Science.gov

Sample records for stability maintenance proteins

  1. Stabilization Pond Operation and Maintenance Manual.

    ERIC Educational Resources Information Center

    Sexauer, Willard N.; Karn, Roger V.

    This manual provides the waste stabilization pond operator with the basics necessary for the treatment of wastewater in stabilization ponds. The material is organized as a comprehensive guide that follows the normal operation and maintenance procedures from the time the wastewater enters the left station until it leaves the pond. A comprehensive…

  2. Stabilization Pond Operation and Maintenance Manual.

    ERIC Educational Resources Information Center

    Sexauer, Willard N.; Karn, Roger V.

    This manual provides the waste stabilization pond operator with the basics necessary for the treatment of wastewater in stabilization ponds. The material is organized as a comprehensive guide that follows the normal operation and maintenance procedures from the time the wastewater enters the left station until it leaves the pond. A comprehensive…

  3. Tumor suppressor protein DAB2IP participates in chromosomal stability maintenance through activating spindle assembly checkpoint and stabilizing kinetochore-microtubule attachments

    PubMed Central

    Yu, Lan; Shang, Zeng-Fu; Abdisalaam, Salim; Lee, Kyung-Jong; Gupta, Arun; Hsieh, Jer-Tsong; Asaithamby, Aroumougame; Chen, Benjamin P.C.; Saha, Debabrata

    2016-01-01

    Defects in kinetochore-microtubule (KT-MT) attachment and the spindle assembly checkpoint (SAC) during cell division are strongly associated with chromosomal instability (CIN). CIN has been linked to carcinogenesis, metastasis, poor prognosis and resistance to cancer therapy. We previously reported that the DAB2IP is a tumor suppressor, and that loss of DAB2IP is often detected in advanced prostate cancer (PCa) and is indicative of poor prognosis. Here, we report that the loss of DAB2IP results in impaired KT-MT attachment, compromised SAC and aberrant chromosomal segregation. We discovered that DAB2IP directly interacts with Plk1 and its loss inhibits Plk1 kinase activity, thereby impairing Plk1-mediated BubR1 phosphorylation. Loss of DAB2IP decreases the localization of BubR1 at the kinetochore during mitosis progression. In addition, the reconstitution of DAB2IP enhances the sensitivity of PCa cells to microtubule stabilizing drugs (paclitaxel, docetaxel) and Plk1 inhibitor (BI2536). Our findings demonstrate a novel function of DAB2IP in the maintenance of KT-MT structure and SAC regulation during mitosis which is essential for chromosomal stability. PMID:27568005

  4. Forces Stabilizing Proteins

    PubMed Central

    Pace, C. Nick; Scholtz, J. Martin; Grimsley, Gerald R.

    2014-01-01

    The goal of this article is to summarize what has been learned about the major forces stabilizing proteins since the late 1980s when site-directed mutagenesis became possible. The following conclusions are derived from experimental studies of hydrophobic and hydrogen bonding variants. 1. Based on studies of 138 hydrophobic interaction variants in 11 proteins, burying a –CH2– group on folding contributes 1.1 ± 0.5 kcal/mol to protein stability. 2. The burial of nonpolar side chains contributes to protein stability in two ways: first, a term that depends on the removal of the side chains from water and, more importantly, the enhanced London dispersion forces that result from the tight packing in the protein interior. 3. Based on studies of 151 hydrogen bonding variants in 15 proteins, forming a hydrogen bond on folding contributes 1.1 ± 0.8 kcal/mol to protein stability. 4. The contribution of hydrogen bonds to protein stability is strongly context dependent. 5. Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. 6. Polar group burial can make a favorable contribution to protein stability even if the polar group is not hydrogen bonded. 7. Hydrophobic interactions and hydrogen bonds both make large contributions to protein stability. PMID:24846139

  5. Getting Folded: Chaperone proteins in muscle development, maintenance and disease

    PubMed Central

    Smith, Daniel A.; Carland, Carmen R.; Guo, Yiming; Bernstein, Sanford I.

    2014-01-01

    Chaperone proteins are critical for protein folding and stability, and hence are necessary for normal cellular organization and function. Recent studies have begun to interrogate the role of this specialized class of proteins in muscle biology. During development, chaperone-mediated folding of client proteins enables their integration into nascent sarcomeres. In addition to assisting with muscle differentiation, chaperones play a key role in maintenance of muscle tissues. Further, disruption of the chaperone network can result in neuromuscular disease. In this review, we discuss how chaperones are involved in myofibrillogenesis, sarcomere maintenance and muscle disorders. We also consider the possibilities of therapeutically targeting chaperones to treat muscle disease. PMID:25125177

  6. Fanconi anemia proteins in telomere maintenance.

    PubMed

    Sarkar, Jaya; Liu, Yie

    2016-07-01

    Mammalian chromosome ends are protected by nucleoprotein structures called telomeres. Telomeres ensure genome stability by preventing chromosome termini from being recognized as DNA damage. Telomere length homeostasis is inevitable for telomere maintenance because critical shortening or over-lengthening of telomeres may lead to DNA damage response or delay in DNA replication, and hence genome instability. Due to their repetitive DNA sequence, unique architecture, bound shelterin proteins, and high propensity to form alternate/secondary DNA structures, telomeres are like common fragile sites and pose an inherent challenge to the progression of DNA replication, repair, and recombination apparatus. It is conceivable that longer the telomeres are, greater is the severity of such challenges. Recent studies have linked excessively long telomeres with increased tumorigenesis. Here we discuss telomere abnormalities in a rare recessive chromosomal instability disorder called Fanconi Anemia and the role of the Fanconi Anemia pathway in telomere biology. Reports suggest that Fanconi Anemia proteins play a role in maintaining long telomeres, including processing telomeric joint molecule intermediates. We speculate that ablation of the Fanconi Anemia pathway would lead to inadequate aberrant structural barrier resolution at excessively long telomeres, thereby causing replicative burden on the cell. Published by Elsevier B.V.

  7. Mdm31 and Mdm32 are inner membrane proteins required for maintenance of mitochondrial shape and stability of mitochondrial DNA nucleoids in yeast.

    PubMed

    Dimmer, Kai Stefan; Jakobs, Stefan; Vogel, Frank; Altmann, Katrin; Westermann, Benedikt

    2005-01-03

    The MDM31 and MDM32 genes are required for normal distribution and morphology of mitochondria in the yeast Saccharomyces cerevisiae. They encode two related proteins located in distinct protein complexes in the mitochondrial inner membrane. Cells lacking Mdm31 and Mdm32 harbor giant spherical mitochondria with highly aberrant internal structure. Mitochondrial DNA (mtDNA) is instable in the mutants, mtDNA nucleoids are disorganized, and their association with Mmm1-containing complexes in the outer membrane is abolished. Mutant mitochondria are largely immotile, resulting in a mitochondrial inheritance defect. Deletion of either one of the MDM31 and MDM32 genes is synthetically lethal with deletion of either one of the MMM1, MMM2, MDM10, and MDM12 genes, which encode outer membrane proteins involved in mitochondrial morphogenesis and mtDNA inheritance. We propose that Mdm31 and Mdm32 cooperate with Mmm1, Mmm2, Mdm10, and Mdm12 in maintenance of mitochondrial morphology and mtDNA.

