of potentially deleterious bacterial metabolites. This could possibly affect epithelial repair as several of these bacterial metabolites are known to inhibit colonic epithelial cell respiration, cell proliferation, and/or to affect barrier function. In this review, we present the available evidence about the impact of the amount of dietary proteins and supplementary amino acids on IBD onset and progression, with a focus on the effects reported in the colon. PMID:28335546

  18. Amino acids in the Tagish Lake Meteorite

    NASA Technical Reports Server (NTRS)

    Kminek, G.; Botta, O.; Glavin, D. P.; Bada, J. L.

    2002-01-01

    High-performance liquid chromatography (HPLC) based amino acid analysis of a Tagish Lake meteorite sample recovered 3 months after the meteorite fell to Earth have revealed that the amino acid composition of Tagish Lake is strikingly different from that of the CM and CI carbonaceous chondrites. We found that the Tagish Lake meteorite contains only trace levels of amino acids (total abundance = 880 ppb), which is much lower than the total abundance of amino acids in the CI Orgueil (4100 ppb) and the CM Murchison (16 900 ppb). Because most of the same amino acids found in the Tagish Lake meteorite are also present in the Tagish Lake ice melt water, we conclude that the amino acids detected in the meteorite are terrestrial contamination. We found that the exposure of a sample of Murchison to cold water lead to a substantial reduction over a period of several weeks in the amount of amino acids that are not strongly bound to the meteorite matrix. However, strongly bound amino acids that are extracted by direct HCl hydrolysis are not affected by the leaching process. Thus even if there had been leaching of amino acids from our Tagish Lake meteorite sample during its 3 month residence in Tagish Lake ice and melt water, a Murchison type abundance of endogenous amino acids in the meteorite would have still been readily detectable. The low amino acid content of Tagish Lake indicates that this meteorite originated fiom a different type of parent body than the CM and CI chondrites. The parent body was apparently devoid of the reagents such as aldehyldes/ketones, HCN and ammonia needed for the effective abiotic synthesis of amino acids. Based on reflectance spectral measurements, Tagish Lake has been associated with P- or D-type asteroids. If the Tagish Lake meteorite was indeed derived fiom these types of parent bodies, our understanding of these primitive asteroids needs to be reevaluated with respect to their potential inventory of biologically important organic compounds.

  19. Pseudomonas fluorescens SBW25 produces furanomycin, a non-proteinogenic amino acid with selective antimicrobial properties.

    PubMed

    Trippe, Kristin; McPhail, Kerry; Armstrong, Donald; Azevedo, Mark; Banowetz, Gary

    2013-05-20

    Pseudomonas fluorescens SBW25 has been extensively studied because of its plant growth promoting properties and potential as a biocontrol agent. The genome of SBW25 has been sequenced, and among sequenced strains of pseudomonads, SBW25 appears to be most closely related to P. fluorescens WH6. In the authors' laboratories, WH6 was previously shown to produce and secrete 4-formylaminooxyvinylglycine (FVG), a non-proteinogenic amino acid with selective herbicidal and antimicrobial activity. Although SBW25 does not have the genetic capacity to produce FVG, we were interested in determining whether this pseudomonad might produce some other type of non-proteinogenic amino acid. P. fluorescens SBW25 was found to produce and secrete a ninhydrin-reactive compound with selective antimicrobial properties. This compound was purified from SBW25 culture filtrate and identified as the non-proteinogenic amino acid L-furanomycin [2S,2'R,5'S)-2-amino-2-(5'methyl-2',5'-dihydrofuran-2'-yl)acetic acid]. The identification of furanomycin as a secondary metabolite of SBW25 is the first report of the production of furanomycin by a pseudomonad. This compound was known previously only as a natural product produced by a strain of Streptomyces. This report adds furanomycin to the small list of non-proteinogenic amino acids that have been identified as secondary products of pseudomonads. This study also extends the list of bacteria that are inhibited by furanomycin to include several plant pathogenic bacteria.

  20. Stimulation of muscle protein synthesis by prolonged parenteral infusion of leucine is dependent on amino acid availability in neonatal pigs

    USDA-ARS?s Scientific Manuscript database

    The postprandial rise in amino acids, particularly leucine, stimulates muscle protein synthesis in neonates. Previously, we showed that a 1-h infusion of leucine increased protein synthesis, but this response was not sustained for 2 h unless the leucine-induced decrease in amino acids was prevented....

  1. Effect of dietary protein and GABA on food intake, growth and tissue amino acids in cats.

    PubMed

    Tews, J K; Rogers, Q R; Morris, J G; Harper, A E

    1984-02-01

    GABA at 5%, but not 3%, of a low protein diet depressed food intake and growth of kittens. Adaptation to high protein prevented these effects. When cats adapted to low or high protein were fed a meal containing GABA, plasma GABA concentration after 2 hr was 8-fold higher in the low than in the high protein group; clearance was almost complete within 6 hr. Concentrations of proline, branched-chain, other large neutral and basic (especially ornithine) amino acids increased more when cats were fed a high rather than a low protein meal; glycine decreased. At 6 hr, concentrations had consistently returned to initial levels only in the low protein group. Feeding the high protein diet ad lib increased tissue concentrations of threonine, proline and the branched-chain amino acids. Hepatic or renal GABA-aminotransferase activity was not altered in kittens fed the high protein diet. Kidney activity was 10-fold that of liver, which may contribute to the better tolerance of GABA by cats than by rats.

  2. iHSP-PseRAAAC: Identifying the heat shock protein families using pseudo reduced amino acid alphabet composition.

    PubMed

    Feng, Peng-Mian; Chen, Wei; Lin, Hao; Chou, Kuo-Chen

    2013-11-01

    Heat shock proteins (HSPs) are a type of functionally related proteins present in all living organisms, both prokaryotes and eukaryotes. They play essential roles in protein-protein interactions such as folding and assisting in the establishment of proper protein conformation and prevention of unwanted protein aggregation. Their dysfunction may cause various life-threatening disorders, such as Parkinson's, Alzheimer's, and cardiovascular diseases. Based on their functions, HSPs are usually classified into six families: (i) HSP20 or sHSP, (ii) HSP40 or J-class proteins, (iii) HSP60 or GroEL/ES, (iv) HSP70, (v) HSP90, and (vi) HSP100. Although considerable progress has been achieved in discriminating HSPs from other proteins, it is still a big challenge to identify HSPs among their six different functional types according to their sequence information alone. With the avalanche of protein sequences generated in the post-genomic age, it is highly desirable to develop a high-throughput computational tool in this regard. To take up such a challenge, a predictor called iHSP-PseRAAAC has been developed by incorporating the reduced amino acid alphabet information into the general form of pseudo amino acid composition. One of the remarkable advantages of introducing the reduced amino acid alphabet is being able to avoid the notorious dimension disaster or overfitting problem in statistical prediction. It was observed that the overall success rate achieved by iHSP-PseRAAAC in identifying the functional types of HSPs among the aforementioned six types was more than 87%, which was derived by the jackknife test on a stringent benchmark dataset in which none of HSPs included has ≥40% pairwise sequence identity to any other in the same subset. It has not escaped our notice that the reduced amino acid alphabet approach can also be used to investigate other protein classification problems. As a user-friendly web server, iHSP-PseRAAAC is accessible to the public at http

  3. The abundant extrachromosomal DNA content of the Spiroplasma citri GII3-3X genome

    PubMed Central

    Saillard, Colette; Carle, Patricia; Duret-Nurbel, Sybille; Henri, Raphaël; Killiny, Nabil; Carrère, Sébastien; Gouzy, Jérome; Bové, Joseph-Marie; Renaudin, Joël; Foissac, Xavier

    2008-01-01

    Background Spiroplama citri, the causal agent of citrus stubborn disease, is a bacterium of the class Mollicutes and is transmitted by phloem-feeding leafhopper vectors. In order to characterize candidate genes potentially involved in spiroplasma transmission and pathogenicity, the genome of S. citri strain GII3-3X is currently being deciphered. Results Assembling 20,000 sequencing reads generated seven circular contigs, none of which fit the 1.8 Mb chromosome map or carried chromosomal markers. These contigs correspond to seven plasmids: pSci1 to pSci6, with sizes ranging from 12.9 to 35.3 kbp and pSciA of 7.8 kbp. Plasmids pSci were detected as multiple copies in strain GII3-3X. Plasmid copy numbers of pSci1-6, as deduced from sequencing coverage, were estimated at 10 to 14 copies per spiroplasma cell, representing 1.6 Mb of extrachromosomal DNA. Genes encoding proteins of the TrsE-TraE, Mob, TraD-TraG, and Soj-ParA protein families were predicted in most of the pSci sequences, in addition to members of 14 protein families of unknown function. Plasmid pSci6 encodes protein P32, a marker of insect transmissibility. Plasmids pSci1-5 code for eight different S. citri adhesion-related proteins (ScARPs) that are homologous to the previously described protein P89 and the S. kunkelii SkARP1. Conserved signal peptides and C-terminal transmembrane alpha helices were predicted in all ScARPs. The predicted surface-exposed N-terminal region possesses the following elements: (i) 6 to 8 repeats of 39 to 42 amino acids each (sarpin repeats), (ii) a central conserved region of 330 amino acids followed by (iii) a more variable domain of about 110 amino acids. The C-terminus, predicted to be cytoplasmic, consists of a 27 amino acid stretch enriched in arginine and lysine (KR) and an optional 23 amino acid stretch enriched in lysine, aspartate and glutamate (KDE). Plasmids pSci mainly present a linear increase of cumulative GC skew except in regions presenting conserved hairpin

  4. Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis

    USDA-ARS?s Scientific Manuscript database

    Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were...

  5. Meal composition and plasma amino acid ratios: Effect of various proteins or carbohydrates, and of various protein concentrations

    NASA Technical Reports Server (NTRS)

    Yokogoshi, Hidehiko; Wurtman, Richard J.

    1986-01-01

    The effects of meals containing various proteins and carbohydrates, and of those containing various proportions of protein (0 percent to 20 percent of a meal, by weight) or of carbohydrate (0 percent to 75 percent), on plasma levels of certain large neutral amino acids (LNAA) in rats previously fasted for 19 hours were examined. Also the plasma tryptophan ratios (the ratio of the plasma trytophan concentration to the summed concentrations of the other large neutral amino acids) and other plasma amino acid ratios were calculated. (The plasma tryptophan ratio has been shown to determine brain tryptophan levels and, thereby, to affect the synthesis and release of the neurotransmitter serotonin). A meal containing 70 percent to 75 percent of an insulin-secreting carbohydrate (dextrose or dextrin) increased plasma insulin levels and the tryptophan ratio; those containing 0 percent or 25 percent carbohydrate failed to do so. Addition of as little as 5 percent casein to a 70 percent carbohydrate meal fully blocked the increase in the plasma tryptophan ratio without affecting the secretion of insulin - probably by contributing much larger quantities of the other LNAA than of tryptophan to the blood. Dietary proteins differed in their ability to suppress the carbohydrate-induced rise in the plasma tryptophan ratio. Addition of 10 percent casein, peanut meal, or gelatin fully blocked this increase, but lactalbumin failed to do so, and egg white did so only partially. (Consumption of the 10 percent gelatin meal also produced a major reduction in the plasma tyrosine ratio, and may thereby have affected brain tyrosine levels and catecholamine synthesis.) These observations suggest that serotonin-releasing neurons in brains of fasted rats are capable of distinguishing (by their metabolic effects) between meals poor in protein but rich in carbohydrates that elicit insulin secretion, and all other meals. The changes in brain serotonin caused by carbohydrate-rich, protein

  6. Quantitation of total protein deposits on contact lenses by means of amino acid analysis.

    PubMed

    Yan, G; Nyquist, G; Caldwell, K D; Payor, R; McCraw, E C

    1993-04-01

    This study was done to characterize and quantify the protein deposits on worn contact lenses and to measure the residual deposits after extraction in 2% sodium dodecyl sulfate and the total protein deposits on worn vifilcon, atlafilcon, and tefilcon lenses (Food and Drug Administration Types IV, II, and I, respectively). Contact lens extracts were separated with gel electrophoresis, and the amount of protein was estimated after silver staining and densitometry. To determine the residual deposits, the contact lenses were hydrolyzed, and amino acid analysis was carried out by reverse-phase high-performance liquid chromatography after precolumn derivatization with phenylisothiocyanate. Refinement of the hydrolysis conditions was undertaken to minimize interference by the lens polymers. The extraction removed only approximately 25% of the protein deposits. Mild hydrolytic conditions, 20 hr in 6 N HCl at 105 degrees C, were found to cause minimal polymer interference. Of the 350, 10, and 20 micrograms of protein typically determined on whole vifilcon, atlafilcon, and tefilcon lenses, the polymers were estimated to account for 4, 0.5, and less than 0.4 micrograms, respectively. Hydrolysis of worn contact lenses with subsequent amino acid separation can be applied to determine the total protein deposits without the uncertainty inherent in extraction of the deposits.

  7. Quality protein maize germplasm characterized for amino acid profiles and endosperm opacity

    USDA-ARS?s Scientific Manuscript database

    Quality protein maize (QPM) is improved over normal maize in grain concentrations of the essential amino acids lysine and tryptophan. QPM has a long history as tropical adapted germplasm but little effort has been made in temperate and sub-tropical adaptation and it is unknown if different modifier ...

  8. Mycoprotein represents a bioavailable and insulinotropic non-animal-derived dietary protein source: a dose-response study.

    PubMed

    Dunlop, Mandy V; Kilroe, Sean P; Bowtell, Joanna L; Finnigan, Tim J A; Salmon, Deborah L; Wall, Benjamin T

    2017-11-01

    The anabolic potential of a dietary protein is determined by its ability to elicit postprandial rises in circulating essential amino acids and insulin. Minimal data exist regarding the bioavailability and insulinotropic effects of non-animal-derived protein sources. Mycoprotein is a sustainable and rich source of non-animal-derived dietary protein. We investigated the impact of mycoprotein ingestion, in a dose-response manner, on acute postprandial hyperaminoacidaemia and hyperinsulinaemia. In all, twelve healthy young men completed five experimental trials in a randomised, single-blind, cross-over design. During each trial, volunteers consumed a test drink containing either 20 g milk protein (MLK20) or a mass matched (not protein matched due to the fibre content) bolus of mycoprotein (20 g; MYC20), a protein matched bolus of mycoprotein (40 g; MYC40), 60 g (MYC60) or 80 g (MYC80) mycoprotein. Circulating amino acid, insulin and uric acid concentrations, and clinical chemistry profiles, were assessed in arterialised venous blood samples during a 4-h postprandial period. Mycoprotein ingestion resulted in slower but more sustained hyperinsulinaemia and hyperaminoacidaemia compared with milk when protein matched, with overall bioavailability equivalent between conditions (P>0·05). Increasing the dose of mycoprotein amplified these effects, with some evidence of a plateau at 60-80 g. Peak postprandial leucine concentrations were 201 (sem 24) (30 min), 118 (sem 10) (90 min), 150 (sem 14) (90 min), 173 (sem 23) (45 min) and 201 (sem 21 (90 min) µmol/l for MLK20, MYC20, MYC40, MYC60 and MYC80, respectively. Mycoprotein represents a bioavailable and insulinotropic dietary protein source. Consequently, mycoprotein may be a useful source of dietary protein to stimulate muscle protein synthesis rates.

  9. Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

    PubMed

    Jong, A Y; Clark, M W; Gilbert, M; Oehm, A; Campbell, J L

    1987-08-01

    To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.

  10. Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

    PubMed Central

    Jong, A Y; Clark, M W; Gilbert, M; Oehm, A; Campbell, J L

    1987-01-01

    To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded. Images PMID:2823109

  11. Distribution of Amino Acids in Lunar Regolith

    NASA Technical Reports Server (NTRS)

    Elsila, J. E.; Callahan, M. P.; Glavin, D. P.; Dworkin, J. P.; Noble, S. K.; Gibson, E. K., Jr.

    2014-01-01

    One of the most eagerly studied questions upon initial return of lunar samples was whether significant amounts of organic compounds, including amino acids, were present. Analyses during the 1970s produced only tentative and inconclusive identifications of indigenous amino acids. Those analyses were hampered by analytical difficulties including relative insensitivity to certain compounds, the inability to separate chiral enantiomers, and the lack of compound-specific isotopic measurements, which made it impossible to determine whether the detected amino acids were indigenous to the lunar samples or the results of contamination. Numerous advances have been made in instrumentation and methodology for amino acid characterization in extraterrestrial samples in the intervening years, yet the origin of amino acids in lunar regolith samples has been revisited only once for a single lunar sample, (3) and remains unclear. Here, we present initial data from the analyses of amino acid abundances in 12 lunar regolith samples. We discuss these abundances in the context of four potential amino acid sources: (1) terrestrial biological contamination; (2) contamination from lunar module (LM) exhaust; (3) derivation from solar windimplanted precursors; and (4) exogenous delivery from meteorites.

  12. A Single Amino Acid Substitution in the Core Protein of West Nile Virus Increases Resistance to Acidotropic Compounds

    PubMed Central

    Martín-Acebes, Miguel A.; Blázquez, Ana-Belén; de Oya, Nereida Jiménez; Escribano-Romero, Estela; Shi, Pei-Yong; Saiz, Juan-Carlos

    2013-01-01

    West Nile virus (WNV) is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH4Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu) on the highly basic internal capsid or core (C) protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A) and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses. PMID:23874963

  13. Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR).

    PubMed

    Gomez, Sandra; Adalid-Peralta, Laura; Palafox-Fonseca, Hector; Cantu-Robles, Vito Adrian; Soberón, Xavier; Sciutto, Edda; Fragoso, Gladis; Bobes, Raúl J; Laclette, Juan P; Yauner, Luis del Pozo; Ochoa-Leyva, Adrián

    2015-05-19

    Excretory/Secretory (ES) proteins play an important role in the host-parasite interactions. Experimental identification of ES proteins is time-consuming and expensive. Alternative bioinformatics approaches are cost-effective and can be used to prioritize the experimental analysis of therapeutic targets for parasitic diseases. Here we predicted and functionally annotated the ES proteins in T. solium genome using an integration of bioinformatics tools. Additionally, we developed a novel measurement to evaluate the potential antigenicity of T. solium secretome using sequence length and number of antigenic regions of ES proteins. This measurement was formalized as the Abundance of Antigenic Regions (AAR) value. AAR value for secretome showed a similar value to that obtained for a set of experimentally determined antigenic proteins and was different to the calculated value for the non-ES proteins of T. solium genome. Furthermore, we calculated the AAR values for known helminth secretomes and they were similar to that obtained for T. solium. The results reveal the utility of AAR value as a novel genomic measurement to evaluate the potential antigenicity of secretomes. This comprehensive analysis of T. solium secretome provides functional information for future experimental studies, including the identification of novel ES proteins of therapeutic, diagnosis and immunological interest.