  8. Hice1, a Novel Microtubule-Associated Protein Required for Maintenance of Spindle Integrity and Chromosomal Stability in Human Cells▿ †

    PubMed Central

    Wu, Guikai; Lin, Yi-Tzu; Wei, Randy; Chen, Yumay; Shan, Zhiyin; Lee, Wen-Hwa

    2008-01-01

    Spindle integrity is critical for efficient mitotic progression and accurate chromosome segregation. Deregulation of spindles often leads to structural and functional aberrations, ultimately promoting segregation errors and aneuploidy, a hallmark of most human cancers. Here we report the characterization of a previously identified human sarcoma antigen (gene located at 19p13.11), Hice1, an evolutionarily nonconserved 46-kDa coiled-coil protein. Hice1 shows distinct cytoplasmic localization and associates with interphase centrosomes and mitotic spindles, preferentially at the spindle pole vicinity. Depletion of Hice1 by RNA interference resulted in abnormal and unstable spindle configurations, mitotic delay at prometaphase and metaphase, and elevated aneuploidy. Conversely, loss of Hice1 had minimal effects on interphase centrosome duplication. We also found that both full-length Hice1 and Hice1-N1, which is composed of 149 amino acids of the N-terminal region, but not the mutant lacking the N-terminal region, exhibited activities of microtubule bundling and stabilization at a near-physiological concentration. Consistently, overexpression of Hice1 rendered microtubule bundles in cells resistant to nocodazole- or cold-treatment-induced depolymerization. These results demonstrate that Hice1 is a novel microtubule-associated protein important for maintaining spindle integrity and chromosomal stability, in part by virtue of its ability to bind, bundle, and stabilize microtubules. PMID:18362163

  9. Protein Stability in Ice

    PubMed Central

    Strambini, Giovanni B.; Gonnelli, Margherita

    2007-01-01

    This study presents an experimental approach, based on the change of Trp fluorescence between native and denatured states of proteins, which permits to monitor unfolding equilibria and the thermodynamic stability (ΔG°) of these macromolecules in frozen aqueous solutions. The results obtained by guanidinium chloride denaturation of the azurin mutant C112S from Pseudomonas aeruginosa, in the temperature range from −8 to −16°C, demonstrate that the stability of the native fold may be significantly perturbed in ice depending mainly on the size of the liquid water pool (VL) in equilibrium with the solid phase. The data establish a threshold, around VL = 1.5%, below which in ice ΔG° decreases progressively relative to liquid state, up to 3 kcal/mole for VL = 0.285%. The sharp dependence of ΔG° on VL is consistent with a mechanism based on adsorption of the protein to the ice surface. The reduction in ΔG° is accompanied by a corresponding decrease in m-value indicating that protein-ice interactions increase the solvent accessible surface area of the native fold or reduce that of the denatured state, or both. The method opens the possibility for examining in a more quantitative fashion the influence of various experimental conditions on the ice perturbation and in particular to test the effectiveness of numerous additives used in formulations to preserve labile pharmaco proteins. PMID:17189314

  10. Mechanisms of RecQ helicases in pathways of DNA metabolism and maintenance of genomic stability

    PubMed Central

    Sharma, Sudha; Doherty, Kevin M.; Brosh, Robert M.

    2006-01-01

    Helicases are molecular motor proteins that couple the hydrolysis of NTP to nucleic acid unwinding. The growing number of DNA helicases implicated in human disease suggests that their vital specialized roles in cellular pathways are important for the maintenance of genome stability. In particular, mutations in genes of the RecQ family of DNA helicases result in chromosomal instability diseases of premature aging and/or cancer predisposition. We will discuss the mechanisms of RecQ helicases in pathways of DNA metabolism. A review of RecQ helicases from bacteria to human reveals their importance in genomic stability by their participation with other proteins to resolve DNA replication and recombination intermediates. In the light of their known catalytic activities and protein interactions, proposed models for RecQ function will be summarized with an emphasis on how this distinct class of enzymes functions in chromosomal stability maintenance and prevention of human disease and cancer. PMID:16925525

  11. Mechanisms of RecQ helicases in pathways of DNA metabolism and maintenance of genomic stability.

    PubMed

    Sharma, Sudha; Doherty, Kevin M; Brosh, Robert M

    2006-09-15

    Helicases are molecular motor proteins that couple the hydrolysis of NTP to nucleic acid unwinding. The growing number of DNA helicases implicated in human disease suggests that their vital specialized roles in cellular pathways are important for the maintenance of genome stability. In particular, mutations in genes of the RecQ family of DNA helicases result in chromosomal instability diseases of premature aging and/or cancer predisposition. We will discuss the mechanisms of RecQ helicases in pathways of DNA metabolism. A review of RecQ helicases from bacteria to human reveals their importance in genomic stability by their participation with other proteins to resolve DNA replication and recombination intermediates. In the light of their known catalytic activities and protein interactions, proposed models for RecQ function will be summarized with an emphasis on how this distinct class of enzymes functions in chromosomal stability maintenance and prevention of human disease and cancer.

  12. Stabilized polyacrylic saccharide protein conjugates

    DOEpatents

    Callstrom, Matthew R.; Bednarski, Mark D.; Gruber, Patrick R.

    1996-01-01

    This invention is directed to water soluble protein polymer conjugates which are stabile in hostile environments. The conjugate comprises a protein which is linked to an acrylic polymer at multiple points through saccharide linker groups.

  13. Septins: Regulators of Protein Stability

    PubMed Central

    Vagin, Olga; Beenhouwer, David O.

    2016-01-01

    Septins are small GTPases that play a role in several important cellular processes. In this review, we focus on the roles of septins in protein stabilization. Septins may regulate protein stability by: (1) interacting with proteins involved in degradation pathways, (2) regulating the interaction between transmembrane proteins and cytoskeletal proteins, (3) affecting the mobility of transmembrane proteins in lipid bilayers, and (4) modulating the interaction of proteins with their adaptor or signaling proteins. In this context, we discuss the role of septins in protecting four different proteins from degradation. First we consider botulinum neurotoxin serotype A (BoNT/A) and the contribution of septins to its extraordinarily long intracellular persistence. Next, we discuss the role of septins in stabilizing the receptor tyrosine kinases EGFR and ErbB2. Finally, we consider the contribution of septins in protecting hypoxia-inducible factor 1α (HIF-1α) from degradation. PMID:28066764

  14. Macromolecular crowding and protein stability.

    PubMed

    Wang, Yaqiang; Sarkar, Mohona; Smith, Austin E; Krois, Alexander S; Pielak, Gary J

    2012-10-10

    An understanding of cellular chemistry requires knowledge of how crowded environments affect proteins. The influence of crowding on protein stability arises from two phenomena, hard-core repulsions and soft (i.e., chemical) interactions. Most efforts to understand crowding effects on protein stability, however, focus on hard-core repulsions, which are inherently entropic and stabilizing. We assessed these phenomena by measuring the temperature dependence of NMR-detected amide proton exchange and used these data to extract the entropic and enthalpic contributions of crowding to the stability of ubiquitin. Contrary to expectations, the contribution of chemical interactions is large and in many cases dominates the contribution from hardcore repulsions. Our results show that both chemical interactions and hard-core repulsions must be considered when assessing the effects of crowding and help explain previous observations about protein stability and dynamics in cells.

  15. Cep164 is a mediator protein required for the maintenance of genomic stability through modulation of MDC1, RPA, and CHK1

    PubMed Central

    Sivasubramaniam, Sudhakar; Sun, Xuemin; Pan, Yen-Ru; Wang, Shaohui; Lee, Eva Y.-H.P.

    2008-01-01

    The activation of the ataxia telangiectasia mutated (ATM) and ATM/Rad3-related (ATR) kinases triggers a diverse cellular response including the initiation of DNA damage-induced cell cycle checkpoints. Mediator of DNA Damage Checkpoint protein, MDC1, and H2AX are chromatin remodeling factors required for the recruitment of DNA repair proteins to the DNA damage sites. We identified a novel mediator protein, Cep164 (KIAA1052), that interacts with both ATR and ATM. Cep164 is phosphorylated upon replication stress, ultraviolet radiation (UV), and ionizing radiation (IR). Ser186 of Cep164 is phosphorylated by ATR/ATM in vitro and in vivo. The phosphorylation of Ser186 is not affected by RPA knockdown but is severely hampered by MDC1 knockdown. siRNA-mediated silencing of Cep164 significantly reduces DNA damage-induced phosphorylation of RPA, H2AX, MDC1, CHK2, and CHK1, but not NBS1. Analyses of Cep164 knockdown cells demonstrate a critical role of Cep164 in G2/M checkpoint and nuclear divisions. These findings reveal that Cep164 is a key player in the DNA damage-activated signaling cascade. PMID:18283122

  16. Protein stability in extremophilic archaea.

    PubMed

    Scandurra, R; Consalvi, V; Chiaraluce, R; Politi, L; Engel, P C

    2000-09-01

    Extremophilic microorganisms have adapted their molecular machinery to grow and thrive under the most adverse environmental conditions. These microorganisms have found their natural habitat at the boiling and freezing point of water, in high salt concentration and at extreme pH values. The extremophilic proteins, selected by Nature to withstand this evolutionary pressure, represent a wide research field for scientists from different disciplines and the study of the determinants of their stability has been an important task for basic and applied research. A surprising conclusion emerges from these studies: there are no general rules to achieve protein stabilization. Each extremophilic protein adopts various strategies and the outstanding adaptation to extreme temperature and solvent conditions is realized through the same weak electrostatic and hydrophobic interactions among the ordinary amino acid residues which are also responsible for the proper balance between protein stability and flexibility in mesophilic proteins.

  17. The role of stabilization centers in protein thermal stability

    SciTech Connect

    Magyar, Csaba; Gromiha, M. Michael; Sávoly, Zoltán; Simon, István

    2016-02-26

    The definition of stabilization centers was introduced almost two decades ago. They are centers of noncovalent long range interaction clusters, believed to have a role in maintaining the three-dimensional structure of proteins by preventing their decay due to their cooperative long range interactions. Here, this hypothesis is investigated from the viewpoint of thermal stability for the first time, using a large protein thermodynamics database. The positions of amino acids belonging to stabilization centers are correlated with available experimental thermodynamic data on protein thermal stability. Our analysis suggests that stabilization centers, especially solvent exposed ones, do contribute to the thermal stabilization of proteins. - Highlights: • Stabilization centers contribute to thermal stabilization of protein structures. • Stabilization center content correlates with melting temperature of proteins. • Exposed stabilization center content correlates with stability even in hyperthermophiles. • Stability changing mutations are frequently found at stabilization centers.

  18. Protein stability: a crystallographer’s perspective

    SciTech Connect

    Deller, Marc C.; Kong, Leopold; Rupp, Bernhard

    2016-01-26

    An understanding of protein stability is essential for optimizing the expression, purification and crystallization of proteins. In this review, discussion will focus on factors affecting protein stability on a somewhat practical level, particularly from the view of a protein crystallographer. Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed.

  19. Monitoring protein stability in vivo.

    PubMed

    Ignatova, Zoya

    2005-08-24

    Reduced protein stability in vivo is a prerequisite to aggregation. While this is merely a nuisance factor in recombinant protein production, it holds a serious impact for man. This review focuses on specific approaches to selectively determine the solubility and/or stability of a target protein within the complex cellular environment using different detection techniques. Noninvasive techniques mapping folding/misfolding events on a fast time scale can be used to unravel the complexity and dynamics of the protein aggregation process and factors altering protein solubility in vivo. The development of approaches to screen for folding and solubility in vivo should facilitate the identification of potential components that improve protein solubility and/or modulate misfolding and aggregation and may provide a therapeutic benefit.

  20. Impact of Cannabis Use During Stabilization on Methadone Maintenance Treatment

    PubMed Central

    Scavone, Jillian L.; Sterling, Robert C.; Weinstein, Stephen P.; Van Bockstaele, Elisabeth J.

    2016-01-01

    Background and Objectives Illicit drug use, particularly of cannabis, is common among opiate-dependent individuals, and has the potential to impact treatment in a negative manner. Methods To examine this, patterns of cannabis use prior to and during methadone maintenance treatment (MMT) were examined to assess possible cannabis-related effects on MMT, particularly during methadone stabilization. Retrospective chart analysis was used to examine outpatient records of patients undergoing MMT (n=91), focusing specifically on past and present cannabis use and its association with opiate abstinence, methadone dose stabilization, and treatment compliance. Results Objective rates of cannabis use were high during methadone induction, dropping significantly following dose stabilization. History of cannabis use correlated with cannabis use during MMT, but did not negatively impact the methadone induction process. Pilot data also suggested that objective ratings of opiate withdrawal decrease in MMT patients using cannabis during stabilization. Conclusions and Scientific Significance The present findings may point to novel interventions to be employed during treatment for opiate dependence that specifically target cannabinoid-opioid system interactions. PMID:23795873

  1. The role of stabilization centers in protein thermal stability.

    PubMed

    Magyar, Csaba; Gromiha, M Michael; Sávoly, Zoltán; Simon, István

    2016-02-26

    The definition of stabilization centers was introduced almost two decades ago. They are centers of noncovalent long range interaction clusters, believed to have a role in maintaining the three-dimensional structure of proteins by preventing their decay due to their cooperative long range interactions. Here, this hypothesis is investigated from the viewpoint of thermal stability for the first time, using a large protein thermodynamics database. The positions of amino acids belonging to stabilization centers are correlated with available experimental thermodynamic data on protein thermal stability. Our analysis suggests that stabilization centers, especially solvent exposed ones, do contribute to the thermal stabilization of proteins.

  2. Kinetic stability of membrane proteins.

    PubMed

    González Flecha, F Luis

    2017-09-18

    Although membrane proteins constitute an important class of biomolecules involved in key cellular processes, study of the thermodynamic and kinetic stability of their structures is far behind that of soluble proteins. It is known that many membrane proteins become unstable when removed by detergent extraction from the lipid environment. In addition, most of them undergo irreversible denaturation, even under mild experimental conditions. This process was found to be associated with partial unfolding of the polypeptide chain exposing hydrophobic regions to water, and it was proposed that the formation of kinetically trapped conformations could be involved. In this review, we will describe some of the efforts toward understanding the irreversible inactivation of membrane proteins. Furthermore, its modulation by phospholipids, ligands, and temperature will be herein discussed.

  3. Protein stability: a crystallographer’s perspective

    PubMed Central

    Deller, Marc C.; Kong, Leopold; Rupp, Bernhard

    2016-01-01

    Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed. PMID:26841758

  4. Cosolvent Effects on Protein Stability

    NASA Astrophysics Data System (ADS)

    Canchi, Deepak R.; García, Angel E.

    2013-04-01

    Proteins are marginally stable, and the folding/unfolding equilibrium of proteins in aqueous solution can easily be altered by the addition of small organic molecules known as cosolvents. Cosolvents that shift the equilibrium toward the unfolded ensemble are termed denaturants, whereas those that favor the folded ensemble are known as protecting osmolytes. Urea is a widely used denaturant in protein folding studies, and the molecular mechanism of its action has been vigorously debated in the literature. Here we review recent experimental as well as computational studies that show an emerging consensus in this problem. Urea has been shown to denature proteins through a direct mechanism, by interacting favorably with the peptide backbone as well as the amino acid side chains. In contrast, the molecular mechanism by which the naturally occurring protecting osmolyte trimethylamine N-oxide (TMAO) stabilizes proteins is not clear. Recent studies have established the strong interaction of TMAO with water. Detailed molecular simulations, when used with force fields that incorporate these interactions, can provide insight into this problem. We present the development of a model for TMAO that is consistent with experimental observations and that provides physical insight into the role of cosolvent-cosolvent interaction in determining its preferential interaction with proteins.

  5. Maintenance of genome stability in plants: repairing DNA double strand breaks and chromatin structure stability.

    PubMed

    Roy, Sujit

    2014-01-01

    Plant cells are subject to high levels of DNA damage resulting from plant's obligatory dependence on sunlight and the associated exposure to environmental stresses like solar UV radiation, high soil salinity, drought, chilling injury, and other air and soil pollutants including heavy metals and metabolic by-products from endogenous processes. The irreversible DNA damages, generated by the environmental and genotoxic stresses affect plant growth and development, reproduction, and crop productivity. Thus, for maintaining genome stability, plants have developed an extensive array of mechanisms for the detection and repair of DNA damages. This review will focus recent advances in our understanding of mechanisms regulating plant genome stability in the context of repairing of double stand breaks and chromatin structure maintenance.