  14. Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR)

    PubMed Central

    Gomez, Sandra; Adalid-Peralta, Laura; Palafox-Fonseca, Hector; Cantu-Robles, Vito Adrian; Soberón, Xavier; Sciutto, Edda; Fragoso, Gladis; Bobes, Raúl J.; Laclette, Juan P.; Yauner, Luis del Pozo; Ochoa-Leyva, Adrián

    2015-01-01

    Excretory/Secretory (ES) proteins play an important role in the host-parasite interactions. Experimental identification of ES proteins is time-consuming and expensive. Alternative bioinformatics approaches are cost-effective and can be used to prioritize the experimental analysis of therapeutic targets for parasitic diseases. Here we predicted and functionally annotated the ES proteins in T. solium genome using an integration of bioinformatics tools. Additionally, we developed a novel measurement to evaluate the potential antigenicity of T. solium secretome using sequence length and number of antigenic regions of ES proteins. This measurement was formalized as the Abundance of Antigenic Regions (AAR) value. AAR value for secretome showed a similar value to that obtained for a set of experimentally determined antigenic proteins and was different to the calculated value for the non-ES proteins of T. solium genome. Furthermore, we calculated the AAR values for known helminth secretomes and they were similar to that obtained for T. solium. The results reveal the utility of AAR value as a novel genomic measurement to evaluate the potential antigenicity of secretomes. This comprehensive analysis of T. solium secretome provides functional information for future experimental studies, including the identification of novel ES proteins of therapeutic, diagnosis and immunological interest. PMID:25989346

  15. Amino acids in a targeted versus a non-targeted metabolomics LC-MS/MS assay. Are the results consistent?

    PubMed

    Klepacki, Jacek; Klawitter, Jost; Klawitter, Jelena; Karimpour-Fard, Anis; Thurman, Joshua; Ingle, Gordon; Patel, Dharmesh; Christians, Uwe

    2016-09-01

    The results of plasma amino acid patterns in samples from kidney transplant patients with good and impaired renal function using a targeted LC-MS/MS amino acid assay and a non-targeted metabolomics assay were compared. EDTA plasma samples were prospectively collected at baseline, 1, 2, 4 and 6months post-transplant (n=116 patients, n=398 samples). Each sample was analyzed using both a commercial amino acid LC-MS/MS assay and a non-targeted metabolomics assay also based on MS/MS ion transitions. The results of both assays were independently statistically analyzed to identify amino acids associated with estimated glomerular filtration rates using correlation and partial least squares-discriminant analysis. Although there was overlap between the results of the targeted and non-targeted metabolomics assays (tryptophan, 1-methyl histidine), there were also substantial inconsistencies, with the non-targeted assay resulting in more "hits" than the targeted assay. Without further verification of the hits detected by the non-targeted discovery assay, this would have led to different interpretation of the results. There were also false negative results when the non-targeted assay was used (hydroxy proline). Several of said discrepancies could be explained by loss of sensitivity during analytical runs for selected amino acids (serine and threonine), retention time shifts, signals above the range of linear detector response and integration of peaks not separated from background and interferences (aspartate) when the non-targeted metabolomics assay was used. Whenever assessment of a specific pathway such as amino acids is the focus of interest, a targeted seems preferable to a non-targeted metabolomics assay. Copyright © 2016. Published by Elsevier Inc.

  16. Study of model systems to test the potential function of Artemia group 1 late embryogenesis abundant (LEA) proteins.

    PubMed

    Warner, Alden H; Guo, Zhi-hao; Moshi, Sandra; Hudson, John W; Kozarova, Anna

    2016-01-01

    Embryos of the brine shrimp, Artemia franciscana, are genetically programmed to develop either ovoviparously or oviparously depending on environmental conditions. Shortly upon their release from the female, oviparous embryos enter diapause during which time they undergo major metabolic rate depression while simultaneously synthesize proteins that permit them to tolerate a wide range of stressful environmental events including prolonged periods of desiccation, freezing, and anoxia. Among the known stress-related proteins that accumulate in embryos entering diapause are the late embryogenesis abundant (LEA) proteins. This large group of intrinsically disordered proteins has been proposed to act as molecular shields or chaperones of macromolecules which are otherwise intolerant to harsh conditions associated with diapause. In this research, we used two model systems to study the potential function of the group 1 LEA proteins from Artemia. Expression of the Artemia group 1 gene (AfrLEA-1) in Escherichia coli inhibited growth in proportion to the number of 20-mer amino acid motifs expressed. As well, clones of E. coli, transformed with the AfrLEA-1 gene, expressed multiple bands of LEA proteins, either intrinsically or upon induction with isopropyl-β-thiogalactoside (IPTG), in a vector-specific manner. Expression of AfrLEA-1 in E. coli did not overcome the inhibitory effects of high concentrations of NaCl and KCl but modulated growth inhibition resulting from high concentrations of sorbitol in the growth medium. In contrast, expression of the AfrLEA-1 gene in Saccharomyces cerevisiae did not alter the growth kinetics or permit yeast to tolerate high concentrations of NaCl, KCl, or sorbitol. However, expression of AfrLEA-1 in yeast improved its tolerance to drying (desiccation) and freezing. Under our experimental conditions, both E. coli and S. cerevisiae appear to be potentially suitable hosts to study the function of Artemia group 1 LEA proteins under environmentally

  17. Catalytic amino acid production from biomass-derived intermediates

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Deng, Weiping; Wang, Yunzhu; Zhang, Sui

    Amino acids are the building blocks for protein biosynthesis and find use in myriad industrial applications including in food for humans, in animal feed, and as precursors for bio-based plastics, among others. However, the development of efficient chemical methods to convert abundant and renewable feedstocks into amino acids has been largely unsuccessful to date. To that end, here we report a heterogeneous catalyst that directly transforms lignocellulosic biomass-derived a-hydroxyl acids into a-amino acids, including alanine, leucine, valine, aspartic acid, and phenylalanine in high yields. The reaction follows a dehydrogenation-reductive amination pathway, with dehydrogenation as the rate-determining step. Ruthenium nanoparticles supportedmore » on carbon nanotubes (Ru/CNT) exhibit exceptional efficiency compared with catalysts based on other metals, due to the unique, reversible enhancement effect of NH 3 on Ru in dehydrogenation. Based on the catalytic system, a two-step chemical process was designed to convert glucose into alanine in 43% yield, comparable with the well-established microbial cultivation process, and therefore, the present strategy enables a route for the production of amino acids from renewable feedstocks. Moreover, a conceptual process design employing membrane distillation to facilitate product purification is proposed and validated. Overall, this study offers a rapid and potentially more efficient chemical method to produce amino acids from woody biomass components.« less

  18. Catalytic amino acid production from biomass-derived intermediates

    PubMed Central

    Deng, Weiping; Zhang, Sui; Gupta, Krishna M.; Hülsey, Max J.; Asakura, Hiroyuki; Liu, Lingmei; Han, Yu; Karp, Eric M.; Jiang, Jianwen; Tanaka, Tsunehiro; Wang, Ye

    2018-01-01

    Amino acids are the building blocks for protein biosynthesis and find use in myriad industrial applications including in food for humans, in animal feed, and as precursors for bio-based plastics, among others. However, the development of efficient chemical methods to convert abundant and renewable feedstocks into amino acids has been largely unsuccessful to date. To that end, here we report a heterogeneous catalyst that directly transforms lignocellulosic biomass-derived α-hydroxyl acids into α-amino acids, including alanine, leucine, valine, aspartic acid, and phenylalanine in high yields. The reaction follows a dehydrogenation-reductive amination pathway, with dehydrogenation as the rate-determining step. Ruthenium nanoparticles supported on carbon nanotubes (Ru/CNT) exhibit exceptional efficiency compared with catalysts based on other metals, due to the unique, reversible enhancement effect of NH3 on Ru in dehydrogenation. Based on the catalytic system, a two-step chemical process was designed to convert glucose into alanine in 43% yield, comparable with the well-established microbial cultivation process, and therefore, the present strategy enables a route for the production of amino acids from renewable feedstocks. Moreover, a conceptual process design employing membrane distillation to facilitate product purification is proposed and validated. Overall, this study offers a rapid and potentially more efficient chemical method to produce amino acids from woody biomass components. PMID:29712826

  19. Catalytic amino acid production from biomass-derived intermediates.

    PubMed

    Deng, Weiping; Wang, Yunzhu; Zhang, Sui; Gupta, Krishna M; Hülsey, Max J; Asakura, Hiroyuki; Liu, Lingmei; Han, Yu; Karp, Eric M; Beckham, Gregg T; Dyson, Paul J; Jiang, Jianwen; Tanaka, Tsunehiro; Wang, Ye; Yan, Ning

    2018-05-15

    Amino acids are the building blocks for protein biosynthesis and find use in myriad industrial applications including in food for humans, in animal feed, and as precursors for bio-based plastics, among others. However, the development of efficient chemical methods to convert abundant and renewable feedstocks into amino acids has been largely unsuccessful to date. To that end, here we report a heterogeneous catalyst that directly transforms lignocellulosic biomass-derived α-hydroxyl acids into α-amino acids, including alanine, leucine, valine, aspartic acid, and phenylalanine in high yields. The reaction follows a dehydrogenation-reductive amination pathway, with dehydrogenation as the rate-determining step. Ruthenium nanoparticles supported on carbon nanotubes (Ru/CNT) exhibit exceptional efficiency compared with catalysts based on other metals, due to the unique, reversible enhancement effect of NH 3 on Ru in dehydrogenation. Based on the catalytic system, a two-step chemical process was designed to convert glucose into alanine in 43% yield, comparable with the well-established microbial cultivation process, and therefore, the present strategy enables a route for the production of amino acids from renewable feedstocks. Moreover, a conceptual process design employing membrane distillation to facilitate product purification is proposed and validated. Overall, this study offers a rapid and potentially more efficient chemical method to produce amino acids from woody biomass components. Copyright © 2018 the Author(s). Published by PNAS.

  20. Catalytic amino acid production from biomass-derived intermediates

    DOE PAGES

    Deng, Weiping; Wang, Yunzhu; Zhang, Sui; ...

    2018-04-30

    Amino acids are the building blocks for protein biosynthesis and find use in myriad industrial applications including in food for humans, in animal feed, and as precursors for bio-based plastics, among others. However, the development of efficient chemical methods to convert abundant and renewable feedstocks into amino acids has been largely unsuccessful to date. To that end, here we report a heterogeneous catalyst that directly transforms lignocellulosic biomass-derived a-hydroxyl acids into a-amino acids, including alanine, leucine, valine, aspartic acid, and phenylalanine in high yields. The reaction follows a dehydrogenation-reductive amination pathway, with dehydrogenation as the rate-determining step. Ruthenium nanoparticles supportedmore » on carbon nanotubes (Ru/CNT) exhibit exceptional efficiency compared with catalysts based on other metals, due to the unique, reversible enhancement effect of NH 3 on Ru in dehydrogenation. Based on the catalytic system, a two-step chemical process was designed to convert glucose into alanine in 43% yield, comparable with the well-established microbial cultivation process, and therefore, the present strategy enables a route for the production of amino acids from renewable feedstocks. Moreover, a conceptual process design employing membrane distillation to facilitate product purification is proposed and validated. Overall, this study offers a rapid and potentially more efficient chemical method to produce amino acids from woody biomass components.« less

  1. Wavelet images and Chou's pseudo amino acid composition for protein classification.

    PubMed

    Nanni, Loris; Brahnam, Sheryl; Lumini, Alessandra

    2012-08-01

    The last decade has seen an explosion in the collection of protein data. To actualize the potential offered by this wealth of data, it is important to develop machine systems capable of classifying and extracting features from proteins. Reliable machine systems for protein classification offer many benefits, including the promise of finding novel drugs and vaccines. In developing our system, we analyze and compare several feature extraction methods used in protein classification that are based on the calculation of texture descriptors starting from a wavelet representation of the protein. We then feed these texture-based representations of the protein into an Adaboost ensemble of neural network or a support vector machine classifier. In addition, we perform experiments that combine our feature extraction methods with a standard method that is based on the Chou's pseudo amino acid composition. Using several datasets, we show that our best approach outperforms standard methods. The Matlab code of the proposed protein descriptors is available at http://bias.csr.unibo.it/nanni/wave.rar .

  2. LEAping to conclusions: a computational reanalysis of late embryogenesis abundant proteins and their possible roles.

    PubMed

    Wise, Michael J

    2003-10-29

    The late embryogenesis abundant (LEA) proteins cover a number of loosely related groups of proteins, originally found in plants but now being found in non-plant species. Their precise function is unknown, though considerable evidence suggests that LEA proteins are involved in desiccation resistance. Using a number of statistically-based bioinformatics tools the classification of a large set of LEA proteins, covering all Groups, is reexamined together with some previous findings. Searches based on peptide composition return proteins with similar composition to different LEA Groups; keyword clustering is then applied to reveal keywords and phrases suggestive of the Groups' properties. Previous research has suggested that glycine is characteristic of LEA proteins, but it is only highly over-represented in Groups 1 and 2, while alanine, thought characteristic of Group 2, is over-represented in Group 3, 4 and 6 but under-represented in Groups 1 and 2. However, for LEA Groups 1 2 and 3 it is shown that glutamine is very significantly over-represented, while cysteine, phenylalanine, isoleucine, leucine and tryptophan are significantly under-represented. There is also evidence that the Group 4 LEA proteins are more appropriately redistributed to Group 2 and Group 3. Similarly, Group 5 is better found among the Group 3 LEA proteins. There is evidence that Group 2 and Group 3 LEA proteins, though distinct, might be related. This relationship is also evident in the overlapping sets of keywords for the two Groups, emphasising alpha-helical structure and, at a larger scale, filaments, all of which fits well with experimental evidence that proteins from both Groups are natively unstructured, but become structured under stress conditions. The keywords support localisation of LEA proteins both in the nucleus and associated with the cytoskeleton, and a mode of action similar to chaperones, perhaps the cold shock chaperones, via a role in DNA-binding. In general, non-globular and

  3. Attenuation of the protein wasting associated with bed rest by branched-chain amino acids

    NASA Technical Reports Server (NTRS)

    Stein, T. P.; Schluter, M. D.; Leskiw, M. J.; Boden, G.

    1999-01-01

    Bed rest is generally accepted as being an appropriate ground-based model for human spaceflight. The objectives of this study were to test the hypothesis that increasing the amount of branched-chain amino acids (BCAAs) in the diet could attenuate the protein loss associated with bed rest. Nineteen healthy subjects were randomized into two groups according to diet. During the 6 d of bed rest, the diets were supplemented with either 30 mmol/d each of three non-essential amino acids, glycine, serine, and alanine (control group), or with 30 mmol/d each of the BCAAs, leucine, isoleucine, and valine (BCAA group). Nutrition was supplied as a commercially available defined formula diet at a rate of 1.3 x REE. Nitrogen (N) balance and urinary 3-MeH excretion were determined for the 6 d. In our results, the urine-based estimate of N balance was 22.2 +/- 14.4 (n = 9) mg N.kg-1.d-1 and 60.5 +/- 10.1 mg (n = 8) N.kg-1.d-1 for the control and BCAA-supplemented groups, respectively (P < 0.05). Urinary 3-MeH excretion was unchanged in both groups with bed rest. We conclude that BCAA supplementation attenuates the N loss during short-term bed rest.

  4. Maternal protein restriction in the rat inhibits placental insulin, mTOR, and STAT3 signaling and down-regulates placental amino acid transporters.

    PubMed

    Rosario, Fredrick J; Jansson, Nina; Kanai, Yoshikatsu; Prasad, Puttur D; Powell, Theresa L; Jansson, Thomas

    2011-03-01

    The mechanisms underlying reduced fetal growth in response to maternal protein restriction are not well established. Maternal levels of insulin, IGF-I, and leptin are decreased in rats fed a low protein (LP) diet. Because these hormones stimulate placental amino acid transporters in vitro, we hypothesized that maternal protein restriction inhibits placental leptin, insulin/IGF-I, and mammalian target of rapamycin signaling and down-regulates the expression and activity of placental amino acid transporters. Pregnant rats were fed either an isocaloric low protein (LP, 4% protein) or control diet (18% protein) and studied at gestational day (GD)15, GD19, or GD21 (term 23). At GD19 and GD21, placental expression of phosphorylated eukaryotic initiation factor 4E binding protein 1 (Thr-36/46 or Thr-70) and phosphorylated S6 ribosomal protein (Ser-235/236) was decreased in the LP group. In addition, placental expression of phosphorylated S6 kinase 1 (Thr-389), phosphorylated Akt (Thr-308), and phosphorylated signal transducer and activator of transcription 3 (Tyr-705) was reduced at GD21. In microvillous plasma membranes (MVM) isolated from placentas of LP animals, protein expression of the sodium-coupled neutral amino acid transporter (SNAT)2 and the large neutral amino acid transporters 1 and 2 was reduced at GD19 and GD21. MVM SNAT1 protein expression was reduced at GD21 in LP rats. SNAT4 and 4F2 heavy chain expression in MVM was unaltered. System A and L amino acid transporter activity was decreased in MVM from LP animals at GD19 and GD21. In conclusion, maternal protein restriction inhibits placental insulin, mammalian target of rapamycin signaling, and signal transducer and activator of transcription 3 signaling, which is associated with a down-regulation of placental amino acid transporters. We speculate that maternal endocrine and metabolic control of placental nutrient transport reduces fetal growth in response to protein restriction.

  5. Proteomics-based identification of differentially abundant proteins reveals adaptation mechanisms of Xanthomonas citri subsp. citri during Citrus sinensis infection.

    PubMed

    Moreira, Leandro M; Soares, Márcia R; Facincani, Agda P; Ferreira, Cristiano B; Ferreira, Rafael M; Ferro, Maria I T; Gozzo, Fábio C; Felestrino, Érica B; Assis, Renata A B; Garcia, Camila Carrião M; Setubal, João C; Ferro, Jesus A; de Oliveira, Julio C F

    2017-07-11

    Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus canker. A proteomic analysis under in planta infectious and non-infectious conditions was conducted in order to increase our knowledge about the adaptive process of Xac during infection. For that, a 2D-based proteomic analysis of Xac at 1, 3 and 5 days after inoculation, in comparison to Xac growth in NB media was carried out and followed by MALDI-TOF-TOF identification of 124 unique differentially abundant proteins. Among them, 79 correspond to up-regulated proteins in at least one of the three stages of infection. Our results indicate an important role of proteins related to biofilm synthesis, lipopolysaccharides biosynthesis, and iron uptake and metabolism as possible modulators of plant innate immunity, and revealed an intricate network of proteins involved in reactive oxygen species adaptation during Plants` Oxidative Burst response. We also identified proteins previously unknown to be involved in Xac-Citrus interaction, including the hypothetical protein XAC3981. A mutant strain for this gene has proved to be non-pathogenic in respect to classical symptoms of citrus canker induced in compatible plants. This is the first time that a protein repertoire is shown to be active and working in an integrated manner during the infection process in a compatible host, pointing to an elaborate mechanism for adaptation of Xac once inside the plant.

  6. Dietary supplementation with an amino acid blend enhances intestinal function in piglets.

    PubMed

    Yi, Dan; Li, Baocheng; Hou, Yongqing; Wang, Lei; Zhao, Di; Chen, Hongbo; Wu, Tao; Zhou, Ying; Ding, Binying; Wu, Guoyao

    2018-05-16

    The traditionally classified nutritionally non-essential amino acids are now known to be insufficiently synthesized for maximal growth and optimal health in piglets. This study determined the effects of dietary supplementation with an amino acid blend (AAB; glutamate:glutamine:glycine:arginine:N-acetylcysteine = 5:2:2:1:0.5) on piglet growth performance and intestinal functions. Sixteen piglets (24-day-old) were randomly assigned to a corn and soybean meal-based diet supplemented with 0.99% alanine (isonitrogenous control) or 1% AAB. On day 20 of the trial, blood and intestinal tissue samples were obtained from piglets. Compared with the control, AAB supplementation reduced (P < 0.05) diarrhoea incidence; plasma alanine aminotransferase and diamine oxidase activities; intestinal concentrations of hydrogen peroxide, malondialdehyde, and heat shock protein-70, and intestinal mRNA levels for interleukin-1β, interferon-γ, and chemokine (C-X-C motif) ligand-9; and the numbers of Enterobacterium family, Enterococcus genus and Clostridium coccoides in the colon digesta. Furthermore, AAB supplementation enhanced (P < 0.05): the plasma concentrations of serine, aspartate, glutamate, cysteine, tyrosine, phenylalanine, tryptophan, lysine, arginine, citrulline, ornithine, taurine, and γ-aminobutyric acid; intestinal villus height and surface area, villus height/crypt depth ratio, antioxidative enzyme activities, and mRNA levels for porcine β-defensin-1, sodium-independent amino acid transporters (b 0,+ AT and y + LAT1), aquaporin (AQP) 3, AQP8, AQP10, nuclear factor erythroid 2-related factor 2 and glutathione S-transferase omega-2, and protein abundances of AQP3, AQP4, claudin-1, occludin and myxovirus resistance 1; and the numbers of Bifidobacterium genus and Lactobacillus genus in the colon digesta. Collectively, these comprehensive results indicate that dietary AAB supplementation plays an important role in improving piglet growth and intestinal function.

  7. Effects of copper and tributyltin on stress protein abundance in the rotifer Brachionus plicatilis.

    PubMed

    Cochrane, B J; Irby, R B; Snell, T W

    1991-01-01

    1. Exposure of the rotifer Brachionus plicatilis to elevated temperature resulted in the synthesis of a number of proteins, including a prominent one of 58,000 Da (SP58). 2. This protein is immunologically crossreactive with the 65,000 Da heat shock protein of the moth Heliothis virescens, which is a member of a highly conserved family of mitochondrial proteins. 3. Exposure of rotifers to sublethal doses of CuSO4 leads to a 4-5-fold increase in abundance of SP58, with maximum increase occurring at a dose that is approximately 5% of the LC50 for that compound. 4. A similar response was seen with tributyl tin (TBT). Kinetics of induction were sigmoidal, with induction occurring in the range of 20-30 micrograms/l. 5. No response was observed when rotifers were exposed to aluminum chloride, mercury chloride, pentachlorophenol, sodium arsenite, sodium azide, sodium dodecyl sulfate, or zinc chloride. 6. These results indicate that changes in stress protein abundance may prove useful as a biomarker of exposure to particular toxicants.

  8. Mass-spectrometric identification of primary biological particle markers: indication for low abundance of primary biological material in the pristine submicron aerosol of Amazonia

    NASA Astrophysics Data System (ADS)

    Schneider, J.; Freutel, F.; Zorn, S. R.; Chen, Q.; Farmer, D. K.; Jimenez, J. L.; Martin, S. T.; Artaxo, P.; Wiedensohler, A.; Borrmann, S.

    2011-07-01

    The abundance of marker compounds for primary biological particles in submicron aerosol was investigated by means of aerosol mass spectrometry. Mass spectra of amino acids, carbohydrates, small peptides, and proteins, all of which are key building blocks of biological particles, were recorded in laboratory experiments. Several characteristic marker peaks were identified. The identified marker peaks were compared with mass spectra recorded during AMAZE-08, a field campaign conducted in the pristine rainforest of the Central Amazon Basin, Brazil, during the wet season of February and March 2008. The low abundance of identified marker peaks places upper limits of 7.5 % for amino acids and 5.6 % for carbohydrates on the contribution of primary biological aerosol particles (PBAPs) to the submicron organic aerosol mass concentration during this time period. Upper limits for the absolute submicron concentrations for both compound classes range from 0.01 to 0.1 μg m-3. Carbohydrates and protein amino acids make up for about two thirds of the dry mass of a biological cell. Thus, our findings suggest an upper limit for the PBAPs mass fraction of about 20 % to the submicron organic aerosol.

  9. The effect of thermal processing on protein quality and free amino acid profile of Terminalia catappa (Indian Almond) seed.

    PubMed

    Adu, O B; Ogundeko, T O; Ogunrinola, O O; Saibu, G M; Elemo, B O

    2015-07-01

    The study examined the effect of various processing methods- boiling, drying and roasting- on the in vitro and in vivo protein digestibility and free amino acid profiles of Terminalia catappa seed. Moisture and crude protein of the various samples were determined. In vitro protein digestibility was determined after pepsin digestion. For the in vivo experiment, defatted T. catappa based diet was fed to 3 weeks old Wistar rats for 4 weeks and compared with animals maintained on casein based and nitrogen- free diets. The biological value (BV), net protein utilisation (NPU) and protein efficiency ratio (PER) of the diets were determined. Free amino acid composition was carried out using thin layer chromatography. Moisture was highest in the boiled T. catappa seed (8.30 ± 0.00 %). The raw, roasted and dried seeds had 5.55 ± 0.07, 3.88 ± 0.22 and 3.75 ± 0.07 % respectively. Crude protein was 19.19, 18.89, 17.62 and 16.36 % in the dried, roasted, boiled and raw seeds respectively. Roasted T. catappa seed had the highest in vitro protein digestibility with 37.52 %, while the dried, boiled and raw samples had digestibility values of 27.57, 27.07 and 24.45 % respectively. All nine essential amino acids were present in T. catappa in high concentrations except methionine and tryptophan. Glutamate was present in the highest concentration. Also, free amino acids were higher in the processed seeds compared to the raw seed. Animals fed T. catappa diet compared favourably with the casein group, thus indicating that the protein is of good quality.

  10. Protein degradation rate is the dominant mechanism accounting for the differences in protein abundance of basal p53 in a human breast and colorectal cancer cell line.

    PubMed

    Lakatos, Eszter; Salehi-Reyhani, Ali; Barclay, Michael; Stumpf, Michael P H; Klug, David R

    2017-01-01

    We determine p53 protein abundances and cell to cell variation in two human cancer cell lines with single cell resolution, and show that the fractional width of the distributions is the same in both cases despite a large difference in average protein copy number. We developed a computational framework to identify dominant mechanisms controlling the variation of protein abundance in a simple model of gene expression from the summary statistics of single cell steady state protein expression distributions. Our results, based on single cell data analysed in a Bayesian framework, lends strong support to a model in which variation in the basal p53 protein abundance may be best explained by variations in the rate of p53 protein degradation. This is supported by measurements of the relative average levels of mRNA which are very similar despite large variation in the level of protein.

  11. Pairwise amino acid secondary structural propensities

    NASA Astrophysics Data System (ADS)

    Chemmama, Ilan E.; Chapagain, Prem P.; Gerstman, Bernard S.

    2015-04-01

    We investigate the propensities for amino acids to form a specific secondary structure when they are paired with other amino acids. Our investigations use molecular dynamics (MD) computer simulations, and we compare the results to those from the Protein Data Bank (PDB). Proper comparison requires weighting of the MD results in a manner consistent with the relative frequency of appearance in the PDB of each possible pair of amino acids. We find that the propensity for an amino acid to assume a secondary structure varies dramatically depending on the amino acid that is before or after it in the primary sequence. This cooperative effect means that when selecting amino acids to facilitate the formation of a secondary structure in peptide engineering experiments, the adjacent amino acids must be considered. We also examine the preference for a secondary structure in bacterial proteins and compare the results to those of human proteins.

  12. Amino Acids and Chirality

    NASA Technical Reports Server (NTRS)

    Cook, Jamie E.

    2012-01-01

    Amino acids are among the most heavily studied organic compound class in carbonaceous chondrites. The abundance, distributions, enantiomeric compositions, and stable isotopic ratios of amino acids have been determined in carbonaceous chondrites fi'om a range of classes and petrographic types, with interesting correlations observed between these properties and the class and typc of the chondritcs. In particular, isomeric distributions appear to correlate with parent bodies (chondrite class). In addition, certain chiral amino acids are found in enantiomeric excess in some chondrites. The delivery of these enantiomeric excesses to the early Earth may have contributed to the origin of the homochirality that is central to life on Earth today. This talk will explore the amino acids in carbonaceous chondritcs and their relevance to the origin of life.

  13. Sulphur-containing Amino Acids: Protective Role Against Free Radicals and Heavy Metals.

    PubMed

    Colovic, Mirjana B; Vasic, Vesna M; Djuric, Dragan M; Krstic, Danijela Z

    2018-01-30

    Sulphur is an abundant element in biological systems, which plays an important role in processes essential for life as a constituent of proteins, vitamins and other crucial biomolecules. The major source of sulphur for humans is plants being able to use inorganic sulphur in the purpose of sulphur-containing amino acids synthesis. Sulphur-containing amino acids include methionine, cysteine, homocysteine, and taurine. Methionine and cysteine are classified as proteinogenic, canonic amino acids incorporated in protein structure. Sulphur amino acids are involved in the synthesis of intracellular antioxidants such as glutathione and N-acetyl cysteine. Moreover, naturally occurring sulphur-containing ligands are effective and safe detoxifying agents, often used in order to prevent toxic metal ions effects and their accumulation in human body. Literature search for peer-reviewed articles was performed using PubMed and Scopus databases, and utilizing appropriate keywords. This review is focused on sulphur-containing amino acids - methionine, cysteine, taurine, and their derivatives - glutathione and N-acetylcysteine, and their defense effects as antioxidant agents against free radicals. Additionally, the protective effects of sulphur-containing ligands against the toxic effects of heavy and transition metal ions, and their reactivation role towards the enzyme inhibition are described. Sulphur-containing amino acids represent a powerful part of cell antioxidant system. Thus, they are essential in the maintenance of normal cellular functions and health. In addition to their worthy antioxidant action, sulphur-containing amino acids may offer a chelating site for heavy metals. Accordingly, they may be supplemented during chelating therapy, providing beneficial effects in eliminating toxic metals. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  14. Amino Acid Composition of Protein-Enriched Dried Pasta:
Is It Suitable for a Low-Carbohydrate Diet?

    PubMed

    Filip, Sebastjan; Vidrih, Rajko

    2015-09-01

    Today, obesity is one of the major health problems, a so-called epidemic of the developed world. Obesity arises through an imbalance between energy intake and energy expenditure, so it is important for products to have a balanced nutritional composition. The aim of this study is to prepare high-protein pasta with high nutritional quality, with emphasis on its amino acid composition, as ordinary durum pasta lacks lysine and threonine. Ordinary durum wheat pasta contains, on average, 77% carbohydrate, and can have even less than 10% protein. It is therefore often excluded from normal energy-restricted diets, and especially from low-carbohydrate diets. In this study pasta that can satisfy the nutritional requirements of a low-carbohydrate diet and is suitable for daily use was developed and evaluated. Protein-enhanced pasta was produced by adding high amounts of plant protein extract (40% dry matter) without (plain high-protein pasta) or with 3% dried spinach powder (high-protein spinach pasta) to durum wheat semolina. According to the sensory analysis data, the addition of 40% of plant protein extract satisfied sensory and nutritional requirements, allowing further development and evaluation for possible marketing. This analysis shows that these high-protein neutral and spinach pasta contain 36.4 and 39.6 g of protein per 100 g of dry mass, 12.07 and 14.70 g of total essential amino acids per 100 g of dry mass, and a high content of branched-chain amino acids, i.e. 5.54 and 6.65 g per 100 g of dry mass, respectively. This therefore represents a true alternative to durum wheat pasta for low-carbohydrate diets.

  15. Amino Acid Composition of Protein-Enriched Dried Pasta:
Is It Suitable for a Low-Carbohydrate Diet?

    PubMed Central

    Vidrih, Rajko

    2015-01-01

    Summary Today, obesity is one of the major health problems, a so-called epidemic of the developed world. Obesity arises through an imbalance between energy intake and energy expenditure, so it is important for products to have a balanced nutritional composition. The aim of this study is to prepare high-protein pasta with high nutritional quality, with emphasis on its amino acid composition, as ordinary durum pasta lacks lysine and threonine. Ordinary durum wheat pasta contains, on average, 77% carbohydrate, and can have even less than 10% protein. It is therefore often excluded from normal energy-restricted diets, and especially from low-carbohydrate diets. In this study pasta that can satisfy the nutritional requirements of a low-carbohydrate diet and is suitable for daily use was developed and evaluated. Protein-enhanced pasta was produced by adding high amounts of plant protein extract (40% dry matter) without (plain high-protein pasta) or with 3% dried spinach powder (high-protein spinach pasta) to durum wheat semolina. According to the sensory analysis data, the addition of 40% of plant protein extract satisfied sensory and nutritional requirements, allowing further development and evaluation for possible marketing. This analysis shows that these high-protein neutral and spinach pasta contain 36.4 and 39.6 g of protein per 100 g of dry mass, 12.07 and 14.70 g of total essential amino acids per 100 g of dry mass, and a high content of branched-chain amino acids, i.e. 5.54 and 6.65 g per 100 g of dry mass, respectively. This therefore represents a true alternative to durum wheat pasta for low-carbohydrate diets. PMID:27904361

  16. Relative and accurate measurement of protein abundance using 15N stable isotope labeling in Arabidopsis (SILIA).

    PubMed

    Guo, Guangyu; Li, Ning

    2011-07-01

    In the quantitative proteomic studies, numerous in vitro and in vivo peptide labeling strategies have been successfully applied to measure differentially regulated protein and peptide abundance. These approaches have been proven to be versatile and repeatable in biological discoveries. (15)N metabolic labeling is one of these widely adopted and economical methods. However, due to the differential incorporation rates of (15)N or (14)N, the labeling results produce imperfectly matched isotopic envelopes between the heavy and light nitrogen-labeled peptides. In the present study, we have modified the solid Arabidopsis growth medium to standardize the (15)N supply, which led to a uniform incorporation of (15)N into the whole plant protein complement. The incorporation rate (97.43±0.11%) of (15)N into (15)N-coded peptides was determined by correlating the intensities of peptide ions with the labeling efficiencies according to Gaussian distribution. The resulting actual incorporation rate (97.44%) and natural abundance of (15)N/(14)N-coded peptides are used to re-calculate the intensities of isotopic envelopes of differentially labeled peptides, respectively. A modified (15)N/(14)N stable isotope labeling strategy, SILIA, is assessed and the results demonstrate that this approach is able to differentiate the fold change in protein abundance down to 10%. The machine dynamic range limitation and purification step will make the precursor ion ratio deriving from the actual ratio fold change. It is suggested that the differentially mixed (15)N-coded and (14)N-coded plant protein samples that are used to establish the protein abundance standard curve should be prepared following a similar protein isolation protocol used to isolate the proteins to be quantitated. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  17. Determination of the D and L isomers of some protein amino acids present in soils

    NASA Technical Reports Server (NTRS)

    Pollock, G. E.; Cheng, C.-N.; Cronin, S. E.

    1977-01-01

    The D and L isomers of some protein amino acids present in soils were measured by using a gas chromatographic technique. The results of two processing procedures were compared to determine the better method. Results of the comparison indicated that the determination of D and L percentages requires amino acid purification if one is to obtain accurate data. It was found that very significant amounts of D-alanine, D-aspartic acid, and D-glutamic acid were present in the contemporary soils studied. Valine, isoleucine, leucine, proline, and phenylalanine generally contained only a trace to very small amounts of the D isomer. It is probable that the D-amino acids from the alanine, aspartic, and glutamic acids are contributed to the soil primarily via microorganisms. The finding of very significant quantities of some D-amino acids (about 5-16%) in present-day soils may alert some investigators of geological sediments to a possible problem in using amino acid racemization as an age-dating technique.

  18. Correlated Amino Acid and Mineralogical Analyses of Milligram and Submilligram Samples of Carbonaceous Chondrite Lonewolf Nunataks 94101

    NASA Technical Reports Server (NTRS)

    Burton, S.; Berger, E. L.; Locke, D. R.; Lewis, E. K.

    2018-01-01

    Amino acids, the building blocks of proteins, have been found to be indigenous in the eight carbonaceous chondrite groups. The abundances, structural, enantiomeric and isotopic compositions of amino acids differ significantly among meteorites of different groups and petrologic types. These results suggest parent-body conditions (thermal or aqueous alteration), mineralogy, and the preservation of amino acids are linked. Previously, elucidating specific relationships between amino acids and mineralogy was not possible because the samples analyzed for amino acids were much larger than the scale at which petrologic heterogeneity is observed (sub mm-scale differences corresponding to sub-mg samples); for example, Pizzarello and coworkers measured amino acid abundances and performed X-ray diffraction (XRD) on several samples of the Murchison meteorite, but these analyses were performed on bulk samples that were 500 mg or larger. Advances in the sensitivity of amino acid measurements by liquid chromatography with fluorescence detection/time-of-flight mass spectrometry (LC-FD/TOF-MS), and application of techniques such as high resolution X-ray diffraction (HR-XRD) and scanning electron microscopy (SEM) with energy dispersive spectroscopy (EDS) for mineralogical characterizations have now enabled coordinated analyses on the scale at which mineral heterogeneity is observed. In this work, we have analyzed samples of the Lonewolf Nunataks (LON) 94101 CM2 carbonaceous chondrite. We are investigating the link(s) between parent body processes, mineralogical context, and amino acid compositions in meteorites on bulk samples (approx. 20mg) and mineral separates (< or = 3mg) from several of spatial locations within our allocated samples. Preliminary results of these analyses are presented here.

  19. Degradation of Amino Acids and Structure in Model Proteins and Bacteriophage MS2 by Chlorine, Bromine, and Ozone.

    PubMed

    Choe, Jong Kwon; Richards, David H; Wilson, Corey J; Mitch, William A

    2015-11-17

    Proteins are important targets of chemical disinfectants. To improve the understanding of disinfectant-protein reactions, this study characterized the disinfectant:protein molar ratios at which 50% degradation of oxidizable amino acids (i.e., Met, Tyr, Trp, His, Lys) and structure were observed during HOCl, HOBr, and O3 treatment of three well-characterized model proteins and bacteriophage MS2. A critical question is the extent to which the targeting of amino acids is driven by their disinfectant rate constants rather than their geometrical arrangement. Across the model proteins and bacteriophage MS2 (coat protein), differing widely in structure, methionine was preferentially targeted, forming predominantly methionine sulfoxide. This targeting concurs with its high disinfectant rate constants and supports its hypothesized role as a sacrificial antioxidant. Despite higher HOCl and HOBr rate constants with histidine and lysine than for tyrosine, tyrosine generally was degraded in preference to histidine, and to a lesser extent, lysine. These results concur with the prevalence of geometrical motifs featuring histidines or lysines near tyrosines, facilitating histidine and lysine regeneration upon Cl[+1] transfer from their chloramines to tyrosines. Lysine nitrile formation occurred at or above oxidant doses where 3,5-dihalotyrosine products began to degrade. For O3, which lacks a similar oxidant transfer pathway, histidine, tyrosine, and lysine degradation followed their relative O3 rate constants. Except for its low reactivity with lysine, the O3 doses required to degrade amino acids were as low as or lower than for HOCl or HOBr, indicating its oxidative efficiency. Loss of structure did not correlate with loss of particular amino acids, suggesting the need to characterize the oxidation of specific geometric motifs to understand structural degradation.

  20. Long-term cadmium exposure influences the abundance of proteins that impact the cell wall structure in medicago sativa stems.

    PubMed

    Gutsch, Annelie; Keunen, Els; Guerriero, Gea; Renaut, Jenny; Cuypers, Ann; Hausman, Jean-François; Sergeant, Kjell

    2018-06-15

    Cadmium (Cd) is a non-essential, toxic heavy metal that poses serious threats to both the ecosystem and the health of humans. Plants employ various cellular and molecular mechanisms to minimize the impact of Cd toxicity and the cell walls function as defensive barrier during Cd exposure. In this study, we adopted a quantitative gel-based proteomic approach (two-dimensional difference gel electrophoresis) to investigate changes in the abundance of cell wall- and soluble proteins in stems of Medicago sativa L. upon long-term exposure to Cd (at 10 mg Cd per kg soil as CdSO 4 ). Obtained protein data were complemented with targeted gene expression analyses. Plants were affected by Cd exposure at an early growth stage but seemed to recover at a more mature plant stage as no difference in biomass was observed. The accumulation of Cd was highest in the roots followed by stems and leaves. Quantitative proteomics revealed a changed abundance for 179 cell wall proteins and 30 proteins in the soluble fraction upon long-term Cd exposure. These proteins are involved in cell wall remodeling, defense response, carbohydrate metabolism and promotion of the lignification process. The data indicate that Cd exposure alters the cell wall proteome and underline the role of cell wall proteins in defense against Cd stress. The identified proteins are linked to alterations in the cell wall structure and lignification process in stems of M. sativa, underpinning the function of the cell wall as an effective barrier against Cd stress. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  1. Incorporating significant amino acid pairs and protein domains to predict RNA splicing-related proteins with functional roles

    NASA Astrophysics Data System (ADS)

    Hsu, Justin Bo-Kai; Huang, Kai-Yao; Weng, Tzu-Ya; Huang, Chien-Hsun; Lee, Tzong-Yi

    2014-01-01

    Machinery of pre-mRNA splicing is carried out through the interaction of RNA sequence elements and a variety of RNA splicing-related proteins (SRPs) (e.g. spliceosome and splicing factors). Alternative splicing, which is an important post-transcriptional regulation in eukaryotes, gives rise to multiple mature mRNA isoforms, which encodes proteins with functional diversities. However, the regulation of RNA splicing is not yet fully elucidated, partly because SRPs have not yet been exhaustively identified and the experimental identification is labor-intensive. Therefore, we are motivated to design a new method for identifying SRPs with their functional roles in the regulation of RNA splicing. The experimentally verified SRPs were manually curated from research articles. According to the functional annotation of Splicing Related Gene Database, the collected SRPs were further categorized into four functional groups including small nuclear Ribonucleoprotein, Splicing Factor, Splicing Regulation Factor and Novel Spliceosome Protein. The composition of amino acid pairs indicates that there are remarkable differences among four functional groups of SRPs. Then, support vector machines (SVMs) were utilized to learn the predictive models for identifying SRPs as well as their functional roles. The cross-validation evaluation presents that the SVM models trained with significant amino acid pairs and functional domains could provide a better predictive performance. In addition, the independent testing demonstrates that the proposed method could accurately identify SRPs in mammals/plants as well as effectively distinguish between SRPs and RNA-binding proteins. This investigation provides a practical means to identifying potential SRPs and a perspective for exploring the regulation of RNA splicing.