  6. Unwinding protein complexes in ALTernative telomere maintenance.

    PubMed

    Bhattacharyya, Saumitri; Sandy, April; Groden, Joanna

    2010-01-01

    Telomeres are composed of specialized chromatin that includes DNA repair/recombination proteins, telomere DNA-binding proteins and a number of three dimensional nucleic acid structures including G-quartets and D-loops. A number of studies suggest that the BLM and WRN recQ-like helicases play important roles in recombination-mediated mechanisms of telomere elongation or Alternative Lengthening of Telomeres (ALT), processes that maintain/elongate telomeres in the absence of telomerase. BLM and WRN localize within ALT-associated nuclear bodies in telomerase-negative immortalized cell lines and interact with the telomere-specific proteins POT1, TRF1 and TRF2. Helicase activity is modulated by these interactions. BLM functions in DNA double-strand break repair processes such as non-homologous end joining, homologous recombination-mediated repair, resolution of stalled replication forks and synthesis-dependent strand annealing, although its precise functions at the telomeres are speculative. WRN also functions in DNA replication, recombination and repair, and in addition to its helicase domain, includes an exonuclease domain not found in other recQ-like helicases. The biochemical properties of BLM and WRN are, therefore, important in biological processes other than DNA replication, recombination and repair. In this review, we discuss some previous and recent findings of human rec-Q-like helicases and their role in telomere elongation during ALT processes.

  7. Protein diets, body weight loss and weight maintenance.

    PubMed

    Martens, Eveline A P; Westerterp-Plantenga, Margriet S

    2014-01-01

    The review addresses briefly the relevance of protein diets for body weight loss and weight maintenance. The addition of recent findings on age-dependent protein requirements, specific effects of protein intake and protein source, the relevance of the other dietary macronutrients, especially of 'low-carb', 'protein leverage', the mechanisms of protein-induced satiety, and food-reward makes the review up-to-date. Different effects of protein diets in different age groups result from age-dependent protein requirements that are primarily related to effects on body composition. A protein intake of 0.8 g/kg/day is sufficient to sustain a negative energy balance in adults, irrespective of the protein source. 'Low-carb' diets trace back to the protein-induced effects. Evidence that protein intake drives energy intake as suggested by the 'Protein leverage hypothesis' is scarce and equivocal. Finally, limited protein-induced food reward may affect compliance to a protein diet. An implication of the findings for clinical practice is that a protein intake of 0.8-1.2 g/kg/day is sufficient to sustain satiety, energy expenditure, and fat-free mass, independent of a dietary 'low-carb' content. Limited protein-induced food reward may affect compliance to a protein diet.

  8. Contribution of Hydrogen Bonds to Protein Stability

    NASA Astrophysics Data System (ADS)

    Pace, Nick

    2014-03-01

    I will discuss the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(Δ G), for a series of hydrogen bonding mutants in four proteins: villin head piece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A (1.1Å), Y51F(1.5Å), and T95A(1.3Å). The structures are very similar to wild type RNase Sa and the hydrogen bonding partners always form intermolecular hydrogen bonds to water in the mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions: 1) Hydrogen bonds contribute favorably to protein stability. 2) The contribution of hydrogen bonds to protein stability is strongly context dependent. 3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. 4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. 5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein.

  9. Contribution of hydrogen bonds to protein stability

    PubMed Central

    Pace, C Nick; Fu, Hailong; Fryar, Katrina Lee; Landua, John; Trevino, Saul R; Schell, David; Thurlkill, Richard L; Imura, Satoshi; Scholtz, J Martin; Gajiwala, Ketan; Sevcik, Jozef; Urbanikova, Lubica; Myers, Jeffery K; Takano, Kazufumi; Hebert, Eric J; Shirley, Bret A; Grimsley, Gerald R

    2014-01-01

    Our goal was to gain a better understanding of the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(ΔG), for a series of hydrogen bonding mutants in four proteins: villin headpiece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A, Y51F, and T95A. The structures are very similar to wild type RNase Sa and the hydrogen bonding partners form intermolecular hydrogen bonds to water in all three mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions. (1) Hydrogen bonds contribute favorably to protein stability. (2) The contribution of hydrogen bonds to protein stability is strongly context dependent. (3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. (5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein. PMID:24591301

  10. Selection of mutations for increased protein stability.

    PubMed

    van den Burg, Bertus; Eijsink, Vincent G H

    2002-08-01

    There are many ways to select mutations that increase the stability of proteins, including rational design, functional screening of randomly generated mutant libraries, and comparison of naturally occurring homologous proteins. The protein engineer's toolbox is expanding and the number of successful examples of engineered protein stability is increasing. Still, the selection of thermostable mutations is not a standard process. Selection is complicated by lack of knowledge of the process that leads to thermal inactivation and by the fact that proteins employ a large variety of structural tricks to achieve stability.

  11. Cytosolic selection systems to study protein stability.

    PubMed

    Malik, Ajamaluddin; Mueller-Schickert, Antje; Bardwell, James C A

    2014-12-01

    Here we describe biosensors that provide readouts for protein stability in the cytosolic compartment of prokaryotes. These biosensors consist of tripartite sandwich fusions that link the in vitro stability or aggregation susceptibility of guest proteins to the in vivo resistance of host cells to the antibiotics kanamycin, spectinomycin, and nourseothricin. These selectable markers confer antibiotic resistance in a wide range of hosts and are easily quantifiable. We show that mutations within guest proteins that affect their stability alter the antibiotic resistances of the cells expressing the biosensors in a manner that is related to the in vitro stabilities of the mutant guest proteins. In addition, we find that polyglutamine tracts of increasing length are associated with an increased tendency to form amyloids in vivo and, in our sandwich fusion system, with decreased resistance to aminoglycoside antibiotics. We demonstrate that our approach allows the in vivo analysis of protein stability in the cytosolic compartment without the need for prior structural and functional knowledge.

  12. A Light Harvesting Complex-Like Protein in Maintenance of Photosynthetic Components in Chlamydomonas.

    PubMed

    Zhao, Lei; Cheng, Dongmei; Huang, Xiahe; Chen, Mei; Dall'Osto, Luca; Xing, Jiale; Gao, Liyan; Li, Lingyu; Wang, Yale; Bassi, Roberto; Peng, Lianwei; Wang, Yingchun; Rochaix, Jean-David; Huang, Fang

    2017-08-01

    Using a genetic approach, we have identified and characterized a novel protein, named Msf1 (Maintenance factor for photosystem I), that is required for the maintenance of specific components of the photosynthetic apparatus in the green alga Chlamydomonas reinhardtii Msf1 belongs to the superfamily of light-harvesting complex proteins with three transmembrane domains and consensus chlorophyll-binding sites. Loss of Msf1 leads to reduced accumulation of photosystem I and chlorophyll-binding proteins/complexes. Msf1is a component of a thylakoid complex containing key enzymes of the tetrapyrrole biosynthetic pathway, thus revealing a possible link between Msf1 and chlorophyll biosynthesis. Protein interaction assays and greening experiments demonstrate that Msf1 interacts with Copper target homolog1 (CHL27B) and accumulates concomitantly with chlorophyll in Chlamydomonas, implying that chlorophyll stabilizes Msf1. Contrary to other light-harvesting complex-like genes, the expression of Msf1 is not stimulated by high-light stress, but its protein level increases significantly under heat shock, iron and copper limitation, as well as in stationary cells. Based on these results, we propose that Msf1 is required for the maintenance of photosystem I and specific protein-chlorophyll complexes especially under certain stress conditions. © 2017 American Society of Plant Biologists. All Rights Reserved.