  2. Incorporating significant amino acid pairs and protein domains to predict RNA splicing-related proteins with functional roles.

    PubMed

    Hsu, Justin Bo-Kai; Huang, Kai-Yao; Weng, Tzu-Ya; Huang, Chien-Hsun; Lee, Tzong-Yi

    2014-01-01

    Machinery of pre-mRNA splicing is carried out through the interaction of RNA sequence elements and a variety of RNA splicing-related proteins (SRPs) (e.g. spliceosome and splicing factors). Alternative splicing, which is an important post-transcriptional regulation in eukaryotes, gives rise to multiple mature mRNA isoforms, which encodes proteins with functional diversities. However, the regulation of RNA splicing is not yet fully elucidated, partly because SRPs have not yet been exhaustively identified and the experimental identification is labor-intensive. Therefore, we are motivated to design a new method for identifying SRPs with their functional roles in the regulation of RNA splicing. The experimentally verified SRPs were manually curated from research articles. According to the functional annotation of Splicing Related Gene Database, the collected SRPs were further categorized into four functional groups including small nuclear Ribonucleoprotein, Splicing Factor, Splicing Regulation Factor and Novel Spliceosome Protein. The composition of amino acid pairs indicates that there are remarkable differences among four functional groups of SRPs. Then, support vector machines (SVMs) were utilized to learn the predictive models for identifying SRPs as well as their functional roles. The cross-validation evaluation presents that the SVM models trained with significant amino acid pairs and functional domains could provide a better predictive performance. In addition, the independent testing demonstrates that the proposed method could accurately identify SRPs in mammals/plants as well as effectively distinguish between SRPs and RNA-binding proteins. This investigation provides a practical means to identifying potential SRPs and a perspective for exploring the regulation of RNA splicing.

  3. Predicting network modules of cell cycle regulators using relative protein abundance statistics.

    PubMed

    Oguz, Cihan; Watson, Layne T; Baumann, William T; Tyson, John J

    2017-02-28

    Parameter estimation in systems biology is typically done by enforcing experimental observations through an objective function as the parameter space of a model is explored by numerical simulations. Past studies have shown that one usually finds a set of "feasible" parameter vectors that fit the available experimental data equally well, and that these alternative vectors can make different predictions under novel experimental conditions. In this study, we characterize the feasible region of a complex model of the budding yeast cell cycle under a large set of discrete experimental constraints in order to test whether the statistical features of relative protein abundance predictions are influenced by the topology of the cell cycle regulatory network. Using differential evolution, we generate an ensemble of feasible parameter vectors that reproduce the phenotypes (viable or inviable) of wild-type yeast cells and 110 mutant strains. We use this ensemble to predict the phenotypes of 129 mutant strains for which experimental data is not available. We identify 86 novel mutants that are predicted to be viable and then rank the cell cycle proteins in terms of their contributions to cumulative variability of relative protein abundance predictions. Proteins involved in "regulation of cell size" and "regulation of G1/S transition" contribute most to predictive variability, whereas proteins involved in "positive regulation of transcription involved in exit from mitosis," "mitotic spindle assembly checkpoint" and "negative regulation of cyclin-dependent protein kinase by cyclin degradation" contribute the least. These results suggest that the statistics of these predictions may be generating patterns specific to individual network modules (START, S/G2/M, and EXIT). To test this hypothesis, we develop random forest models for predicting the network modules of cell cycle regulators using relative abundance statistics as model inputs. Predictive performance is assessed by the

  4. Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats

    PubMed Central

    Pelassa, Ilaria; Fiumara, Ferdinando

    2015-01-01

    Homopolymeric amino acids repeats (AARs), which are widespread in proteomes, have often been viewed simply as spacers between protein domains, or even as “junk” sequences with no obvious function but with a potential to cause harm upon expansion as in genetic diseases associated with polyglutamine or polyalanine expansions, including Huntington disease and cleidocranial dysplasia. A growing body of evidence indicates however that at least some AARs can form organized, functional protein structures, and can regulate protein function. In particular, certain AARs can mediate protein-protein interactions, either through homotypic AAR-AAR contacts or through heterotypic contacts with other protein domains. It is still unclear however, whether AARs may have a generalized, proteome-wide role in shaping protein-protein interaction networks. Therefore, we have undertaken here a bioinformatics screening of the human proteome and interactome in search of quantitative evidence of such a role. We first identified the sets of proteins that contain repeats of any one of the 20 amino acids, as well as control sets of proteins chosen at random in the proteome. We then analyzed the connectivity between the proteins of the AAR-containing protein sets and we compared it with that observed in the corresponding control networks. We find evidence for different degrees of connectivity in the different AAR-containing protein networks. Indeed, networks of proteins containing polyglutamine, polyglutamate, polyproline, and other AARs show significantly increased levels of connectivity, whereas networks containing polyleucine and other hydrophobic repeats show lower degrees of connectivity. Furthermore, we observed that numerous protein-protein, -nucleic acid, and -lipid interaction domains are significantly enriched in specific AAR protein groups. These findings support the notion of a generalized, combinatorial role of AARs, together with conventional protein interaction domains, in

  5. Abscisic acid-regulated protein degradation causes osmotic stress-induced accumulation of branched-chain amino acids in Arabidopsis thaliana.

    PubMed

    Huang, Tengfang; Jander, Georg

    2017-10-01

    Whereas proline accumulates through de novo biosynthesis in plants subjected to osmotic stress, leucine, isoleucine, and valine accumulation in drought-stressed Arabidopsis thaliana is caused by abscisic acid-regulated protein degradation. In response to several kinds of abiotic stress, plants greatly increase their accumulation of free amino acids. Although stress-induced proline increases have been studied the most extensively, the fold-increase of other amino acids, in particular branched-chain amino acids (BCAAs; leucine, isoleucine, and valine), is often higher than that of proline. In Arabidopsis thaliana (Arabidopsis), BCAAs accumulate in response to drought, salt, mannitol, polyethylene glycol, herbicide treatment, and nitrogen starvation. Plants that are deficient in abscisic acid signaling accumulate lower amounts of BCAAs, but not proline and most other amino acids. Previous bioinformatic studies had suggested that amino acid synthesis, rather than protein degradation, is responsible for the observed BCAA increase in osmotically stressed Arabidopsis. However, whereas treatment with the protease inhibitor MG132 decreased drought-induced BCAA accumulation, inhibition of BCAA biosynthesis with the acetolactate synthase inhibitors chlorsulfuron and imazapyr did not. Additionally, overexpression of BRANCHED-CHAIN AMINO ACID TRANSFERASE2 (BCAT2), which is upregulated in response to osmotic stress and functions in BCAA degradation, decreased drought-induced BCAA accumulation. Together, these results demonstrate that BCAA accumulation in osmotically stressed Arabidopsis is primarily the result of protein degradation. After relief of the osmotic stress, BCAA homeostasis is restored over time by amino acid degradation involving BCAT2. Thus, drought-induced BCAA accumulation is different from that of proline, which is accumulated due to de novo synthesis in an abscisic acid-independent manner and remains elevated for a more prolonged period of time after removal of

  6. The Curious Case of NH_2OH: Hunting a Direct Amino Acid Precursor Species in the Interstellar Medium

    NASA Astrophysics Data System (ADS)

    McGuire, Brett A.; Carroll, Brandon; Dollhopf, Niklaus M.; Crockett, Nathan; Blake, Geoffrey; Remijan, Anthony

    2015-06-01

    Despite the detection of amino acids, the building blocks of the proteins that support life, in cometary and meteoritic samples, we do not yet understand the conditions under which these life-essential species have formed. Hydroxylamine (NH_2OH) is potentially a direct precursor to the formation of the amino acids glycine and alanine in the ISM, through reaction with acetic and propionic acids. Recent laboratory and modeling work has shown that there are a variety of pathways to the formation of NH_2OH in interstellar ices both efficiently and in high abundance. Here, we present the result of a deep, multi-telescope search for NH_2OH in the shocked, complex molecular source L1157. We find no evidence suggesting the presence of this important precursor, and discuss the implications of this non-detection on the reactivity of NH_2OH both within the ices, and in the gas-phase ISM. We will also discuss how these observations should inform the direction of future studies, both in the laboratory and with state-of-the-art telescopes such as ALMA.

  7. Mutations in y-aminobutyric acid (GABA) transaminase genes in plants or Pseudomonas syringae reduce bacterial virulence

    USDA-ARS?s Scientific Manuscript database

    Pseudomonas syringae pv. tomato DC3000 is a bacterial pathogen of Arabidopsis and tomato that grows in the apoplast. The non-protein amino acid '-amino butyric acid (GABA) is produced by Arabidopsis and tomato and is the most abundant amino acid in the apoplastic fluid of tomato. The DC3000 genome h...

  8. Multiple marker abundance profiling: combining selected reaction monitoring and data-dependent acquisition for rapid estimation of organelle abundance in subcellular samples

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hooper, Cornelia M.; Stevens, Tim J.; Saukkonen, Anna

    Measuring changes in protein or organelle abundance in the cell is an essential, but challenging aspect of cell biology. Frequently-used methods for determining organelle abundance typically rely on detection of a very few marker proteins, so are unsatisfactory. In silico estimates of protein abundances from publicly available protein spectra can provide useful standard abundance values but contain only data from tissue proteomes, and are not coupled to organelle localization data. A new protein abundance score, the normalized protein abundance scale (NPAS), expands on the number of scored proteins and the scoring accuracy of lower-abundance proteins in Arabidopsis. NPAS was combinedmore » with subcellular protein localization data, facilitating quantitative estimations of organelle abundance during routine experimental procedures. A suite of targeted proteomics markers for subcellular compartment markers was developed, enabling independent verification of in silico estimates for relative organelle abundance. Estimation of relative organelle abundance was found to be reproducible and consistent over a range of tissues and growth conditions. In silico abundance estimations and localization data have been combined into an online tool, multiple marker abundance profiling, available in the SUBA4 toolbox (http://suba.live).« less

  9. Multiple marker abundance profiling: combining selected reaction monitoring and data-dependent acquisition for rapid estimation of organelle abundance in subcellular samples

    DOE PAGES

    Hooper, Cornelia M.; Stevens, Tim J.; Saukkonen, Anna; ...

    2017-10-12

    Measuring changes in protein or organelle abundance in the cell is an essential, but challenging aspect of cell biology. Frequently-used methods for determining organelle abundance typically rely on detection of a very few marker proteins, so are unsatisfactory. In silico estimates of protein abundances from publicly available protein spectra can provide useful standard abundance values but contain only data from tissue proteomes, and are not coupled to organelle localization data. A new protein abundance score, the normalized protein abundance scale (NPAS), expands on the number of scored proteins and the scoring accuracy of lower-abundance proteins in Arabidopsis. NPAS was combinedmore » with subcellular protein localization data, facilitating quantitative estimations of organelle abundance during routine experimental procedures. A suite of targeted proteomics markers for subcellular compartment markers was developed, enabling independent verification of in silico estimates for relative organelle abundance. Estimation of relative organelle abundance was found to be reproducible and consistent over a range of tissues and growth conditions. In silico abundance estimations and localization data have been combined into an online tool, multiple marker abundance profiling, available in the SUBA4 toolbox (http://suba.live).« less

  10. Discovering amino acid patterns on binding sites in protein complexes

    PubMed Central

    Kuo, Huang-Cheng; Ong, Ping-Lin; Lin, Jung-Chang; Huang, Jen-Peng

    2011-01-01

    Discovering amino acid (AA) patterns on protein binding sites has recently become popular. We propose a method to discover the association relationship among AAs on binding sites. Such knowledge of binding sites is very helpful in predicting protein-protein interactions. In this paper, we focus on protein complexes which have protein-protein recognition. The association rule mining technique is used to discover geographically adjacent amino acids on a binding site of a protein complex. When mining, instead of treating all AAs of binding sites as a transaction, we geographically partition AAs of binding sites in a protein complex. AAs in a partition are treated as a transaction. For the partition process, AAs on a binding site are projected from three-dimensional to two-dimensional. And then, assisted with a circular grid, AAs on the binding site are placed into grid cells. A circular grid has ten rings: a central ring, the second ring with 6 sectors, the third ring with 12 sectors, and later rings are added to four sectors in order. As for the radius of each ring, we examined the complexes and found that 10Å is a suitable range, which can be set by the user. After placing these recognition complexes on the circular grid, we obtain mining records (i.e. transactions) from each sector. A sector is regarded as a record. Finally, we use the association rule to mine these records for frequent AA patterns. If the support of an AA pattern is larger than the predetermined minimum support (i.e. threshold), it is called a frequent pattern. With these discovered patterns, we offer the biologists a novel point of view, which will improve the prediction accuracy of protein-protein recognition. In our experiments, we produced the AA patterns by data mining. As a result, we found that arginine (arg) most frequently appears on the binding sites of two proteins in the recognition protein complexes, while cysteine (cys) appears the fewest. In addition, if we discriminate the shape

  11. Differential response to sulfur nutrition of two common bean genotypes differing in storage protein composition.

    PubMed

    Pandurangan, Sudhakar; Sandercock, Mark; Beyaert, Ronald; Conn, Kenneth L; Hou, Anfu; Marsolais, Frédéric

    2015-01-01

    It has been hypothesized that the relatively low concentration of sulfur amino acids in legume seeds might be an ecological adaptation to nutrient poor, marginal soils. SARC1 and SMARC1N-PN1 are genetically related lines of common bean (dry bean, Phaseolus vulgaris) differing in seed storage protein composition. In SMARC1N-PN1, the lack of phaseolin and major lectins is compensated by increased levels of sulfur-rich proteins, resulting in an enhanced concentration of cysteine and methionine, mostly at the expense of the abundant non-protein amino acid, S-methylcysteine. To identify potential effects associated with an increased concentration of sulfur amino acids in the protein pool, the response of the two genotypes to low and high sulfur nutrition was evaluated under controlled conditions. Seed yield was increased by the high sulfate treatment in SMARC1N-PN1. The seed concentrations of sulfur, sulfate, and S-methylcysteine were altered by the sulfur treatment in both genotypes. The concentration of total cysteine and extractible globulins was increased specifically in SMARC1N-PN1. Proteomic analysis identified arcelin-like protein 4, lipoxygenase-3, albumin-2, and alpha amylase inhibitor beta chain as having increased levels under high sulfur conditions. Lipoxygenase-3 accumulation was sensitive to sulfur nutrition only in SMARC1N-PN1. Under field conditions, both SARC1 and SMARC1N-PN1 exhibited a slight increase in yield in response to sulfur treatment, typical for common bean.

  12. Nutritive evaluation of a non-conventional leafy vegetable (Pereskia aculeata Miller).

    PubMed

    Takeiti, Cristina Y; Antonio, Graziella C; Motta, Eliana M P; Collares-Queiroz, Fernanda P; Park, Kil J

    2009-01-01

    Pereskia aculeata Miller is a native cactus that can be found in Brazil and is called 'ora-pro-nobis' (OPN). Many people from poor communities consume the dark green leaves of OPN as a vegetable. The objective of the present work was to evaluate the nutritional components in terms of proximate composition, minerals, vitamins, protein content and their in vitro protein digestibility. OPN leaves showed remarkable levels of total dietary fiber (39.1% dry basis), minerals (calcium, magnesium, manganese and zinc) and vitamins (vitamin A, vitamin C and folic acid). Among amino acids, tryptophan was the most abundant (20.5% of the total amino acids) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed small peptides, inferior to 6.5 kDa, and four major bands (61 kDa, 53 kDa, 33 kDa, and 15 kDa). The protein digestibility corrected amino acid score showed the lowest value of sulfur-amino acids (Met+Cys). OPN leaves could be considered a good source of minerals, vitamins and amino acids, and may serve as a potential functional ingredient.

  13. Complete nucleotide and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of a mitochondrial targeting presequence.

    PubMed Central

    Ridley, R G; Patel, H V; Gerber, G E; Morton, R C; Freeman, K B

    1986-01-01

    A cDNA clone spanning the entire amino acid sequence of the nuclear-encoded uncoupling protein of rat brown adipose tissue mitochondria has been isolated and sequenced. With the exception of the N-terminal methionine the deduced N-terminus of the newly synthesized uncoupling protein is identical to the N-terminal 30 amino acids of the native uncoupling protein as determined by protein sequencing. This proves that the protein contains no N-terminal mitochondrial targeting prepiece and that a targeting region must reside within the amino acid sequence of the mature protein. Images PMID:3012461

  14. Docking of oxalyl aryl amino benzoic acid derivatives into PTP1B

    PubMed Central

    Verma, Neelam; Mittal, Minakshi; Verma, Raman kumar

    2008-01-01

    Protein Tyrosine Phosphatases (PTPs) that function as negative regulators of the insulin signaling cascade have been identified as novel targets for the therapeutic enhancement of insulin action in insulin resistant disease states. Reducing Protein Tyrosine Phosphatase1B (PTP1B) abundance not only enhances insulin sensitivity and improves glucose metabolism but also protects against obesity induced by high fat feeding. PTP1B inhibitors such as Formylchromone derivatives, 1, 2-Naphthoquinone derivatives and Oxalyl aryl amino benzoic derivatives may eventually find an important clinical role as insulin sensitizers in the management of Type-II Diabetes and metabolic syndrome. We have carried out docking of modified oxalyl aryl amino benzoic acid derivatives into three dimensional structure of PTP1B using BioMed CAChe 6.1. These compounds exhibit good selectivity for PTP1B over most of phosphatases in selectivity panel such as SHP-2, LAR, CD45 and TCPTP found in literature. This series of compounds identified the amino acid residues such as Gly220 and Arg221 are important for achieving specificity via H-bonding interactions. Lipophilic side chain of methionine in modified oxalyl aryl amino benzoic acid derivative [1b (a2, b2, c1, d)] lies in closer vicinity of hydrophobic region of protein consisted of Meth258 and Phe52 in comparison to active ligand. Docking Score in [1b (a2, b2, c1, d)] is -131.740Kcal/mol much better than active ligand score -98.584Kcal/mol. This information can be exploited to design PTP1B specific inhibitors. PMID:19238234

  15. Non-LTE gallium abundance in HgMn stars

    NASA Astrophysics Data System (ADS)

    Zboril, M.; Berrington, K. A.

    2001-07-01

    We present, for the first time, the Non-LTE gallium equivalent widths for the most prominent gallium transitions as identified in real spectra and in (hot) mercury-manganese star. The common feature of the departure coefficients is to decrease near the stellar surface, the collision rates are dominant in many cases and the Non-LTE equivalent widths are generally smaller. In particular, the abundance difference as derived from UV and visual lines is reduced. The photoionization cross sections were computed by means of standard R-matrix formalism. The gallium cross-sections are only available in electronic form at the CDS via anonymous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via http://cdsweb.u-strasbg.fr/cgi-bin/qcat?J/A+A/373/987

  16. Improving predictions of protein-protein interfaces by combining amino acid-specific classifiers based on structural and physicochemical descriptors with their weighted neighbor averages.