  13. Contribution of Hydrophobic Interactions to Protein Stability

    PubMed Central

    Pace, C. Nick; Fu, Hailong; Fryar, Katrina Lee; Landua, John; Trevino, Saul R.; Shirley, Bret A.; Hendricks, Marsha McNutt; Iimura, Satoshi; Gajiwala, Ketan; Scholtz, J. Martin; Grimsley, Gerald R.

    2011-01-01

    Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin head piece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compare our results with previous studies and reach the following conclusions. 1. Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6 ± 0.3 kcal/mole per –CH2– group), than to the stability of a large protein, VlsE (1.6 ± 0.3 kcal/mol per –CH2– group). 2. Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kcal/mol): Phe 18 (3.9), Met 13 (3.1), Phe 7 (2.9), Phe 11 (2.7), and Leu 21 (2.7). 3. Based on Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a –CH2– group on folding contributes, on average, 1.1 ± 0.5 kcal/mol to protein stability. 4. The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔGtr values from water to cyclohexane. 5. For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60 ± 4% and hydrogen bonds 40 ± 4% to protein stability. 6. Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominately by hydrophobic interactions. PMID:21377472

  14. Contribution of hydrophobic interactions to protein stability.

    PubMed

    Pace, C Nick; Fu, Hailong; Fryar, Katrina Lee; Landua, John; Trevino, Saul R; Shirley, Bret A; Hendricks, Marsha McNutt; Iimura, Satoshi; Gajiwala, Ketan; Scholtz, J Martin; Grimsley, Gerald R

    2011-05-06

    Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin headpiece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compared our results with those of previous studies and reached the following conclusions: (1) Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6±0.3 kcal/mol per -CH(2)- group), than to the stability of a large protein, VlsE (1.6±0.3 kcal/mol per -CH(2)- group). (2) Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kilocalories per mole) Phe18 (3.9), Met13 (3.1), Phe7 (2.9), Phe11 (2.7), and Leu21 (2.7). (3) Based on the Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a -CH(2)- group on folding contributes, on average, 1.1±0.5 kcal/mol to protein stability. (4) The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔG(tr) values from water to cyclohexane. (5) For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60±4% and hydrogen bonds contribute 40±4% to protein stability. (6) Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominantly by hydrophobic interactions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. Protein interactions in genome maintenance as novel antibacterial targets.

    PubMed

    Marceau, Aimee H; Bernstein, Douglas A; Walsh, Brian W; Shapiro, Walker; Simmons, Lyle A; Keck, James L

    2013-01-01

    Antibacterial compounds typically act by directly inhibiting essential bacterial enzyme activities. Although this general mechanism of action has fueled traditional antibiotic discovery efforts for decades, new antibiotic development has not kept pace with the emergence of drug resistant bacterial strains. These limitations have severely restricted the therapeutic tools available for treating bacterial infections. Here we test an alternative antibacterial lead-compound identification strategy in which essential protein-protein interactions are targeted rather than enzymatic activities. Bacterial single-stranded DNA-binding proteins (SSBs) form conserved protein interaction "hubs" that are essential for recruiting many DNA replication, recombination, and repair proteins to SSB/DNA nucleoprotein substrates. Three small molecules that block SSB/protein interactions are shown to have antibacterial activity against diverse bacterial species. Consistent with a model in which the compounds target multiple SSB/protein interactions, treatment of Bacillus subtilis cultures with the compounds leads to rapid inhibition of DNA replication and recombination, and ultimately to cell death. The compounds also have unanticipated effects on protein synthesis that could be due to a previously unknown role for SSB/protein interactions in translation or to off-target effects. Our results highlight the potential of targeting protein-protein interactions, particularly those that mediate genome maintenance, as a powerful approach for identifying new antibacterial compounds.

  16. Protein stabilization utilizing a redefined codon

    PubMed Central

    Ohtake, Kazumasa; Yamaguchi, Atsushi; Mukai, Takahito; Kashimura, Hiroki; Hirano, Nobutaka; Haruki, Mitsuru; Kohashi, Sosuke; Yamagishi, Kenji; Murayama, Kazutaka; Tomabechi, Yuri; Itagaki, Takashi; Akasaka, Ryogo; Kawazoe, Masahito; Takemoto, Chie; Shirouzu, Mikako; Yokoyama, Shigeyuki; Sakamoto, Kensaku

    2015-01-01

    Recent advances have fundamentally changed the ways in which synthetic amino acids are incorporated into proteins, enabling their efficient and multiple-site incorporation, in addition to the 20 canonical amino acids. This development provides opportunities for fresh approaches toward addressing fundamental problems in bioengineering. In the present study, we showed that the structural stability of proteins can be enhanced by integrating bulky halogenated amino acids at multiple selected sites. Glutathione S-transferase was thus stabilized significantly (by 5.2 and 5.6 kcal/mol) with 3-chloro- and 3-bromo-l-tyrosines, respectively, incorporated at seven selected sites. X-ray crystallographic analyses revealed that the bulky halogen moieties filled internal spaces within the molecules, and formed non-canonical stabilizing interactions with the neighboring residues. This new mechanism for protein stabilization is quite simple and applicable to a wide range of proteins, as demonstrated by the rapid stabilization of the industrially relevant azoreductase. PMID:25985257

  17. Protein stabilization utilizing a redefined codon.

    PubMed

    Ohtake, Kazumasa; Yamaguchi, Atsushi; Mukai, Takahito; Kashimura, Hiroki; Hirano, Nobutaka; Haruki, Mitsuru; Kohashi, Sosuke; Yamagishi, Kenji; Murayama, Kazutaka; Tomabechi, Yuri; Itagaki, Takashi; Akasaka, Ryogo; Kawazoe, Masahito; Takemoto, Chie; Shirouzu, Mikako; Yokoyama, Shigeyuki; Sakamoto, Kensaku

    2015-05-18

    Recent advances have fundamentally changed the ways in which synthetic amino acids are incorporated into proteins, enabling their efficient and multiple-site incorporation, in addition to the 20 canonical amino acids. This development provides opportunities for fresh approaches toward addressing fundamental problems in bioengineering. In the present study, we showed that the structural stability of proteins can be enhanced by integrating bulky halogenated amino acids at multiple selected sites. Glutathione S-transferase was thus stabilized significantly (by 5.2 and 5.6 kcal/mol) with 3-chloro- and 3-bromo-l-tyrosines, respectively, incorporated at seven selected sites. X-ray crystallographic analyses revealed that the bulky halogen moieties filled internal spaces within the molecules, and formed non-canonical stabilizing interactions with the neighboring residues. This new mechanism for protein stabilization is quite simple and applicable to a wide range of proteins, as demonstrated by the rapid stabilization of the industrially relevant azoreductase.

  18. Stability and solubility of proteins from extremophiles.

    PubMed

    Greaves, Richard B; Warwicker, Jim

    2009-03-13

    Charges are important for hyperthermophile protein structure and function. However, the number of charges and their predicted contributions to folded state stability are not correlated, implying that more charge does not imply greater stability. The charge properties that distinguish hyperthermophile proteins also differentiate psychrophile proteins from mesophile proteins, but in the opposite direction and to a smaller extent. We conclude that charge number relates to solubility, whereas protein stability is determined by charge location. Most other structural properties are poorly separated over the ambient temperature range, apart from the burial of certain amino acids. Of particular interest are large non-polar sidechains that tend to increased exposure in proteins evolved to function at higher temperatures. Looking at tryptophan in more detail, this increase is often located close to the termini of secondary structure elements, and is discussed in terms of a novel potential role in protein thermostabilisation.

  19. Stabilized polyacrylic saccharide protein conjugates

    DOEpatents

    Callstrom, M.R.; Bednarski, M.D.; Gruber, P.R.

    1996-02-20

    This invention is directed to water soluble protein polymer conjugates which are stable in hostile environments. The conjugate comprises a protein which is linked to an acrylic polymer at multiple points through saccharide linker groups. 16 figs.

  20. Amphiphiles for protein solubilization and stabilization

    DOEpatents

    Gellman, Samuel Helmer; Chae, Pil Seok; Laible, Phillip D; Wander, Marc J

    2014-11-04

    The invention provides amphiphiles for manipulating membrane proteins. The amphiphiles can feature carbohydrate-derived hydrophilic groups and branchpoints in the hydrophilic moiety and/or in a lipophilic moiety. Such amphiphiles are useful as detergents for solubilization and stabilization of membrane proteins, including photosynthetic protein superassemblies obtained from bacterial membranes.

  1. Amphiphiles for protein solubilization and stabilization

    DOEpatents

    Gellman, Samuel Helmer; Chae, Pil Seok; Laible, Philip D.; Wander, Marc J.

    2012-09-11

    The invention provides amphiphiles for manipulating membrane proteins. The amphiphiles can feature carbohydrate-derived hydrophilic groups and branchpoints in the hydrophilic moiety and/or in a lipophilic moiety. Such amphiphiles are useful as detergents for solubilization and stabilization of membrane proteins, including photosynthetic protein superassemblies obtained from bacterial membranes.

  2. A prevalent intraresidue hydrogen bond stabilizes proteins

    PubMed Central

    Newberry, Robert W.; Raines, Ronald T.

    2016-01-01

    Current limitations in de novo protein structure prediction and design suggest an incomplete understanding of the interactions that govern protein folding. Here we demonstrate that previously unappreciated hydrogen bonds occur within proteins between the amide proton and carbonyl oxygen of the same residue. Quantum calculations, infrared spectroscopy, and nuclear magnetic resonance spectroscopy show that these interactions share hallmark features of canonical hydrogen bonds. Biophysical analyses demonstrate that selective attenuation or enhancement of these C5 hydrogen bonds affects the stability of synthetic β-sheets. These interactions are common, affecting approximately 5% of all residues and 94% of proteins, and their cumulative impact provides several kcal/mol of conformational stability to a typical protein. C5 hydrogen bonds stabilize, especially, the flat β-sheets of the amyloid state, which is linked with Alzheimer’s disease and other neurodegenerative disorders. Inclusion of these interactions in computational force fields would improve models of protein folding, function, and dysfunction. PMID:27748749

  3. Protein-stabilized magnetic fluids

    NASA Astrophysics Data System (ADS)

    Soenen, S. J. H.; Hodenius, M.; Schmitz-Rode, T.; De Cuyper, M.

    The adsorption of bovine serum albumin (BSA) and egg yolk phosvitin on magnetic fluid particles was investigated. Incubation mixtures were prepared by mixing an alkaline suspension of tetramethylammonium-coated magnetite cores with protein solutions at various protein/Fe 3O 4 ratios, followed by dialysis against a 5 mM TES buffer (pH 7.0), after which separation of bound and non-bound protein by high-gradient magnetophoresis was executed. Both the kinetic profiles as well as the isotherms of adsorption strongly differed for both proteins. In case of the spherical BSA, initially, abundant adsorption occurred, then it decreased and—at high protein concentrations—it slowly raised again. In contrast, with the highly phosphorylated phosvitin, binding slowly started and the extent of protein adsorption remained unchanged both as a function of time and phosvitin concentration. Competition binding studies, using binary protein mixtures composed of equal weight amounts of BSA and phosvitin, showed that binding of the latter protein is 'unrealistically' high. Based on the geometry of the two proteins, putative pictures on their orientation on the particle's surface in the various experimental conditions were deduced.

  4. Improvement of interfacial protein stability by CHAPS.

    PubMed

    Sah, Hongkee; Kim, Kil-Soo

    2006-04-01

    Emulsification of aqueous protein solutions in methylene chloride triggered the formation of water-insoluble aggregates at a water/methylene chloride interface. As a result, the amounts of beta-lactoglobulin and ovalbumin recovered in water were 36 and 44%, respectively. Addition of 5 mM: CHAPS in the aqueous phase raised the degree of beta-lactoglobulin recovery to 96%. Sodium taurocholate, however, failed to improve protein recovery. The stabilizing effect of CHAPS was also protein-specific and concentration-dependent: at >or=5 mM: , the surfactant caused unfolding of ovalbumin to make a water-soluble oligomer. CHAPS thus stabilizes proteins at an interface.

  5. Maintenance and stability of introduced genotypes in groundwater aquifer material

    SciTech Connect

    Jain, R.K.; Sayler, G.S.; Wilson, J.T.; Houston, L.; Pacia, D.

    1987-05-01

    Three indigenous groundwater bacterial strains and Pseudomonas putida harboring plasmids TOL (pWWO) and RK2 were introduced into experimentally contaminated groundwater aquifer microcosms. Maintenance of the introduced genotypes was measured over time by colony hybridization with gene probes of various specificity. On the basis of the results of colony hybridization quantitation of the introduced organisms and genes, all introduced genotypes were stably maintained at approximately 10/sup 5/ positive hybrid colonies g/sup -1/ of aquifer microcosm material throughout an 8-week incubation period. Concomitant removal of the environmental contaminants, viz., toluene, chlorobenzene, and styrene, in both natural (uninoculated) and inoculated aquifer microcosms was also demonstrated. The results indicate that introduced catabolic plasmids, as well as indigenous organisms, can be stably maintained in groundwater aquifer material without specific selective pressure for the introduced genotypes. These results have positive implications for in situ treatment and biodegradation in contaminated aerobic groundwater aquifers.

  6. Maintenance and stability of introduced genotypes in groundwater aquifer material.

    PubMed Central

    Jain, R K; Sayler, G S; Wilson, J T; Houston, L; Pacia, D

    1987-01-01

    Three indigenous groundwater bacterial strains and Pseudomonas putida harboring plasmids TOL (pWWO) and RK2 were introduced into experimentally contaminated groundwater aquifer microcosms. Maintenance of the introduced genotypes was measured over time by colony hybridization with gene probes of various specificity. On the basis of the results of colony hybridization quantitation of the introduced organisms and genes, all introduced genotypes were stably maintained at approximately 10(5) positive hybrid colonies g-1 of aquifer microcosm material throughout an 8-week incubation period. Concomitant removal of the environmental contaminants, viz., toluene, chlorobenzene, and styrene, in both natural (uninoculated) and inoculated aquifer microcosms was also demonstrated. The results indicate that introduced catabolic plasmids, as well as indigenous organisms, can be stably maintained in groundwater aquifer material without specific selective pressure for the introduced genotypes. These results have positive implications for in situ treatment and biodegradation in contaminated aerobic groundwater aquifers. Images PMID:3300546

  7. Enhancing recombinant protein quality and yield by protein stability profiling.

    PubMed

    Mezzasalma, Tara M; Kranz, James K; Chan, Winnie; Struble, Geoffrey T; Schalk-Hihi, Céline; Deckman, Ingrid C; Springer, Barry A; Todd, Matthew J

    2007-04-01

    The reliable production of large amounts of stable, high-quality proteins is a major challenge facing pharmaceutical protein biochemists, necessary for fulfilling demands from structural biology, for high-throughput screening, and for assay purposes throughout early discovery. One strategy for bypassing purification challenges in problematic systems is to engineer multiple forms of a particular protein to optimize expression, purification, and stability, often resulting in a nonphysiological sub-domain. An alternative strategy is to alter process conditions to maximize wild-type construct stability, based on a specific protein stability profile (PSP). ThermoFluor, a miniaturized 384-well thermal stability assay, has been implemented as a means of monitoring solution-dependent changes in protein stability, complementing the protein engineering and purification processes. A systematic analysis of pH, buffer or salt identity and concentration, biological metals, surfactants, and common excipients in terms of an effect on protein stability rapidly identifies conditions that might be used (or avoided) during protein production. Two PSPs are presented for the kinase catalytic domains of Akt-3 and cFMS, in which information derived from a ThermoFluor PSP led to an altered purification strategy, improving the yield and quality of the protein using the primary sequences of the catalytic domains.