    PubMed

    de Moraes, Fábio R; Neshich, Izabella A P; Mazoni, Ivan; Yano, Inácio H; Pereira, José G C; Salim, José A; Jardine, José G; Neshich, Goran

    2014-01-01

    Protein-protein interactions are involved in nearly all regulatory processes in the cell and are considered one of the most important issues in molecular biology and pharmaceutical sciences but are still not fully understood. Structural and computational biology contributed greatly to the elucidation of the mechanism of protein interactions. In this paper, we present a collection of the physicochemical and structural characteristics that distinguish interface-forming residues (IFR) from free surface residues (FSR). We formulated a linear discriminative analysis (LDA) classifier to assess whether chosen descriptors from the BlueStar STING database (http://www.cbi.cnptia.embrapa.br/SMS/) are suitable for such a task. Receiver operating characteristic (ROC) analysis indicates that the particular physicochemical and structural descriptors used for building the linear classifier perform much better than a random classifier and in fact, successfully outperform some of the previously published procedures, whose performance indicators were recently compared by other research groups. The results presented here show that the selected set of descriptors can be utilized to predict IFRs, even when homologue proteins are missing (particularly important for orphan proteins where no homologue is available for comparative analysis/indication) or, when certain conformational changes accompany interface formation. The development of amino acid type specific classifiers is shown to increase IFR classification performance. Also, we found that the addition of an amino acid conservation attribute did not improve the classification prediction. This result indicates that the increase in predictive power associated with amino acid conservation is exhausted by adequate use of an extensive list of independent physicochemical and structural parameters that, by themselves, fully describe the nano-environment at protein-protein interfaces. The IFR classifier developed in this study is now

  17. Improving Predictions of Protein-Protein Interfaces by Combining Amino Acid-Specific Classifiers Based on Structural and Physicochemical Descriptors with Their Weighted Neighbor Averages

    PubMed Central

    de Moraes, Fábio R.; Neshich, Izabella A. P.; Mazoni, Ivan; Yano, Inácio H.; Pereira, José G. C.; Salim, José A.; Jardine, José G.; Neshich, Goran

    2014-01-01

    Protein-protein interactions are involved in nearly all regulatory processes in the cell and are considered one of the most important issues in molecular biology and pharmaceutical sciences but are still not fully understood. Structural and computational biology contributed greatly to the elucidation of the mechanism of protein interactions. In this paper, we present a collection of the physicochemical and structural characteristics that distinguish interface-forming residues (IFR) from free surface residues (FSR). We formulated a linear discriminative analysis (LDA) classifier to assess whether chosen descriptors from the BlueStar STING database (http://www.cbi.cnptia.embrapa.br/SMS/) are suitable for such a task. Receiver operating characteristic (ROC) analysis indicates that the particular physicochemical and structural descriptors used for building the linear classifier perform much better than a random classifier and in fact, successfully outperform some of the previously published procedures, whose performance indicators were recently compared by other research groups. The results presented here show that the selected set of descriptors can be utilized to predict IFRs, even when homologue proteins are missing (particularly important for orphan proteins where no homologue is available for comparative analysis/indication) or, when certain conformational changes accompany interface formation. The development of amino acid type specific classifiers is shown to increase IFR classification performance. Also, we found that the addition of an amino acid conservation attribute did not improve the classification prediction. This result indicates that the increase in predictive power associated with amino acid conservation is exhausted by adequate use of an extensive list of independent physicochemical and structural parameters that, by themselves, fully describe the nano-environment at protein-protein interfaces. The IFR classifier developed in this study is now

  18. Multiple Amino Acid Supplementations to Reduce Dietary Protein in Plant-Based Rainbow Trout, Oncorhynchus mykiss, Feeds

    USDA-ARS?s Scientific Manuscript database

    Reducing dietary protein in trout feeds will reduce production costs if growth performance can be maintained. A study was conducted to determine if balancing plant-based diets on an available amino acid basis would result in a reduction in total protein level. The diets were formulated to contain ...

  19. The amino-terminal region of the retinoblastoma gene product binds a novel nuclear matrix protein that co-localizes to centers for RNA processing

    PubMed Central

    1994-01-01

    The tumor suppressing capacity of the retinoblastoma protein (p110RB) is dependent on interactions made with cellular proteins through its carboxy-terminal domains. How the p110RB amino-terminal region contributes to this activity is unclear, though evidence now indicates it is important for both growth suppression and regulation of the full- length protein. We have used the yeast two-hybrid system to screen for cellular proteins which bind to the first 300 amino acids of p110RB. The only gene isolated from this screen encodes a novel 84-kD nuclear matrix protein that localizes to subnuclear regions associated with RNA processing. This protein, p84, requires a structurally defined domain in the amino terminus of p110RB for binding. Furthermore, both in vivo and in vitro experiments demonstrate that p84 binds preferentially to the functionally active, hypophosphorylated form of p110RB. Thus, the amino terminus of p110RB may function in part to facilitate the binding of growth promoting factors at subnuclear regions actively involved in RNA metabolism. PMID:7525595

  20. Effect of specific amino acids on hepatic lipid metabolism in fructose-induced non-alcoholic fatty liver disease.

    PubMed

    Jegatheesan, Prasanthi; Beutheu, Stéphanie; Ventura, Gabrielle; Sarfati, Gilles; Nubret, Esther; Kapel, Nathalie; Waligora-Dupriet, Anne-Judith; Bergheim, Ina; Cynober, Luc; De-Bandt, Jean-Pascal

    2016-02-01

    Fructose diets have been shown to induce insulin resistance and to alter liver metabolism and gut barrier function, ultimately leading to non-alcoholic fatty liver disease. Citrulline, Glutamine and Arginine may improve insulin sensitivity and have beneficial effects on gut trophicity. Our aim was to evaluate their effects on liver and gut functions in a rat model of fructose-induced non-alcoholic fatty liver disease. Male Sprague-Dawley rats (n = 58) received a 4-week fructose (60%) diet or standard chow with or without Citrulline (0.15 g/d) or an isomolar amount of Arginine or Glutamine. All diets were made isonitrogenous by addition of non-essential amino acids. At week 4, nutritional and metabolic status (plasma glucose, insulin, cholesterol, triglycerides and amino acids, net intestinal absorption) was determined; steatosis (hepatic triglycerides content, histological examination) and hepatic function (plasma aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, bilirubin) were assessed; and gut barrier integrity (myeloperoxidase activity, portal endotoxemia, tight junction protein expression and localization) and intestinal and hepatic inflammation were evaluated. We also assessed diets effects on caecal microbiota. In these experimental isonitrogenous fructose diet conditions, fructose led to steatosis with dyslipidemia but without altering glucose homeostasis, liver function or gut permeability. Fructose significantly decreased Bifidobacterium and Lactobacillus and tended to increase endotoxemia. Arginine and Glutamine supplements were ineffective but Citrulline supplementation prevented hypertriglyceridemia and attenuated liver fat accumulation. While nitrogen supply alone can attenuate fructose-induced non-alcoholic fatty liver disease, Citrulline appears to act directly on hepatic lipid metabolism by partially preventing hypertriglyceridemia and steatosis. Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition

  1. Prediction of glutathionylation sites in proteins using minimal sequence information and their experimental validation.

    PubMed

    Pal, Debojyoti; Sharma, Deepak; Kumar, Mukesh; Sandur, Santosh K

    2016-09-01

    S-glutathionylation of proteins plays an important role in various biological processes and is known to be protective modification during oxidative stress. Since, experimental detection of S-glutathionylation is labor intensive and time consuming, bioinformatics based approach is a viable alternative. Available methods require relatively longer sequence information, which may prevent prediction if sequence information is incomplete. Here, we present a model to predict glutathionylation sites from pentapeptide sequences. It is based upon differential association of amino acids with glutathionylated and non-glutathionylated cysteines from a database of experimentally verified sequences. This data was used to calculate position dependent F-scores, which measure how a particular amino acid at a particular position may affect the likelihood of glutathionylation event. Glutathionylation-score (G-score), indicating propensity of a sequence to undergo glutathionylation, was calculated using position-dependent F-scores for each amino-acid. Cut-off values were used for prediction. Our model returned an accuracy of 58% with Matthew's correlation-coefficient (MCC) value of 0.165. On an independent dataset, our model outperformed the currently available model, in spite of needing much less sequence information. Pentapeptide motifs having high abundance among glutathionylated proteins were identified. A list of potential glutathionylation hotspot sequences were obtained by assigning G-scores and subsequent Protein-BLAST analysis revealed a total of 254 putative glutathionable proteins, a number of which were already known to be glutathionylated. Our model predicted glutathionylation sites in 93.93% of experimentally verified glutathionylated proteins. Outcome of this study may assist in discovering novel glutathionylation sites and finding candidate proteins for glutathionylation.

  2. Dynamic Fluctuations of Protein-Carbohydrate Interactions Promote Protein Aggregation

    PubMed Central

    Voynov, Vladimir; Chennamsetty, Naresh; Kayser, Veysel; Helk, Bernhard; Forrer, Kurt; Zhang, Heidi; Fritsch, Cornelius; Heine, Holger; Trout, Bernhardt L.

    2009-01-01

    Protein-carbohydrate interactions are important for glycoprotein structure and function. Antibodies of the IgG class, with increasing significance as therapeutics, are glycosylated at a conserved site in the constant Fc region. We hypothesized that disruption of protein-carbohydrate interactions in the glycosylated domain of antibodies leads to the exposure of aggregation-prone motifs. Aggregation is one of the main problems in protein-based therapeutics because of immunogenicity concerns and decreased efficacy. To explore the significance of intramolecular interactions between aromatic amino acids and carbohydrates in the IgG glycosylated domain, we utilized computer simulations, fluorescence analysis, and site-directed mutagenesis. We find that the surface exposure of one aromatic amino acid increases due to dynamic fluctuations. Moreover, protein-carbohydrate interactions decrease upon stress, while protein-protein and carbohydrate-carbohydrate interactions increase. Substitution of the carbohydrate-interacting aromatic amino acids with non-aromatic residues leads to a significantly lower stability than wild type, and to compromised binding to Fc receptors. Our results support a mechanism for antibody aggregation via decreased protein-carbohydrate interactions, leading to the exposure of aggregation-prone regions, and to aggregation. PMID:20037630

  3. Distribution and Origin of Amino Acids in Lunar Regolith Samples

    NASA Technical Reports Server (NTRS)

    Elsila, J. E.; Callahan, M. P.; Glavin, D. P.; Dworkin, J. P.; McLain, H. L.; Noble, S. K.; Gibson, E. K., Jr.

    2015-01-01

    The existence of organic compounds on the lunar surface has been a question of interest from the Apollo era to the present. Investigations of amino acids immediately after collection of lunar samples yielded inconclusive identifications, in part due to analytical limitations including insensitivity to certain compounds, an inability to separate enantiomers, and lack of compound-specific isotopic measurements. It was not possible to determine if the detected amino acids were indigenous to the lunar samples or the result of terrestrial contamination. Recently, we presented initial data from the analysis of amino acid abundances in 12 lunar regolith samples and discussed those results in the context of four potential amino acid sources [5]. Here, we expand on our previous work, focusing on amino acid abundances and distributions in seven regolith samples and presenting the first compound-specific carbon isotopic ratios measured for amino acids in a lunar sample.

  4. Supplementing a low-protein diet with dibasic amino acids increases urinary calcium excretion in young women.

    PubMed

    Bihuniak, Jessica D; Sullivan, Rebecca R; Simpson, Christine A; Caseria, Donna M; Huedo-Medina, Tania B; O'Brien, Kimberly O; Kerstetter, Jane E; Insogna, Karl L

    2014-03-01

    Increasing dietary protein within a physiologic range stimulates intestinal calcium absorption, but it is not known if specific amino acids or dietary protein as a whole are responsible for this effect. Therefore, we selectively supplemented a low-protein (0.7 g/kg) diet with either the calcium-sensing receptor-activating amino acids (CaSR-AAAs) L-tryptophan, L-phenylalanine, and L-histidine, or the dibasic amino acids (DAAs) L-arginine and L-lysine, to achieve intakes comparable to the content of a high-protein diet (2.1 g/kg) and measured intestinal calcium absorption. Fourteen young women took part in a placebo-controlled, double-blind, crossover feeding trial in which each participant ingested a 6-d low-protein diet supplemented with CaSR-AAAs, DAAs, or methylcellulose capsules (control) after an 11-d adjustment period. All participants ingested all 3 diets in random order. Intestinal calcium absorption was measured between days 5 and 6 using dual-stable calcium isotopes ((42)Ca, (43)Ca, and (44)Ca). There was no difference in calcium absorption between the diet supplemented with CaSR-AAAs (22.9 ± 2.0%) and the control diet (22.3 ± 1.4%) (P = 0.64). However, calcium absorption tended to be greater during the DAA supplementation period (25.2 ± 1.4%) compared with the control diet period (22.3 ± 1.4%) (P < 0.10). Larger and longer clinical trials are needed to clarify the possible benefit of arginine and lysine on calcium absorption.

  5. Unprecedented concentrations of indigenous amino acids in primitive CR meteorites

    NASA Astrophysics Data System (ADS)

    Ehrenfreund, Pascale; Martins, Zita; Alexander, Conel; Orzechowska, Grazyna; Fogel, Marylin

    CR meteorites are among the most primitive meteorites. We have performed pioneering work determining the compositional characteristics of amino acids in this type of carbonaceous chondrites. We report the first measurements of amino acids in Antarctic CR meteorites, two of which show the highest amino acid concentrations ever found in a chondrite. We have analyzed the amino acid content of the Antarctic CRs EET92042, GRA95229 and GRO95577 using high performance liquid chromatography with UV fluorescence detection (HPLC-FD) and gas chromatography-mass spectrometry (GC-MS). Additionally, compound-specific carbon isotopic measurements for most of the individual amino acids from the EET92042 and GRA95229 meteorites were achieved by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Our data show that EET92042 and GRA95229 are the most amino acid-rich chondrites ever analyzed, with total amino acid concentrations of 180 and 249 parts-per-million (ppm), respectively. GRO95577, however, is depleted in amino acids (<1 ppm). The most abundant amino acids present in the EET92042 and GRA95229 meteorites are the α-amino acids glycine, isovaline, α-aminoisobutyric acid (α-AIB), and alanine, with δ 13 C values ranging from +31.6% to +50.5%. The highly enriched carbon isotope results together with racemic enantiomeric ratios determined for most amino acids indicate that primitive organic matter was preserved in these meteorites. In addition, the relative abundances of α-AIB and β-alanine amongst Antarctic CR meteorites appear to correspond to the degree of aqueous alteration on their respective parent body. Investigating the abundances and isotopic composition of amino acids in primitive chondrites helps to understand the role of meteorites as a source of extraterrestrial prebiotic organic compounds to the early Earth.

  6. Protein Requirements Are Elevated in Endurance Athletes after Exercise as Determined by the Indicator Amino Acid Oxidation Method

    PubMed Central

    Kato, Hiroyuki; Suzuki, Katsuya; Bannai, Makoto; Moore, Daniel R.

    2016-01-01

    A higher protein intake has been recommended for endurance athletes compared with healthy non-exercising individuals based primarily on nitrogen balance methodology. The aim of this study was to determine the estimated average protein requirement and recommended protein intake in endurance athletes during an acute 3-d controlled training period using the indicator amino acid oxidation method. After 2-d of controlled diet (1.4 g protein/kg/d) and training (10 and 5km/d, respectively), six male endurance-trained adults (28±4 y of age; Body weight, 64.5±10.0 kg; VO2peak, 60.3±6.7 ml·kg-1·min-1; means±SD) performed an acute bout of endurance exercise (20 km treadmill run) prior to consuming test diets providing variable amounts of protein (0.2–2.8 g·kg-1·d-1) and sufficient energy. Protein was provided as a crystalline amino acid mixture based on the composition of egg protein with [1-13C]phenylalanine provided to determine whole body phenylalanine flux, 13CO2 excretion, and phenylalanine oxidation. The estimated average protein requirement was determined as the breakpoint after biphasic linear regression analysis with a recommended protein intake defined as the upper 95% confidence interval. Phenylalanine flux (68.8±8.5 μmol·kg-1·h-1) was not affected by protein intake. 13CO2 excretion displayed a robust bi-phase linear relationship (R2 = 0.86) that resulted in an estimated average requirement and a recommended protein intake of 1.65 and 1.83 g protein·kg-1·d-1, respectively, which was similar to values based on phenylalanine oxidation (1.53 and 1.70 g·kg-1·d-1, respectively). We report a recommended protein intake that is greater than the RDA (0.8 g·kg-1·d-1) and current recommendations for endurance athletes (1.2–1.4 g·kg-1·d-1). Our results suggest that the metabolic demand for protein in endurance-trained adults on a higher volume training day is greater than their sedentary peers and current recommendations for athletes based primarily on

  7. Evaluating the efficacy of a structure-derived amino acid substitution matrix in detecting protein homologs by BLAST and PSI-BLAST.

    PubMed

    Goonesekere, Nalin Cw

    2009-01-01

    The large numbers of protein sequences generated by whole genome sequencing projects require rapid and accurate methods of annotation. The detection of homology through computational sequence analysis is a powerful tool in determining the complex evolutionary and functional relationships that exist between proteins. Homology search algorithms employ amino acid substitution matrices to detect similarity between proteins sequences. The substitution matrices in common use today are constructed using sequences aligned without reference to protein structure. Here we present amino acid substitution matrices constructed from the alignment of a large number of protein domain structures from the structural classification of proteins (SCOP) database. We show that when incorporated into the homology search algorithms BLAST and PSI-blast, the structure-based substitution matrices enhance the efficacy of detecting remote homologs.

  8. Amino acid signature enables proteins to recognize modified tRNA.

    PubMed

    Spears, Jessica L; Xiao, Xingqing; Hall, Carol K; Agris, Paul F

    2014-02-25

    Human tRNA(Lys3)UUU is the primer for HIV replication. The HIV-1 nucleocapsid protein, NCp7, facilitates htRNA(Lys3)UUU recruitment from the host cell by binding to and remodeling the tRNA structure. Human tRNA(Lys3)UUU is post-transcriptionally modified, but until recently, the importance of those modifications in tRNA recognition by NCp7 was unknown. Modifications such as the 5-methoxycarbonylmethyl-2-thiouridine at anticodon wobble position-34 and 2-methylthio-N(6)-threonylcarbamoyladenosine, adjacent to the anticodon at position-37, are important to the recognition of htRNA(Lys3)UUU by NCp7. Several short peptides selected from phage display libraries were found to also preferentially recognize these modifications. Evolutionary algorithms (Monte Carlo and self-consistent mean field) and assisted model building with energy refinement were used to optimize the peptide sequence in silico, while fluorescence assays were developed and conducted to verify the in silico results and elucidate a 15-amino acid signature sequence (R-W-Q/N-H-X2-F-Pho-X-G/A-W-R-X2-G, where X can be most amino acids, and Pho is hydrophobic) that recognized the tRNA's fully modified anticodon stem and loop domain, hASL(Lys3)UUU. Peptides of this sequence specifically recognized and bound modified htRNA(Lys3)UUU with an affinity 10-fold higher than that of the starting sequence. Thus, this approach provides an effective means of predicting sequences of RNA binding peptides that have better binding properties. Such peptides can be used in cell and molecular biology as well as biochemistry to explore RNA binding proteins and to inhibit those protein functions.

  9. Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis.

    PubMed

    Gan, Qinglei; Fan, Chenguang

    2017-11-01

    Cell-free protein synthesis provides a robust platform for co-translational incorporation of noncanonical amino acid (ncAA) into proteins to facilitate biological studies and biotechnological applications. Recently, eliminating the activity of release factor 1 has been shown to increase ncAA incorporation in response to amber codons. However, this approach could promote mis-incorporation of canonical amino acids by near cognate suppression. We performed a facile protocol to remove near cognate tRNA isoacceptors of the amber codon from total tRNAs, and used the phosphoserine (Sep) incorporation system as validation. By manipulating codon usage of target genes and tRNA species introduced into the cell-free protein synthesis system, we increased the fidelity of Sep incorporation at a specific position. By removing three near cognate tRNA isoacceptors of the amber stop codon [tRNA Lys , tRNA Tyr , and tRNA Gln (CUG)] from the total tRNA, the near cognate suppression decreased by 5-fold without impairing normal protein synthesis in the cell-free protein synthesis system. Mass spectrometry analyses indicated that the fidelity of ncAA incorporation was improved. Removal of near cognate tRNA isoacceptors of the amber codon could increase ncAA incorporation fidelity towards the amber stop codon in release factor deficiency systems. We provide a general strategy to improve fidelity of ncAA incorporation towards stop, quadruplet and sense codons in cell-free protein synthesis systems. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Characterization of pea (Pisum sativum) seed protein fractions.