  8. Stability of proteins inside a hydrophobic cavity

    NASA Astrophysics Data System (ADS)

    Radhakrishna, Mithun; Sharma, Sumit; Kumar, Sanat K.

    2011-03-01

    Previous studies have shown that enclosing a protein in an athermal cavity stabilizes the protein against reversible unfolding by virtue of eliminating many open chain conformations. Examples of such confined spaces include pores in chromatographic columns, Anfinsen's cage in Chaperonins, interiors of Ribosomes or regions of steric occlusion inside cells. However, the situation is more complex inside a hydrophobic cavity. The protein has a tendency to adsorb on the surface of the hydrophobic cavity, but at the same time it loses conformational entropy because of confinement. We study this system using a simple Hydrophobic Polar (HP) lattice protein model. Canonical Monte Carlo (MC) simulations at different temperatures and surface hydrophobicity show that proteins are stabilized at low and moderate hydrophobicity upon adsorption. The range of surface hydrophobicity over which a protein is stable increases with a decrease in radius of the cavity.

  9. Stabilizing effect of knots on proteins.

    PubMed

    Sułkowska, Joanna I; Sulkowski, Piotr; Szymczak, P; Cieplak, Marek

    2008-12-16

    Molecular dynamics studies within a coarse-grained, structure-based model were used on two similar proteins belonging to the transcarbamylase family to probe the effects of the knot in the native structure of a protein. The first protein, N-acetylornithine transcarbamylase, contains no knot, whereas human ormithine transcarbamylase contains a trefoil knot located deep within the sequence. In addition, we also analyzed a modified transferase with the knot removed by the appropriate change of a knot-making crossing of the protein chain. The studies of thermally and mechanically induced unfolding processes suggest a larger intrinsic stability of the protein with the knot.

  10. Stabilizing effect of knots on proteins

    PubMed Central

    Sułkowska, Joanna I.; Sułkowski, Piotr; Szymczak, P.; Cieplak, Marek

    2008-01-01

    Molecular dynamics studies within a coarse-grained, structure-based model were used on two similar proteins belonging to the transcarbamylase family to probe the effects of the knot in the native structure of a protein. The first protein, N-acetylornithine transcarbamylase, contains no knot, whereas human ormithine transcarbamylase contains a trefoil knot located deep within the sequence. In addition, we also analyzed a modified transferase with the knot removed by the appropriate change of a knot-making crossing of the protein chain. The studies of thermally and mechanically induced unfolding processes suggest a larger intrinsic stability of the protein with the knot. PMID:19064918

  11. Biochemical function of mouse minichromosome maintenance 2 protein.

    PubMed

    Ishimi, Y; Komamura, Y; You, Z; Kimura, H

    1998-04-03

    Minichromosome maintenance (MCM) proteins play an essential role in eukaryotic DNA replication and bind to chromatin before the initiation of DNA replication. We reported that MCM protein complexes consisting of MCM2, -4, -6, and -7 bind strongly to a histone-Sepharose column (Ishimi, Y., Ichinose, S., Omori, A., Sato, K., and Kimura, H. (1996) J. Biol. Chem. 271, 24115-24122). Here, we have analyzed this interaction at the molecular level. We found that among six mouse MCM proteins, only MCM2 binds to histone; amino acid residues 63-153 are responsible for this binding. The region required for nuclear localization of MCM2 was mapped near this histone-binding domain. Far-Western blotting analysis of truncated forms of H3 histone indicated that amino acid residues 26-67 of H3 histone are required for binding to MCM2. We have also shown that mouse MCM2 can inhibit the DNA helicase activity of the human MCM4, -6, and -7 protein complex. These results suggest that MCM2 plays a different role in the initiation of DNA replication than the other MCM proteins.

  12. Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids.

    PubMed

    Panas, Michael W; Jain, Paras; Yang, Hui; Mitra, Shimontini; Biswas, Debasis; Wattam, Alice Rebecca; Letvin, Norman L; Jacobs, William R

    2014-09-16

    Research on tuberculosis and leprosy was revolutionized by the development of a plasmid transformation system in the fast-growing surrogate, Mycobacterium smegmatis. This transformation system was made possible by the successful isolation of a M. smegmatis mutant strain mc(2)155, whose efficient plasmid transformation (ept) phenotype supported the replication of Mycobacterium fortuitum pAL5000 plasmids. In this report, we identified the EptC gene, the loss of which confers the ept phenotype. EptC shares significant amino acid sequence homology and domain structure with the MukB protein of Escherichia coli, a structural maintenance of chromosomes (SMC) protein. Surprisingly, M. smegmatis has three paralogs of SMC proteins: EptC and MSMEG_0370 both share homology with Gram-negative bacterial MukB; and MSMEG_2423 shares homology with Gram-positive bacterial SMCs, including the single SMC protein predicted for Mycobacterium tuberculosis and Mycobacterium leprae. Purified EptC was shown to bind ssDNA and stabilize negative supercoils in plasmid DNA. Moreover, an EptC-mCherry fusion protein was constructed and shown to bind to DNA in live mycobacteria, and to prevent segregation of plasmid DNA to daughter cells. To our knowledge, this is the first report of impaired plasmid maintenance caused by a SMC homolog, which has been canonically known to assist the segregation of genetic materials.

  13. Pressure Stabilization of Proteins from Extreme Thermophiles

    PubMed Central

    Hei, Derek J.; Clark, Douglas S.

    1994-01-01

    We describe the stabilization by pressure of enzymes, including a hydrogenase from Methanococcus jannaschii, an extremely thermophilic deep-sea methanogen. This is the first published report of proteins from thermophiles being stabilized by pressure. Inactivation studies of partially purified hydrogenases from an extreme thermophile (Methanococcus igneus), a moderate thermophile (Methanococcus thermolithotrophicus), and a mesophile (Methanococcus maripaludis), all from shallow marine sites, show that pressure stabilization is not unique to enzymes isolated from high-pressure environments. These studies suggest that pressure stabilization of an enzyme may be related to its thermophilicity. Further experiments comparing the effects of increased pressure on the stability of α-glucosidases from the hyperthermophile Pyrococcus furiosus and Saccharomyces cerevisiae support this possibility. We have also examined pressure effects on several highly homologous glyceraldehyde-3-phosphate dehydrogenases from mesophilic and thermophilic sources and a rubredoxin from P. furiosus. The results suggest that hydrophobic interactions, which have been implicated in the stabilization of many thermophilic proteins, contribute to the pressure stabilization of enzymes from thermophiles. PMID:16349220

  14. Stabilizer-Guided Inhibition of Protein-Protein Interactions.

    PubMed

    Milroy, Lech-Gustav; Bartel, Maria; Henen, Morkos A; Leysen, Seppe; Adriaans, Joris M C; Brunsveld, Luc; Landrieu, Isabelle; Ottmann, Christian

    2015-12-21

    The discovery of novel protein-protein interaction (PPI) modulators represents one of the great molecular challenges of the modern era. PPIs can be modulated by either inhibitor or stabilizer compounds, which target different though proximal regions of the protein interface. In principle, protein-stabilizer complexes can guide the design of PPI inhibitors (and vice versa). In the present work, we combine X-ray crystallographic data from both stabilizer and inhibitor co-crystal complexes of the adapter protein 14-3-3 to characterize, down to the atomic scale, inhibitors of the 14-3-3/Tau PPI, a potential drug target to treat Alzheimer's disease. The most potent compound notably inhibited the binding of phosphorylated full-length Tau to 14-3-3 according to NMR spectroscopy studies. Our work sets a precedent for the rational design of PPI inhibitors guided by PPI stabilizer-protein complexes while potentially enabling access to new synthetically tractable stabilizers of 14-3-3 and other PPIs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Stabilization of protein-protein interactions by small molecules.