    PubMed

    Rubio, Luis A; Pérez, Alicia; Ruiz, Raquel; Guzmán, M Ángeles; Aranda-Olmedo, Isabel; Clemente, Alfonso

    2014-01-30

    Legume seed proteins have to be chemically characterized in order to properly link their nutritional effects with their chemical structure. Vicilin and albumin fractions devoid of cross-contamination, as assessed by mass peptide fingerprinting analysis, were obtained from defatted pea (Pisum sativum cv. Bilbo) meal. The extracted protein fractions contained 56.7-67.7 g non-starch polysaccharides kg⁻¹. The vicilin fraction was higher than legumins in arginine, isoleucine, leucine, phenylalanine and lysine. The most abundant amino acids in the albumin fraction were aspartic acid, glutamic acid, lysine and arginine, and the amounts of methionine were more than double than those in legumins and vicilins. The pea albumin fraction showed a clear enrichment of protease inhibitory activity when compared with the seed meal. In vitro digestibility values for pea proteins were 0.63 ±  0.04, 0.88 ±  0.04 and 0.41 ±  0.23 for legumins, vicilins and albumins respectively. Vicilin and albumin fractions devoid of cross-contamination with other proteins were obtained from pea seed meal. The vicilin fraction also contained low amounts of soluble non-starch polysaccharides and was enriched in isoleucine, leucine, phenylalanine and lysine. In vitro digestibility values for pea proteins were similar or even numerically higher than those for control proteins. © 2013 Society of Chemical Industry.

  11. Screening and identification of resistance related proteins from apple leaves inoculated with Marssonina coronaria (EII. & J. J. Davis)

    PubMed Central

    2014-01-01

    Background Apple, an invaluable fruit crop worldwide, is often prone to infection by pathogenic fungi. Identification of potentially resistance-conferring apple proteins is one of the most important aims for studying apple resistance mechanisms and promoting the development of disease-resistant apple strains. In order to find proteins which promote resistance to Marssonina coronaria, a deadly pathogen which has been related to premature apple maturation, proteomes from apple leaves inoculated with M. coronaria were characterized at 3 and 6 days post-inoculation by two dimensional electrophoresis (2-DE). Results Overall, 59 differentially accumulated protein spots between inoculation and non-inoculation were successfully identified and aligned as 35 different proteins or protein families which involved in photosynthesis, amino acid metabolism, transport, energy metabolism, carbohydrate metabolism, binding, antioxidant, defense and stress. Quantitative real-time PCR (qRT-PCR) was also used to examine the change of some defense and stress related genes abundance under inoculated conditions. Conclusions In a conclusion, different proteins in response to Marssonina coronaria were identified by proteomic analysis. Among of these proteins, there are some PR proteins, for example class III endo-chitinase, beta-1,3-glucanase and thaumatine-like protein, and some antioxidant related proteins including aldo/keto reductase AKR, ascorbate peroxidase and phi class glutathione S-transferase protein that were associated with disease resistance. The transcription levels of class III endo-chitinase (L13) and beta-1, 3-glucanase (L17) have a good relation with the abundance of the encoded protein’s accumulation, however, the mRNA abundance of thaumatine-like protein (L22) and ascorbate peroxidase (L28) are not correlated with their protein abundance of encoded protein. To elucidate the resistant mechanism, the data in the present study will promote us to investigate further the

  12. Complement factor H family proteins in their non-canonical role as modulators of cellular functions.

    PubMed

    Józsi, Mihály; Schneider, Andrea E; Kárpáti, Éva; Sándor, Noémi

    2018-01-04

    Complement factor H is a major regulator of the alternative pathway of the complement system. The factor H-related proteins are less characterized, but recent data indicate that they rather promote complement activation. These proteins have some common ligands with factor H and have both overlapping and distinct functions depending on domain composition and the degree of conservation of amino acid sequence. Factor H and some of the factor H-related proteins also appear in a non-canonical function that is beyond their role in the modulation of complement activation. This review covers our current understanding on this emerging role of factor H family proteins in modulating the activation and function of various cells by binding to receptors or receptor ligands. Copyright © 2018 Elsevier Ltd. All rights reserved.

  13. Fuzzy cluster analysis of simple physicochemical properties of amino acids for recognizing secondary structure in proteins.

    PubMed Central

    Mocz, G.

    1995-01-01

    Fuzzy cluster analysis has been applied to the 20 amino acids by using 65 physicochemical properties as a basis for classification. The clustering products, the fuzzy sets (i.e., classical sets with associated membership functions), have provided a new measure of amino acid similarities for use in protein folding studies. This work demonstrates that fuzzy sets of simple molecular attributes, when assigned to amino acid residues in a protein's sequence, can predict the secondary structure of the sequence with reasonable accuracy. An approach is presented for discriminating standard folding states, using near-optimum information splitting in half-overlapping segments of the sequence of assigned membership functions. The method is applied to a nonredundant set of 252 proteins and yields approximately 73% matching for correctly predicted and correctly rejected residues with approximately 60% overall success rate for the correctly recognized ones in three folding states: alpha-helix, beta-strand, and coil. The most useful attributes for discriminating these states appear to be related to size, polarity, and thermodynamic factors. Van der Waals volume, apparent average thickness of surrounding molecular free volume, and a measure of dimensionless surface electron density can explain approximately 95% of prediction results. hydrogen bonding and hydrophobicity induces do not yet enable clear clustering and prediction. PMID:7549882

  14. Amino acids as antioxidants for frying oil

    USDA-ARS?s Scientific Manuscript database

    Amino acids, proteins and hydrolysates of proteins have been known to protect edible oils from oxidation. While amino acids and related materials have high potential as antioxidants for frying oil, effectiveness of each amino acid and mechanisms of their activities are not well understood yet. Propo...

  15. Combining affinity enrichment, cross-linking with photo-amino acids, and mass spectrometry for probing protein kinase D2 interactions.

    PubMed

    Häupl, Björn; Ihling, Christian H; Sinz, Andrea

    2017-04-07

    We present a novel approach that relies on the affinity capture of protein interaction partners from a complex mixture, followed by covalent fixation via UV-induced activation of incorporated diazirine photo-reactive amino acids (photo-methionine and photo-leucine). The captured protein complexes are enzymatically digested and interacting proteins are identified and quantified by label-free LC/MS analysis. Using HeLa cell lysates with photo-methionine and photo-leucine-labeled proteins, we were able to capture and preserve protein interactions that are otherwise elusive in conventional pull-down experiments. Our approach is exemplified for mapping the protein interaction network of protein kinase D2, but has the potential be applied to any protein system. Data are available via ProteomeXchange with identifiers PXD005346 (photo-amino acid incorporation) and PXD005349 (enrichment experiments). This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  16. Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence, and complementary protein chemical studies

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Peters, J.; Peters, M.; Lottspeich, F.

    1987-11-01

    The complete nucleotide sequence of the gene encoding the surface (hexagonally packed intermediate (HPI))-layer polypeptide of Deinococcus radiodurans Sark was determined and found to encode a polypeptide of 1036 amino acids. Amino acid sequence analysis of about 30% of the residues revealed that the mature polypeptide consists of at least 978 amino acids. The N terminus was blocked to Edman degradation. The results of proteolytic modification of the HPI layer in situ and M/sub r/ estimations of the HPI polypeptide expressed in Escherichia coli indicated that there is a leader sequence. The N-terminal region contained a very high percentage (29%)more » of threonine and serine, including a cluster of nine consecutive serine or threonine residues, whereas a stretch near the C terminus was extremely rich in aromatic amino acids (29%). The protein contained at least two disulfide bridges, as well as tightly bound reducing sugars and fatty acids.« less

  17. Identification of multiply charged proteins and amino acid clusters by liquid nitrogen assisted spray ionization mass spectrometry.

    PubMed

    Kumar Kailasa, Suresh; Hasan, Nazim; Wu, Hui-Fen

    2012-08-15

    The development of liquid nitrogen assisted spray ionization mass spectrometry (LNASI MS) for the analysis of multiply charged proteins (insulin, ubiquitin, cytochrome c, α-lactalbumin, myoglobin and BSA), peptides (glutathione, HW6, angiotensin-II and valinomycin) and amino acid (arginine) clusters is described. The charged droplets are formed by liquid nitrogen assisted sample spray through a stainless steel nebulizer and transported into mass analyzer for the identification of multiply charged protein ions. The effects of acids and modifier volumes for the efficient ionization of the above analytes in LNASI MS were carefully investigated. Multiply charged proteins and amino acid clusters were effectively identified by LNASI MS. The present approach can effectively detect the multiply charged states of cytochrome c at 400 nM. A comparison between LNASI and ESI, CSI, SSI and V-EASI methods on instrumental conditions, applied temperature and observed charge states for the multiply charged proteins, shows that the LNASI method produces the good quality spectra of amino acid clusters at ambient conditions without applied any electric field and heat. To date, we believe that the LNASI method is the most simple, low cost and provided an alternative paradigm for production of multiply charged ions by LNASI MS, just as ESI-like ions yet no need for applying any electrical field and it could be operated at low temperature for generation of highly charged protein/peptide ions. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. The Next Generation MOD: A Microchip Amino Acid Analyzer for Detecting Extraterrestrial Life

    NASA Technical Reports Server (NTRS)

    Mathies, R. A.; Hutt, L. D.; Bada, J. L.; Glavin, D.; Grunthaner, F. J.; Grunthaner, P. J.

    2000-01-01

    The MOD (Mars Organic Detector) instrument which has selected for the definition phase of the BEDS package on the 2005 Mars Explorer Program spacecraft is designed to simply detect the presence of amino acids in Martian surface samples at a sensitivity of a few parts per billion (ppb). An additional important aspect of amino acid analyses of Martian samples is identifying and quantifying which compounds are present, and also distinguishing those produced abiotically from those synthesized by either extinct or extant life. Amino acid homochirality provides an unambiguous way of distinguishing between abiotic vs. biotic origins. Proteins made up of mixed D- and L-amino acids would not likely have been efficient catalysts in early organisms because they could not fold into bioactive configurations such as the a-helix. However, enzymes made up of all D-amino acids function just as well as those made up of only L-amino acids, but the two enzymes use the opposite stereoisomeric substrates. There are no biochemical reasons why L-amino acids would be favored over Damino acids. On Earth, the use of only L-amino acids in proteins by life is probably simply a matter of chance. We assume that if proteins and enzymes were a component of extinct or extant life on Mars, then amino acid homochirality would have been a requirement. However, the possibility that Martian life was (or is) based on D-amino acids would be equal to that based on L-amino acids. The detection of a nonracemic mixture of amino acids in a Martian sample would be strong evidence for the presence of an extinct or extant biota on Mars. The finding of an excess of D-amino acids would provide irrefutable evidence of unique Martian life that could not have been derived from seeding the planet with terrestrial life (or the seeding of the Earth with Martian life). In contrast, the presence of racemic amino acids, along with non-protein amino acids such as alpha-aminoisobutyric acid and isovaline, would be indicative

  19. Lactobacillus kefiri shows inter-strain variations in the amino acid sequence of the S-layer proteins.

    PubMed

    Malamud, Mariano; Carasi, Paula; Bronsoms, Sílvia; Trejo, Sebastián A; Serradell, María de Los Angeles

    2017-04-01

    The S-layer is a proteinaceous envelope constituted by subunits that self-assemble to form a two-dimensional lattice that covers the surface of different species of Bacteria and Archaea, and it could be involved in cell recognition of microbes among other several distinct functions. In this work, both proteomic and genomic approaches were used to gain knowledge about the sequences of the S-layer protein (SLPs) encoding genes expressed by six aggregative and sixteen non-aggregative strains of potentially probiotic Lactobacillus kefiri. Peptide mass fingerprint (PMF) analysis confirmed the identity of SLPs extracted from L. kefiri, and based on the homology with phylogenetically related species, primers located outside and inside the SLP-genes were employed to amplify genomic DNA. The O-glycosylation site SASSAS was found in all L. kefiri SLPs. Ten strains were selected for sequencing of the complete genes. The total length of the mature proteins varies from 492 to 576 amino acids, and all SLPs have a calculated pI between 9.37 and 9.60. The N-terminal region is relatively conserved and shows a high percentage of positively charged amino acids. Major differences among strains are found in the C-terminal region. Different groups could be distinguished regarding the mature SLPs and the similarities observed in the PMF spectra. Interestingly, SLPs of the aggregative strains are 100% homologous, although these strains were isolated from different kefir grains. This knowledge provides relevant data for better understanding of the mechanisms involved in SLPs functionality and could contribute to the development of products of biotechnological interest from potentially probiotic bacteria.

  20. CDSbank: taxonomy-aware extraction, selection, renaming and formatting of protein-coding DNA or amino acid sequences.

    PubMed

    Hazes, Bart

    2014-02-28

    Protein-coding DNA sequences and their corresponding amino acid sequences are routinely used to study relationships between sequence, structure, function, and evolution. The rapidly growing size of sequence databases increases the power of such comparative analyses but it makes it more challenging to prepare high quality sequence data sets with control over redundancy, quality, completeness, formatting, and labeling. Software tools for some individual steps in this process exist but manual intervention remains a common and time consuming necessity. CDSbank is a database that stores both the protein-coding DNA sequence (CDS) and amino acid sequence for each protein annotated in Genbank. CDSbank also stores Genbank feature annotation, a flag to indicate incomplete 5' and 3' ends, full taxonomic data, and a heuristic to rank the scientific interest of each species. This rich information allows fully automated data set preparation with a level of sophistication that aims to meet or exceed manual processing. Defaults ensure ease of use for typical scenarios while allowing great flexibility when needed. Access is via a free web server at http://hazeslab.med.ualberta.ca/CDSbank/. CDSbank presents a user-friendly web server to download, filter, format, and name large sequence data sets. Common usage scenarios can be accessed via pre-programmed default choices, while optional sections give full control over the processing pipeline. Particular strengths are: extract protein-coding DNA sequences just as easily as amino acid sequences, full access to taxonomy for labeling and filtering, awareness of incomplete sequences, and the ability to take one protein sequence and extract all synonymous CDS or identical protein sequences in other species. Finally, CDSbank can also create labeled property files to, for instance, annotate or re-label phylogenetic trees.

  1. Insulinotropic and muscle protein synthetic effects of branched-chain amino acids: potential therapy for type 2 diabetes and sarcopenia.

    PubMed

    Manders, Ralph J; Little, Jonathan P; Forbes, Scott C; Candow, Darren G

    2012-11-08

    The loss of muscle mass and strength with aging (i.e., sarcopenia) has a negative effect on functional independence and overall quality of life. One main contributing factor to sarcopenia is the reduced ability to increase skeletal muscle protein synthesis in response to habitual feeding, possibly due to a reduction in postprandial insulin release and an increase in insulin resistance. Branched-chain amino acids (BCAA), primarily leucine, increases the activation of pathways involved in muscle protein synthesis through insulin-dependent and independent mechanisms, which may help counteract the "anabolic resistance" to feeding in older adults. Leucine exhibits strong insulinotropic characteristics, which may increase amino acid availability for muscle protein synthesis, reduce muscle protein breakdown, and enhance glucose disposal to help maintain blood glucose homeostasis.

  2. Amino acid and structural variability of Yersinia pestis LcrV protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Anisimov, A P; Dentovskaya, S V; Panfertsev, E A

    2009-11-09

    The LcrV protein is a multifunctional virulence factor and protective antigen of the plague bacterium which is generally conserved between the epidemic strains of Yersinia pestis. They investigated the diversity in the LcrV sequences among non-epidemic Y. pestis strains which have a limited virulence in selected animal models and for humans. Sequencing of lcrV genes from ten Y. pestis strains belonging to different phylogenetic groups (subspecies) showed that the LcrV proteins possess four major variable hotspots at positions 18, 72, 273, and 324-326. These major variations, together with other minor substitutions in amino acid sequences, allowed them to classify themore » LcrV alleles into five sequence types (A-E). They observed that the strains of different Y. pestis subspecies can have the same typ of LcrV, and different types of LcrV can exist within the same natural plague focus. The LcrV polymorphisms were structurally analyzed by comparing the modeled structures of LcrV from all available strains. All changes except one occurred either in flexible regions or on the surface of the protein, but local chemical properties (i.e. those of a hydrophobic, hydrophilic, amphipathic, or charged nature) were conserved across all of the strains. Polymorphisms in flexible and surface regions are likely subject to less selective pressure, and have a limited impact on the structure. In contrast, the substitution of tryptophan at position 113 with either glutamic acid or glycine likely has a serious influence on the regional structure of the protein, and these mutations might have an effect on the function of LcrV. The polymorphisms at positions 18, 72 and 273 were accountable for differences in oligomerization of LcrV. The importance of the latter property in emergence of epidemic strains of Y. pestis during evolution of this pathogen will need to be further investigated.« less

  3. Non-invasive detection of candidate pregnancy protein biomarkers in the feces of captive polar bears (Ursus maritimus).

    PubMed

    Curry, E; Stoops, M A; Roth, T L

    2012-07-15

    Currently, there is no method of accurately and non-invasively diagnosing pregnancy in polar bears. Specific proteins may exhibit altered profiles in the feces of pregnant bears, but predicting appropriate candidate proteins to investigate is speculative at best. The objective of this study was to identify potential pregnancy biomarker proteins based on their increased abundance in the feces of pregnant polar bears compared to pseudopregnant females (controls) using two-dimensional in-gel electrophoresis (2D-DIGE) and mass spectrometry (MS). Three 2D-DIGE gels were performed to evaluate fecal protein profiles from controls (n=3) and pregnant polar bears (n=3). There were 2224.67±52.39 (mean±SEM) spots resolved per gel. Of these, only five proteins were elevated in the pregnant group (P<0.05), and seven additional spots tended to be higher (0.0599.9% confidence interval. The 11 spots represented seven distinct proteins, five of which were significantly more abundant in the pregnant group: IgGFc-binding protein, filamin-C, carboxypeptidase B, transthyretin, and immunoglobulin heavy chain variable region. To our knowledge, this was the first study that employed 2D-DIGE to identify differentially expressed proteins in fecal samples to characterize a physiological condition other than those related to gastrointestinal disorders. These promising results provided a strong foundation for ensuing efforts to develop a non-invasive pregnancy assay for use in both captive and wild polar bears. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Whey protein ingestion in elderly persons results in greater muscle protein accrual than ingestion of its constituent essential amino acid content.

    PubMed

    Katsanos, Christos S; Chinkes, David L; Paddon-Jones, Douglas; Zhang, Xiao-jun; Aarsland, Asle; Wolfe, Robert R

    2008-10-01

    It is recognized that both whey protein (WY) and essential amino acids (EAA) are stimuli for muscle protein anabolism. The aim of the present study was to determine if the effects of WY ingestion on muscle protein accrual in elderly persons are due solely to its constituent EAA content. Fifteen elderly persons were randomly assigned to ingest a bolus of either 15 g of WY, 6.72 g of EAA, or 7.57 g of nonessential amino acids (NEAA). We used the leg arteriovenous model to measure the leg phenylalanine balance, which is an index of muscle protein accrual. Phenylalanine balance (nmol x min(-1) kg lean leg mass(-1)) during the 3.5 hours after the bolus ingestion improved in the WY (-216 +/- 14 vs -105 +/- 19; P < .05) but not in the EAA (-203 +/- 21 vs -172 +/- 38; P > .05) or NEAA groups (-203 +/- 19 vs -204 +/- 21; P > .05). The insulin response (uIU x mL(-1) 210 min(-1)) during the same period was lower in both the NEAA (48 +/- 40) and EAA (213 +/- 127) when compared to the WY (1073 +/- 229; P < .05). In conclusion, WY ingestion improves skeletal muscle protein accrual through mechanisms that are beyond those attributed to its EAA content. This finding may have practical implications for the formulation of nutritional supplements to enhance muscle anabolism in older individuals.

  5. Amino acids of the Murchison meteorite. II - Five carbon acyclic primary beta-, gamma-, and delta-amino alkanoic acids

    NASA Technical Reports Server (NTRS)

    Cronin, J. R.; Pizzarello, S.; Yuen, G. U.

    1985-01-01

    The five-carbon acyclic primary beta, gamma, and delta amino alkanoic acids of the Murchison meteorite are studied using gas chromatography-mass spectrometry and ion exchange chromatography. The chromatograms reveal that alpha is the most abundant monoamino alkanoic acid followed by gamma and beta, and an exponential increase in the amount of amino acid is observed as the carbon number increases in the homologous series. The influence of frictional heating, spontaneous thermal decomposition, and radiation of the synthesis of amino acids is examined. The data obtained support an amino acid synthesis process involving random combination of single-carbon precursors.

  6. Potential impact of the digestible indispensable amino acid score as a measure of protein quality on dietary regulations and health

    PubMed Central

    Marinangeli, Christopher P F; House, James D

    2017-01-01

    Abstract Regulatory frameworks for protein content claims in Canada and the United States are underpinned by the protein efficiency ratio and protein digestibility-corrected amino acid score (PDCAAS), respectively, which are used to assess the protein quality of a given food. The digestible indispensable amino acid score (DIAAS) is a novel approach to measuring the protein quality of foods and is supported by the Food and Agriculture Organization of the United Nations. Methodological concerns about the PDCAAS are addressed by the DIAAS through introduction of the use of ileal amino acid digestibility coefficients and untruncated protein scores. However, before the DIAAS is widely adopted within regulatory frameworks, a comprehensive assessment is required. Accordingly, this review addresses the potential impact of the DIAAS on regulation, communication, and public health, as well as knowledge gaps, analytical challenges, and cost of implementation. A pragmatic approach to addressing protein quality is advocated by suggesting the use of conservative coefficients of digestibility that are derived from in vitro methods. Before adopting the DIAAS as a framework for supporting protein content claims, updated food-related regulations and policies should also be evaluated through a lens that anticipates the impact on consumer-facing nutrition communication, the adoption of dietary patterns that are nutritionally adequate, and a food value chain that fosters a spirit of food and nutritional innovation. PMID:28969364

  7. Absolute Quantification of Middle- to High-Abundant Plasma Proteins via Targeted Proteomics.

    PubMed

    Dittrich, Julia; Ceglarek, Uta

    2017-01-01

    The increasing number of peptide and protein biomarker candidates requires expeditious and reliable quantification strategies. The utilization of liquid chromatography coupled to quadrupole tandem mass spectrometry (LC-MS/MS) for the absolute quantitation of plasma proteins and peptides facilitates the multiplexed verification of tens to hundreds of biomarkers from smallest sample quantities. Targeted proteomics assays derived from bottom-up proteomics principles rely on the identification and analysis of proteotypic peptides formed in an enzymatic digestion of the target protein. This protocol proposes a procedure for the establishment of a targeted absolute quantitation method for middle- to high-abundant plasma proteins waiving depletion or enrichment steps. Essential topics as proteotypic peptide identification and LC-MS/MS method development as well as sample preparation and calibration strategies are described in detail.

  8. α-Amino Acid-Isosteric α-Amino Tetrazoles

    PubMed Central

    Zhao, Ting; Kurpiewska, Katarzyna; Kalinowska-Tłuścik, Justyna; Herdtweck, Eberhardt

    2016-01-01

    The synthesis of all 20 common natural proteinogenic and 4 otherα-amino acid-isosteric α-amino tetrazoles has been accomplished, whereby the carboxyl group is replaced by the isosteric 5-tetrazolyl group. The short process involves the use of the key Ugi tetrazole reaction followed by deprotection chemistries. The tetrazole group is bioisosteric to the carboxylic acid and is widely used in medicinal chemistry and drug design. Surprisingly, several of the common α-amino acid-isosteric α-amino tetrazoles are unknown up to now. Therefore a rapid synthetic access to this compound class and non-natural derivatives is of high interest to advance the field. PMID:26817531

  9. Comparative Study of Human and Mouse Postsynaptic Proteomes Finds High Compositional Conservation and Abundance Differences for Key Synaptic Proteins

    PubMed Central

    Bayés, Àlex; Collins, Mark O.; Croning, Mike D. R.; van de Lagemaat, Louie N.; Choudhary, Jyoti S.; Grant, Seth G. N.

    2012-01-01

    Direct comparison of protein components from human and mouse excitatory synapses is important for determining the suitability of mice as models of human brain disease and to understand the evolution of the mammalian brain. The postsynaptic density is a highly complex set of proteins organized into molecular networks that play a central role in behavior and disease. We report the first direct comparison of the proteome of triplicate isolates of mouse and human cortical postsynaptic densities. The mouse postsynaptic density comprised 1556 proteins and the human one 1461. A large compositional overlap was observed; more than 70% of human postsynaptic density proteins were also observed in the mouse postsynaptic density. Quantitative analysis of postsynaptic density components in both species indicates a broadly similar profile of abundance but also shows that there is higher abundance variation between species than within species. Well known components of this synaptic structure are generally more abundant in the mouse postsynaptic density. Significant inter-species abundance differences exist in some families of key postsynaptic density proteins including glutamatergic neurotransmitter receptors and adaptor proteins. Furthermore, we have identified a closely interacting set of molecules enriched in the human postsynaptic density that could be involved in dendrite and spine structural plasticity. Understanding synapse proteome diversity within and between species will be important to further our understanding of brain complexity and disease. PMID:23071613

  10. Genome-scale prediction of proteins with long intrinsically disordered regions.

    PubMed

    Peng, Zhenling; Mizianty, Marcin J; Kurgan, Lukasz

    2014-01-01

    Proteins with long disordered regions (LDRs), defined as having 30 or more consecutive disordered residues, are abundant in eukaryotes, and these regions are recognized as a distinct class of biologically functional domains. LDRs facilitate various cellular functions and are important for target selection in structural genomics. Motivated by the lack of methods that directly predict proteins with LDRs, we designed Super-fast predictor of proteins with Long Intrinsically DisordERed regions (SLIDER). SLIDER utilizes logistic regression that takes an empirically chosen set of numerical features, which consider selected physicochemical properties of amino acids, sequence complexity, and amino acid composition, as its inputs. Empirical tests show that SLIDER offers competitive predictive performance combined with low computational cost. It outperforms, by at least a modest margin, a comprehensive set of modern disorder predictors (that can indirectly predict LDRs) and is 16 times faster compared to the best currently available disorder predictor. Utilizing our time-efficient predictor, we characterized abundance and functional roles of proteins with LDRs over 110 eukaryotic proteomes. Similar to related studies, we found that eukaryotes have many (on average 30.3%) proteins with LDRs with majority of proteomes having between 25 and 40%, where higher abundance is characteristic to proteomes that have larger proteins. Our first-of-its-kind large-scale functional analysis shows that these proteins are enriched in a number of cellular functions and processes including certain binding events, regulation of catalytic activities, cellular component organization, biogenesis, biological regulation, and some metabolic and developmental processes. A webserver that implements SLIDER is available at http://biomine.ece.ualberta.ca/SLIDER/. Copyright © 2013 Wiley Periodicals, Inc.

  11. Roles of fragile X mental retardation protein in dopaminergic stimulation-induced synapse-associated protein synthesis and subsequent alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) receptor internalization.

    PubMed

    Wang, Hansen; Kim, Susan S; Zhuo, Min

    2010-07-09

    Fragile X syndrome, the most common form of inherited mental retardation, is caused by the absence of the RNA-binding protein fragile X mental retardation protein (FMRP). FMRP regulates local protein synthesis in dendritic spines. Dopamine (DA) is involved in the modulation of synaptic plasticity. Activation of DA receptors can regulate higher brain functions in a protein synthesis-dependent manner. Our recent study has shown that FMRP acts as a key messenger for DA modulation in forebrain neurons. Here, we demonstrate that FMRP is critical for DA D1 receptor-mediated synthesis of synapse-associated protein 90/PSD-95-associated protein 3 (SAPAP3) in the prefrontal cortex (PFC). DA D1 receptor stimulation induced dynamic changes of FMRP phosphorylation. The changes in FMRP phosphorylation temporally correspond with the expression of SAPAP3 after D1 receptor stimulation. Protein phosphatase 2A, ribosomal protein S6 kinase, and mammalian target of rapamycin are the key signaling molecules for FMRP linking DA D1 receptors to SAPAP3. Knockdown of SAPAP3 did not affect surface expression of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) GluR1 receptors induced by D1 receptor activation but impaired their subsequent internalization in cultured PFC neurons; the subsequent internalization of GluR1 was also impaired in Fmr1 knock-out PFC neurons, suggesting that FMRP may be involved in subsequent internalization of GluR1 through regulating the abundance of SAPAP3 after DA D1 receptor stimulation. Our study thus provides further insights into FMRP involvement in DA modulation and may help to reveal the molecular mechanisms underlying impaired learning and memory in fragile X syndrome.

  12. Revealing the role of oxidation state in interaction between nitro/amino-derived particulate matter and blood proteins

    PubMed Central

    Liu, Zhen; Li, Ping; Bian, Weiwei; Yu, Jingkai; Zhan, Jinhua

    2016-01-01

    Surface oxidation states of ultrafine particulate matter can influence the proinflammatory responses and reactive oxygen species levels in tissue. Surface active species of vehicle-emission soot can serve as electron transfer-mediators in mitochondrion. Revealing the role of surface oxidation state in particles-proteins interaction will promote the understanding on metabolism and toxicity. Here, the surface oxidation state was modeled by nitro/amino ligands on nanoparticles, the interaction with blood proteins were evaluated by capillary electrophoresis quantitatively. The nitro shown larger affinity than amino. On the other hand, the affinity to hemoglobin is 103 times larger than that to BSA. Further, molecular docking indicated the difference of binding intensity were mainly determined by hydrophobic forces and hydrogen bonds. These will deepen the quantitative understanding of protein-nanoparticles interaction from the perspective of surface chemical state. PMID:27181651

  13. Protein cold adaptation strategy via a unique seven-amino acid domain in the icefish (Chionodraco hamatus) PEPT1 transporter

    PubMed Central

    Rizzello, Antonia; Romano, Alessandro; Kottra, Gabor; Acierno, Raffaele; Storelli, Carlo; Verri, Tiziano; Daniel, Hannelore; Maffia, Michele

    2013-01-01

    Adaptation of organisms to extreme environments requires proteins to work at thermodynamically unfavorable conditions. To adapt to subzero temperatures, proteins increase the flexibility of parts of, or even the whole, 3D structure to compensate for the lower thermal kinetic energy available at low temperatures. This may be achieved through single-site amino acid substitutions in regions of the protein that undergo large movements during the catalytic cycle, such as in enzymes or transporter proteins. Other strategies of cold adaptation involving changes in the primary amino acid sequence have not been documented yet. In Antarctic icefish (Chionodraco hamatus) peptide transporter 1 (PEPT1), the first transporter cloned from a vertebrate living at subzero temperatures, we came upon a unique principle of cold adaptation. A de novo domain composed of one to six repeats of seven amino acids (VDMSRKS), placed as an extra stretch in the cytosolic COOH-terminal region, contributed per se to cold adaptation. VDMSRKS was in a protein region uninvolved in transport activity and, notably, when transferred to the COOH terminus of a warm-adapted (rabbit) PEPT1, it conferred cold adaptation to the receiving protein. Overall, we provide a paradigm for protein cold adaptation that relies on insertion of a unique domain that confers greater affinity and maximal transport rates at low temperatures. Due to its ability to transfer a thermal trait, the VDMSRKS domain represents a useful tool for future cell biology or biotechnological applications. PMID:23569229

  14. Amino Acid Selective 13C Labeling and 13C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance.

    PubMed

    Sugiki, Toshihiko; Furuita, Kyoko; Fujiwara, Toshimichi; Kojima, Chojiro

    2018-06-20

    Amino acid selective isotope labeling is an important nuclear magnetic resonance technique, especially for larger proteins, providing strong bases for the unambiguous resonance assignments and information concerning the structure, dynamics, and intermolecular interactions. Amino acid selective 15 N labeling suffers from isotope dilution caused by metabolic interconversion of the amino acids, resulting in isotope scrambling within the target protein. Carbonyl 13 C atoms experience less isotope scrambling than the main-chain 15 N atoms do. However, little is known about the side-chain 13 C atoms. Here, the 13 C scrambling profiles of the Cα and side-chain carbons were investigated for 15 N scrambling-prone amino acids, such as Leu, Ile, Tyr, Phe, Thr, Val, and Ala. The level of isotope scrambling was substantially lower in 13 Cα and 13 C side-chain labeling than in 15 N labeling. We utilized this reduced scrambling-prone character of 13 C as a simple and efficient method for amino acid selective 13 C labeling using an Escherichia coli cold-shock expression system and high-cell density fermentation. Using this method, the 13 C labeling efficiency was >80% for Leu and Ile, ∼60% for Tyr and Phe, ∼50% for Thr, ∼40% for Val, and 30-40% for Ala. 1 H- 15 N heteronuclear single-quantum coherence signals of the 15 N scrambling-prone amino acid were also easily filtered using 15 N-{ 13 Cα} spin-echo difference experiments. Our method could be applied to the assignment of the 55 kDa protein.

  15. Disordered nucleiome: Abundance of intrinsic disorder in the DNA- and RNA-binding proteins in 1121 species from Eukaryota, Bacteria and Archaea.

    PubMed

    Wang, Chen; Uversky, Vladimir N; Kurgan, Lukasz

    2016-05-01

    Intrinsically disordered proteins (IDPs) are abundant in various proteomes, where they play numerous important roles and complement biological activities of ordered proteins. Among functions assigned to IDPs are interactions with nucleic acids. However, often, such assignments are made based on the guilty-by-association principle. The validity of the extension of these correlations to all nucleic acid binding proteins has never been analyzed on a large scale across all domains of life. To fill this gap, we perform a comprehensive computational analysis of the abundance of intrinsic disorder and intrinsically disordered domains in nucleiomes (∼548 000 nucleic acid binding proteins) of 1121 species from Archaea, Bacteria and Eukaryota. Nucleiome is a whole complement of proteins involved in interactions with nucleic acids. We show that relative to other proteins in the corresponding proteomes, the DNA-binding proteins have significantly increased disorder content and are significantly enriched in disordered domains in Eukaryotes but not in Archaea and Bacteria. The RNA-binding proteins are significantly enriched in the disordered domains in Bacteria, Archaea and Eukaryota, while the overall abundance of disorder in these proteins is significantly increased in Bacteria, Archaea, animals and fungi. The high abundance of disorder in nucleiomes supports the notion that the nucleic acid binding proteins often require intrinsic disorder for their functions and regulation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. A Simple Method for Rapid Depletion of Rubisco from Soybean (Glycine max) Leaf for Proteomic Analysis of Lower Abundance Proteins

    USDA-ARS?s Scientific Manuscript database

    2-DE analysis of complex plant proteomes has limited dynamic resolution because only abundant proteins can be detected. Proteomic assessment of the low abundance proteins within leaf tissue is difficult when it is comprised of 30 – 50% of the CO2 fixation enzyme Rubisco. Resolution can be improved t...

  17. Fermentable soluble fibres spare amino acids in healthy dogs fed a low-protein diet.

    PubMed

    Wambacq, Wendy; Rybachuk, Galena; Jeusette, Isabelle; Rochus, Kristel; Wuyts, Brigitte; Fievez, Veerle; Nguyen, Patrick; Hesta, Myriam

    2016-06-28

    Research in cats has shown that increased fermentation-derived propionic acid and its metabolites can be used as alternative substrates for gluconeogenesis, thus sparing amino acids for other purposes. This amino acid sparing effect could be of particular interest in patients with kidney or liver disease, where this could reduce the kidneys'/liver's burden of N-waste removal. Since dogs are known to have a different metabolism than the obligatory carnivorous cat, the main objective of this study was to assess the possibility of altering amino acid metabolism through intestinal fermentation in healthy dogs. This was studied by supplementing a low-protein diet with fermentable fibres, hereby providing an initial model for future studies in dogs suffering from renal/liver disease. Eight healthy dogs were randomly assigned to one of two treatment groups: sugar beet pulp and guar gum mix (SF: soluble fibre, estimated to mainly stimulate propionic acid production) or cellulose (IF: insoluble fibre). Treatments were incorporated into a low-protein (17 %) extruded dry diet in amounts to obtain similar total dietary fibre (TDF) contents for both diets (9.4 % and 8.2 % for the SF and IF diet, respectively) and were tested in a 4-week crossover feeding trial. Apparent faecal nitrogen digestibility and post-prandial fermentation metabolites in faeces and plasma were evaluated. Dogs fed the SF diet showed significantly higher faecal excretion of acetic and propionic acid, resulting in a higher total SCFA excretion compared to IF. SF affected the three to six-hour postprandial plasma acylcarnitine profile by significantly increasing AUC of acetyl-, propionyl-, butyryl- + isobutyryl-, 3-OH-butyryl-, 3-OH-isovaleryl- and malonyl-L-carnitine. Moreover, the amino acid plasma profile at that time was modified as leucine + isoleucine concentrations were significantly increased by SF, and a similar trend for phenylalanine and tyrosine's AUC was found. These results indicate

  18. Protein Degradation Rate in Arabidopsis thaliana Leaf Growth and Development[OPEN

    PubMed Central

    Nelson, Clark J.; Castleden, Ian

    2017-01-01

    We applied 15N labeling approaches to leaves of the Arabidopsis thaliana rosette to characterize their protein degradation rate and understand its determinants. The progressive labeling of new peptides with 15N and measuring the decrease in the abundance of >60,000 existing peptides over time allowed us to define the degradation rate of 1228 proteins in vivo. We show that Arabidopsis protein half-lives vary from several hours to several months based on the exponential constant of the decay rate for each protein. This rate was calculated from the relative isotope abundance of each peptide and the fold change in protein abundance during growth. Protein complex membership and specific protein domains were found to be strong predictors of degradation rate, while N-end amino acid, hydrophobicity, or aggregation propensity of proteins were not. We discovered rapidly degrading subunits in a variety of protein complexes in plastids and identified the set of plant proteins whose degradation rate changed in different leaves of the rosette and correlated with leaf growth rate. From this information, we have calculated the protein turnover energy costs in different leaves and their key determinants within the proteome. PMID:28138016

  19. Predicting membrane protein types by fusing composite protein sequence features into pseudo amino acid composition.

    PubMed

    Hayat, Maqsood; Khan, Asifullah

    2011-02-21

    Membrane proteins are vital type of proteins that serve as channels, receptors, and energy transducers in a cell. Prediction of membrane protein types is an important research area in bioinformatics. Knowledge of membrane protein types provides some valuable information for predicting novel example of the membrane protein types. However, classification of membrane protein types can be both time consuming and susceptible to errors due to the inherent similarity of membrane protein types. In this paper, neural networks based membrane protein type prediction system is proposed. Composite protein sequence representation (CPSR) is used to extract the features of a protein sequence, which includes seven feature sets; amino acid composition, sequence length, 2 gram exchange group frequency, hydrophobic group, electronic group, sum of hydrophobicity, and R-group. Principal component analysis is then employed to reduce the dimensionality of the feature vector. The probabilistic neural network (PNN), generalized regression neural network, and support vector machine (SVM) are used as classifiers. A high success rate of 86.01% is obtained using SVM for the jackknife test. In case of independent dataset test, PNN yields the highest accuracy of 95.73%. These classifiers exhibit improved performance using other performance measures such as sensitivity, specificity, Mathew's correlation coefficient, and F-measure. The experimental results show that the prediction performance of the proposed scheme for classifying membrane protein types is the best reported, so far. This performance improvement may largely be credited to the learning capabilities of neural networks and the composite feature extraction strategy, which exploits seven different properties of protein sequences. The proposed Mem-Predictor can be accessed at http://111.68.99.218/Mem-Predictor. Copyright © 2010 Elsevier Ltd. All rights reserved.

  20. Motivated Proteins: A web application for studying small three-dimensional protein motifs

    PubMed Central

    Leader, David P; Milner-White, E James

    2009-01-01

    Background Small loop-shaped motifs are common constituents of the three-dimensional structure of proteins. Typically they comprise between three and seven amino acid residues, and are defined by a combination of dihedral angles and hydrogen bonding partners. The most abundant of these are αβ-motifs, asx-motifs, asx-turns, β-bulges, β-bulge loops, β-turns, nests, niches, Schellmann loops, ST-motifs, ST-staples and ST-turns. We have constructed a database of such motifs from a range of high-quality protein structures and built a web application as a visual interface to this. Description The web application, Motivated Proteins, provides access to these 12 motifs (with 48 sub-categories) in a database of over 400 representative proteins. Queries can be made for specific categories or sub-categories of motif, motifs in the vicinity of ligands, motifs which include part of an enzyme active site, overlapping motifs, or motifs which include a particular amino acid sequence. Individual proteins can be specified, or, where appropriate, motifs for all proteins listed. The results of queries are presented in textual form as an (X)HTML table, and may be saved as parsable plain text or XML. Motifs can be viewed and manipulated either individually or in the context of the protein in the Jmol applet structural viewer. Cartoons of the motifs imposed on a linear representation of protein secondary structure are also provided. Summary information for the motifs is available, as are histograms of amino acid distribution, and graphs of dihedral angles at individual positions in the motifs. Conclusion Motivated Proteins is a publicly and freely accessible web application that enables protein scientists to study small three-dimensional motifs without requiring knowledge of either Structured Query Language or the underlying database schema. PMID:19210785

  1. Analysis of the Plasma Proteome in COPD: Novel Low Abundance Proteins Reflect the Severity of Lung Remodeling

    PubMed Central

    Merali, Salim; Barrero, Carlos A.; Bowler, Russell P.; Chen, Diane Er; Criner, Gerard; Braverman, Alan; Litwin, Samuel; Yeung, Anthony; Kelsen, Steven G.

    2015-01-01

    The search for COPD biomarkers has largely employed a targeted approach that focuses on plasma proteins involved in the systemic inflammatory response and in lung injury and repair. This proof of concept study was designed to test the idea that an open, unbiased, in-depth proteomics approach could identify novel, low abundance plasma proteins i.e., ng/mL concentration, which could serve as potential biomarkers. Differentially expressed proteins were identified in a discovery group with severe COPD (FEV1 <45% predicted; n = 10). Subjects with normal lung function matched for age, sex, ethnicity and smoking history served as controls (n = 10). Pooled plasma from each group was exhaustively immunodepleted of abundant proteins, d separated by 1-D gel electrophoresis and extensively fractionated prior to LC-tandem mass spectroscopy (GeLC-MS). Thirty one differentially expressed proteins were identified in the discovery group including markers of lung defense against oxidant stress, alveolar macrophage activation, and lung tissue injury and repair. Four of the 31 proteins (i.e., GRP78, soluble CD163, IL1AP and MSPT9) were measured in a separate verification group of 80 subjects with varying COPD severity by immunoassay. All 4 were significantly altered in COPD and 2 (GRP78 and soluble CD163) correlated with both FEV1 and the extent of emphysema. In-depth, plasma proteomic analysis identified a group of novel, differentially expressed, low abundance proteins that reflect known pathogenic mechanisms and the severity of lung remodeling in COPD. These proteins may also prove useful as COPD biomarkers. PMID:24111704

  2. Analysis of the plasma proteome in COPD: Novel low abundance proteins reflect the severity of lung remodeling.

    PubMed

    Merali, Salim; Barrero, Carlos A; Bowler, Russell P; Chen, Diane Er; Criner, Gerard; Braverman, Alan; Litwin, Samuel; Yeung, Anthony; Kelsen, Steven G

    2014-04-01

    The search for COPD biomarkers has largely employed a targeted approach that focuses on plasma proteins involved in the systemic inflammatory response and in lung injury and repair. This proof of concept study was designed to test the idea that an open, unbiased, in-depth proteomics approach could identify novel, low abundance plasma proteins i.e., ng/mL concentration, which could serve as potential biomarkers. Differentially expressed proteins were identified in a discovery group with severe COPD (FEV1 <45% predicted; n = 10). Subjects with normal lung function matched for age, sex, ethnicity and smoking history served as controls (n = 10). Pooled plasma from each group was exhaustively immunodepleted of abundant proteins, d separated by 1-D gel electrophoresis and extensively fractionated prior to LC-tandem mass spectroscopy (GeLC-MS). Thirty one differentially expressed proteins were identified in the discovery group including markers of lung defense against oxidant stress, alveolar macrophage activation, and lung tissue injury and repair. Four of the 31 proteins (i.e., GRP78, soluble CD163, IL1AP and MSPT9) were measured in a separate verification group of 80 subjects with varying COPD severity by immunoassay. All 4 were significantly altered in COPD and 2 (GRP78 and soluble CD163) correlated with both FEV1 and the extent of emphysema. In-depth, plasma proteomic analysis identified a group of novel, differentially expressed, low abundance proteins that reflect known pathogenic mechanisms and the severity of lung remodeling in COPD. These proteins may also prove useful as COPD biomarkers.

  3. Unnatural reactive amino acid genetic code additions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Deiters, Alexander; Cropp, T. Ashton; Chin, Jason W.

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  4. Unnatural reactive amino acid genetic code additions

    DOEpatents

    Deiters, Alexander; Cropp, Ashton T; Chin, Jason W; Anderson, Christopher J; Schultz, Peter G

    2013-05-21

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  5. Unnatural reactive amino acid genetic code additions

    DOEpatents

    Deiters, Alexander [La Jolla, CA; Cropp, T Ashton [San Diego, CA; Chin, Jason W [Cambridge, GB; Anderson, J Christopher [San Francisco, CA; Schultz, Peter G [La Jolla, CA

    2011-02-15

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  6. Unnatural reactive amino acid genetic code additions

    DOEpatents

    Deiters, Alexander; Cropp, T. Ashton; Chin, Jason W.; Anderson, J. Christopher; Schultz, Peter G.

    2014-08-26

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  7. Unnatural reactive amino acid genetic code additions

    DOEpatents

    Deiters, Alexander [La Jolla, CA; Cropp, T Ashton [Bethesda, MD; Chin, Jason W [Cambridge, GB; Anderson, J Christopher [San Francisco, CA; Schultz, Peter G [La Jolla, CA

    2011-08-09

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNAsyn-thetases, pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  8. Quantifying protein synthesis and degradation in Arabidopsis by dynamic 13CO2 labeling and analysis of enrichment in individual amino acids in their free pools and in protein.

    PubMed

    Ishihara, Hirofumi; Obata, Toshihiro; Sulpice, Ronan; Fernie, Alisdair R; Stitt, Mark

    2015-05-01

    Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied (13)CO2 to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%-4% d(-1)), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark. © 2015 American Society of Plant Biologists. All Rights Reserved.

  9. Non-Arrhenius protein aggregation.

    PubMed

    Wang, Wei; Roberts, Christopher J

    2013-07-01

    Protein aggregation presents one of the key challenges in the development of protein biotherapeutics. It affects not only product quality but also potentially impacts safety, as protein aggregates have been shown to be linked with cytotoxicity and patient immunogenicity. Therefore, investigations of protein aggregation remain a major focus in pharmaceutical companies and academic institutions. Due to the complexity of the aggregation process and temperature-dependent conformational stability, temperature-induced protein aggregation is often non-Arrhenius over even relatively small temperature windows relevant for product development, and this makes low-temperature extrapolation difficult based simply on accelerated stability studies at high temperatures. This review discusses the non-Arrhenius nature of the temperature dependence of protein aggregation, explores possible causes, and considers inherent hurdles for accurately extrapolating aggregation rates from conventional industrial approaches for selecting accelerated conditions and from conventional or more advanced methods of analyzing the resulting rate data.

  10. Next-Generation Sequencing of Protein-Coding and Long Non-protein-Coding RNAs in Two Types of Exosomes Derived from Human Whole Saliva.

    PubMed

    Ogawa, Yuko; Tsujimoto, Masafumi; Yanoshita, Ryohei

    2016-01-01

    Exosomes are small extracellular vesicles containing microRNAs and mRNAs that are produced by various types of cells. We previously used ultrafiltration and size-exclusion chromatography to isolate two types of human salivary exosomes (exosomes I, II) that are different in size and proteomes. We showed that salivary exosomes contain large repertoires of small RNAs. However, precise information regarding long RNAs in salivary exosomes has not been fully determined. In this study, we investigated the compositions of protein-coding RNAs (pcRNAs) and long non-protein-coding RNAs (lncRNAs) of exosome I, exosome II and whole saliva (WS) by next-generation sequencing technology. Although 11% of all RNAs were commonly detected among the three samples, the compositions of reads mapping to known RNAs were similar. The most abundant pcRNA is ribosomal RNA protein, and pcRNAs of some salivary proteins such as S100 calcium-binding protein A8 (protein S100-A8) were present in salivary exosomes. Interestingly, lncRNAs of pseudogenes (presumably, processed pseudogenes) were abundant in exosome I, exosome II and WS. Translationally controlled tumor protein gene, which plays an important role in cell proliferation, cell death and immune responses, was highly expressed as pcRNA and pseudogenes in salivary exosomes. Our results show that salivary exosomes contain various types of RNAs such as pseudogenes and small RNAs, and may mediate intercellular communication by transferring these RNAs to target cells as gene expression regulators.

  11. Beta-and gamma-turns in proteins revisited: a new set of amino acid turn-type dependent positional preferences and potentials.

    PubMed

    Guruprasad, K; Rajkumar, S

    2000-06-01

    The number of beta-turns in a representative set of 426 protein three-dimensional crystal structures selected from the recent Protein Data Bank has nearly doubled and the number of gamma-turns in a representative set of 320 proteins has increased over seven times since the previous analysis. Beta-turns (7153) and gamma-turns (911) extracted from these proteins were used to derive a revised set of type-dependent amino acid positional preferences and potentials. Compared with previous results, the preference for proline, methionine and tryptophan has increased and the preference for glutamine, valine, glutamic acid and alanine has decreased for beta-turns. Certain new amino acid preferences were observed for both turn types and individual amino acids showed turn-type dependent positional preferences. The rationale for new amino acid preferences are discussed in the light of hydrogen bonds and other interactions involving the turns. Where main-chain hydrogen bonds of the type NH(i + 3) --> CO(i) were not observed for some beta-turns, other main-chain hydrogen bonds or solvent interactions were observed that possibly stabilize such beta-turns. A number of unexpected isolated beta-turns with proline at i + 2 position were also observed. The NH(i + 2) --> CO(i) hydrogen bond was observed for almost all gamma-turns. Nearly 20% classic gamma-turns and 43% inverse gamma-turns are isolated turns.

  12. Regulation of the proteome by amino acids.

    PubMed

    Bourgoin-Voillard, Sandrine; Goron, Arthur; Seve, Michel; Moinard, Christophe

    2016-03-01

    Besides their main contribution as substrates for protein synthesis, amino acids as signaling molecules could exert some regulatory functions on protein synthesis and/or proteolysis that have been emphasized in a number of recent studies. Several publications have highlighted supplemental roles of those amino acids in protein metabolism as well as in immunity, heat shock response, or apoptosis processes. In this way, via their regulatory properties, selected amino acids (such as leucine, glutamine, arginine, citrulline, or methionine) directly influence the proteome. In this review, we are proposing an overview of the regulation of the proteome by amino acids in mammals. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Non-natural amino acid peptide microarrays to discover Ebola virus glycoprotein ligands.

    PubMed

    Rabinowitz, Joshua A; Lainson, John C; Johnston, Stephen Albert; Diehnelt, Chris W

    2018-02-06

    We demonstrate a platform to screen a virus pseudotyped with Ebola virus glycoprotein (GP) against a library of peptides that contain non-natural amino acids to develop GP affinity ligands. This system could be used for rapid development of peptide-based antivirals for other emerging or neglected tropical infectious diseases.

  14. Characterizing hydrophobicity of amino acid side chains in a protein environment via measuring contact angle of a water nanodroplet on planar peptide network

    PubMed Central

    Zhu, Chongqin; Gao, Yurui; Li, Hui; Meng, Sheng; Li, Lei; Francisco, Joseph S.; Zeng, Xiao Cheng

    2016-01-01

    Hydrophobicity of macroscopic planar surface is conventionally characterized by the contact angle of water droplets. However, this engineering measurement cannot be directly extended to surfaces of proteins, due to the nanometer scale of amino acids and inherent nonplanar structures. To measure the hydrophobicity of side chains of proteins quantitatively, numerous parameters were developed to characterize behavior of hydrophobic solvation. However, consistency among these parameters is not always apparent. Herein, we demonstrate an alternative way of characterizing hydrophobicity of amino acid side chains in a protein environment by constructing a monolayer of amino acids (i.e., artificial planar peptide network) according to the primary and the β-sheet secondary structures of protein so that the conventional engineering measurement of the contact angle of a water droplet can be brought to bear. Using molecular dynamics simulations, contact angles θ of a water nanodroplet on the planar peptide network, together with excess chemical potentials of purely repulsive methane-sized Weeks−Chandler−Andersen solute, are computed. All of the 20 types of amino acids and the corresponding planar peptide networks are studied. Expectedly, all of the planar peptide networks with nonpolar amino acids are hydrophobic due to θ > 90°, whereas all of the planar peptide networks of the polar and charged amino acids are hydrophilic due to θ < 90°. Planar peptide networks of the charged amino acids exhibit complete-wetting behavior due to θ = 0°. This computational approach for characterization of hydrophobicity can be extended to artificial planar networks of other soft matter. PMID:27803319

  15. Characterizing hydrophobicity of amino acid side chains in a protein environment via measuring contact angle of a water nanodroplet on planar peptide network.

    PubMed

    Zhu, Chongqin; Gao, Yurui; Li, Hui; Meng, Sheng; Li, Lei; Francisco, Joseph S; Zeng, Xiao Cheng

    2016-11-15

    Hydrophobicity of macroscopic planar surface is conventionally characterized by the contact angle of water droplets. However, this engineering measurement cannot be directly extended to surfaces of proteins, due to the nanometer scale of amino acids and inherent nonplanar structures. To measure the hydrophobicity of side chains of proteins quantitatively, numerous parameters were developed to characterize behavior of hydrophobic solvation. However, consistency among these parameters is not always apparent. Herein, we demonstrate an alternative way of characterizing hydrophobicity of amino acid side chains in a protein environment by constructing a monolayer of amino acids (i.e., artificial planar peptide network) according to the primary and the β-sheet secondary structures of protein so that the conventional engineering measurement of the contact angle of a water droplet can be brought to bear. Using molecular dynamics simulations, contact angles θ of a water nanodroplet on the planar peptide network, together with excess chemical potentials of purely repulsive methane-sized Weeks-Chandler-Andersen solute, are computed. All of the 20 types of amino acids and the corresponding planar peptide networks are studied. Expectedly, all of the planar peptide networks with nonpolar amino acids are hydrophobic due to θ [Formula: see text] 90°, whereas all of the planar peptide networks of the polar and charged amino acids are hydrophilic due to θ [Formula: see text] 90°. Planar peptide networks of the charged amino acids exhibit complete-wetting behavior due to θ [Formula: see text] 0°. This computational approach for characterization of hydrophobicity can be extended to artificial planar networks of other soft matter.

  16. Protein digestibility-corrected amino acid scores for bean and bean-rice infant weaning food products.

    PubMed

    Kannan, S; Nielsen, S S; Mason, A C

    2001-10-01

    Vegetable proteins are an integral part of infant weaning diets in Latin America. Protein quality in plant-based products, however, is constrained by amino acid composition and intrinsically present antinutritional factors. The goal of this study was to improve bean protein quality by utilizing fermentation and germination processing. The objectives were to determine if protein quality, as measured by Food and Agricultural Organization (FAO) approved True Protein Digestibility (TPD) and Protein Digestibility-Corrected Amino Acid Scores (PDCAAS), of formulated bean-based weaning products could be improved upon fermentation and germination and if protein quality could be further improved when processed beans were combined with cooked rice. Results showed that the highest TPD and PDCAAS values were obtained for cooked germinated beans combined with rice. The TPD values for products ranged from 80 to 91%, and the PDCAAS values were 0.38-0.51. There was no significant increase (P < 0.05) of either TPD or PDCAAS values upon fermentation. Germination increased TPD of cooked bean products; this increase was not, however, accompanied by an increase in PDCAAS. When combined with rice, the PDCAAS values for all bean products improved significantly, thus supporting the concept of cereal-legume complementation. In conclusion, this study showed the range of PDCAAS in processed black bean and bean-rice infant weaning food products. The potential for incorporation of these products into the diets of weaning age Latin American children would, however, be confirmed only after validation with growth or metabolic balance studies in human infants.

  17. Seasonal proteomic changes reveal molecular adaptations to preserve and replenish liver proteins during ground squirrel hibernation

    PubMed Central

    Epperson, L. Elaine; Rose, James C.; Carey, Hannah V.

    2010-01-01

    Hibernators are unique among mammals in their ability to survive extended periods of time with core body temperatures near freezing and with dramatically reduced heart, respiratory, and metabolic rates in a state known as torpor. To gain insight into the molecular events underlying this remarkable physiological phenotype, we applied a proteomic screening approach to identify liver proteins that differ between the summer active (SA) and the entrance (Ent) phase of winter hibernation in 13-lined ground squirrels. The relative abundance of 1,600 protein spots separated on two-dimensional gels was quantitatively determined using fluorescence difference gel electrophoresis, and 74 unique proteins exhibiting significant differences between the two states were identified using liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). Proteins elevated in Ent hibernators included liver fatty acid-binding protein, fatty acid transporter, and 3-hydroxy-3-methylglutaryl-CoA synthase, which support the known metabolic fuel switch to lipid and ketone body utilization in winter. Several proteins involved in protein stability and protein folding were also elevated in the Ent phase, consistent with previous findings. In contrast to transcript screening results, there was a surprising increase in the abundance of proteins involved in protein synthesis during Ent hibernation, including several initiation and elongation factors. This finding, coupled with decreased abundance of numerous proteins involved in amino acid and nitrogen metabolism, supports the intriguing hypothesis that the mechanism of protein preservation and resynthesis is used by hibernating ground squirrels to help avoid nitrogen toxicity and ensure preservation of essential amino acids throughout the long winter fast. PMID:19923364

  18. Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR.

    PubMed

    Varkey, Jobin; Langen, Ralf

    2017-07-01

    The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single

  19. Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR

    NASA Astrophysics Data System (ADS)

    Varkey, Jobin; Langen, Ralf

    2017-07-01

    The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single

  20. Metals in proteins: correlation between the metal-ion type, coordination number and the amino-acid residues involved in the coordination.

    PubMed

    Dokmanić, Ivan; Sikić, Mile; Tomić, Sanja

    2008-03-01

    Metal ions are constituents of many metalloproteins, in which they have either catalytic (metalloenzymes) or structural functions. In this work, the characteristics of various metals were studied (Cu, Zn, Mg, Mn, Fe, Co, Ni, Cd and Ca in proteins with known crystal structure) as well as the specificity of their environments. The analysis was performed on two data sets: the set of protein structures in the Protein Data Bank (PDB) determined with resolution <1.5 A and the set of nonredundant protein structures from the PDB. The former was used to determine the distances between each metal ion and its electron donors and the latter was used to assess the preferred coordination numbers and common combinations of amino-acid residues in the neighbourhood of each metal. Although the metal ions considered predominantly had a valence of two, their preferred coordination number and the type of amino-acid residues that participate in the coordination differed significantly from one metal ion to the next. This study concentrates on finding the specificities of a metal-ion environment, namely the distribution of coordination numbers and the amino-acid residue types that frequently take part in coordination. Furthermore, the correlation between the coordination number and the occurrence of certain amino-acid residues (quartets and triplets) in a metal-ion coordination sphere was analysed. The results obtained are of particular value for the identification and modelling of metal-binding sites in protein structures derived by homology modelling. Knowledge of the geometry and characteristics of the metal-binding sites in metalloproteins of known function can help to more closely determine the biological activity of proteins of unknown function and to aid in design of proteins with specific affinity for certain metals.