    PubMed

    Giordanetto, Fabrizio; Schäfer, Anja; Ottmann, Christian

    2014-11-01

    Protein-protein interactions (PPIs) are implicated in every disease and mastering the ability to influence PPIs with small molecules would considerably enlarge the druggable genome. Whereas inhibition of PPIs has repeatedly been shown to work successfully, targeted stabilization of PPIs is underrepresented in the literature. This is all the more surprising because natural products like FK506, rapamycin, brefeldin, forskolin and fusicoccin confer their physiological activity by stabilizing specific PPIs. However, recently a number of very interesting synthetic molecules have been reported from drug discovery projects that indeed achieve their desired activities by stabilizing either homo- or hetero-oligomeric complexes of their target proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Effects of confinement on protein folding and protein stability

    NASA Astrophysics Data System (ADS)

    Ping, G.; Yuan, J. M.; Vallieres, M.; Dong, H.; Sun, Z.; Wei, Y.; Li, F. Y.; Lin, S. H.

    2003-05-01

    In a cell, proteins exist in crowded environments; these environments influence their stability and dynamics. Similarly, for an enzyme molecule encapsulated in an inorganic cavity as in biosensors or biocatalysts, confinement and even surface effects play important roles in its stability and dynamics. Using a minimalist model (two-dimensional HP lattice model), we have carried out Monte Carlo simulations to study confinement effects on protein stability. We have calculated heat capacity as a function of temperature using the histogram method and results obtained show that confinement tends to stabilize the folded conformations, consistent with experimental results (some reported here) and previous theoretical analyses. Furthermore, for a protein molecule tethered to a solid surface the stabilization effect can be even greater. We have also investigated the effects of confinement on the kinetics of the refolding and unfolding processes as functions of temperature and box size. As expected, unfolding time increases as box size decreases, however, confinement affects folding times in a more complicated way. Our theoretical results agree with our experimentally observed trends that thermal stability of horseradish peroxidase and acid phosphatase, encapsulated in mesoporous silica, increases as the pore size of the silica matrix decreases.

  17. Long Astral Microtubules and RACK-1 Stabilize Polarity Domains during Maintenance Phase in Caenorhabditis elegans Embryos

    PubMed Central

    Ai, Erkang; Poole, Daniel S.; Skop, Ahna R.

    2011-01-01

    Cell polarity is a very well conserved process important for cell differentiation, cell migration, and embryonic development. After the establishment of distinct cortical domains, polarity cues have to be stabilized and maintained within a fluid and dynamic membrane to achieve proper cell asymmetry. Microtubules have long been thought to deliver the signals required to polarize a cell. While previous studies suggest that microtubules play a key role in the establishment of polarity, the requirement of microtubules during maintenance phase remains unclear. In this study, we show that depletion of Caenorhabditis elegans RACK-1, which leads to short astral microtubules during prometaphase, specifically affects maintenance of cortical PAR domains and Dynamin localization. We then investigated the consequence of knocking down other factors that also abolish astral microtubule elongation during polarity maintenance phase. We found a correlation between short astral microtubules and the instability of PAR-6 and PAR-2 domains during maintenance phase. Our data support a necessary role for astral microtubules in the maintenance phase of cell polarity. PMID:21533050

  18. Multifunctional Role of ATM/Tel1 Kinase in Genome Stability: From the DNA Damage Response to Telomere Maintenance

    PubMed Central

    2014-01-01

    The mammalian protein kinase ataxia telangiectasia mutated (ATM) is a key regulator of the DNA double-strand-break response and belongs to the evolutionary conserved phosphatidylinositol-3-kinase-related protein kinases. ATM deficiency causes ataxia telangiectasia (AT), a genetic disorder that is characterized by premature aging, cerebellar neuropathy, immunodeficiency, and predisposition to cancer. AT cells show defects in the DNA damage-response pathway, cell-cycle control, and telomere maintenance and length regulation. Likewise, in Saccharomyces cerevisiae, haploid strains defective in the TEL1 gene, the ATM ortholog, show chromosomal aberrations and short telomeres. In this review, we outline the complex role of ATM/Tel1 in maintaining genomic stability through its control of numerous aspects of cellular survival. In particular, we describe how ATM/Tel1 participates in the signal transduction pathways elicited by DNA damage and in telomere homeostasis and its importance as a barrier to cancer development. PMID:25247188

  19. Enhancing protein stability with extended disulfide bonds

    DOE PAGES

    Liu, Tao; Wang, Yan; Luo, Xiaozhou; ...

    2016-05-09

    Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. In this paper, we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a librarymore » of random β-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ~9 °C was identified. Finally, this result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.« less

  20. Axonal maintenance, glia, exosomes, and heat shock proteins

    PubMed Central

    Tytell, Michael; Lasek, Raymond J.; Gainer, Harold

    2016-01-01

    Of all cellular specializations, the axon is especially distinctive because it is a narrow cylinder of specialized cytoplasm called axoplasm with a length that may be orders of magnitude greater than the diameter of the cell body from which it originates. Thus, the volume of axoplasm can be much greater than the cytoplasm in the cell body. This fact raises a logistical problem with regard to axonal maintenance. Many of the components of axoplasm, such as soluble proteins and cytoskeleton, are slowly transported, taking weeks to months to travel the length of axons longer than a few millimeters after being synthesized in the cell body. Furthermore, this slow rate of supply suggests that the axon itself might not have the capacity to respond fast enough to compensate for damage to transported macromolecules. Such damage is likely in view of the mechanical fragility of an axon, especially those innervating the limbs, as rapid limb motion with high impact, like running, subjects the axons in the limbs to considerable mechanical force. Some researchers have suggested that local, intra-axonal protein synthesis is the answer to this problem. However, the translational state of axonal RNAs remains controversial. We suggest that glial cells, which envelop all axons, whether myelinated or not, are the local sources of replacement and repair macromolecules for long axons. The plausibility of this hypothesis is reinforced by reviewing several decades of work on glia-axon macromolecular transfer, together with recent investigations of exosomes and other extracellular vesicles, as vehicles for the transmission of membrane and cytoplasmic components from one cell to another. PMID:26962444

  1. Maintenance of native-like protein dynamics may not be required for engineering functional proteins.

    PubMed

    Gobeil, Sophie M C; Clouthier, Christopher M; Park, Jaeok; Gagné, Donald; Berghuis, Albert M; Doucet, Nicolas; Pelletier, Joelle N

    2014-10-23

    Proteins are dynamic systems, and understanding dynamics is critical for fully understanding protein function. Therefore, the question of whether laboratory engineering has an impact on protein dynamics is of general interest. Here, we demonstrate that two homologous, naturally evolved enzymes with high degrees of structural and functional conservation also exhibit conserved dynamics. Their similar set of slow timescale dynamics is highly restricted, consistent with evolutionary conservation of a functionally important feature. However, we also show that dynamics of a laboratory-engineered chimeric enzyme obtained by recombination of the two homologs exhibits striking difference on the millisecond timescale, despite function and high-resolution crystal structure (1.05 Å) being conserved. The laboratory-engineered chimera is thus functionally tolerant to modified dynamics on the timescale of catalytic turnover. Tolerance to dynamic variation implies that maintenance of native-like protein dynamics may not be required when engineering functional proteins.

  2. Protein stabilization by urea and guanidine hydrochloride.

    PubMed

    Bhuyan, Abani K

    2002-11-12

    stability of ferrocytochrome c up to the limit of the subdenaturing concentrations of the additives. NaCl and Na(2)SO(4), which stabilize proteins through their salting-in effect, also decrease the rate with a corresponding increase in activation entropy of CO dissociation from CO-bound native ferrocytochrome c, lending support to the view that low concentrations of GdnHCl and urea stabilize proteins. These results have direct relevance to the understanding and interpretation of the free energy-denaturant relationship and protein folding chevrons.

  3. Stability analysis of an autocatalytic protein model

    NASA Astrophysics Data System (ADS)

    Lee, Julian

    2016-05-01

    A self-regulatory genetic circuit, where a protein acts as a positive regulator of its own production, is known to be the simplest biological network with a positive feedback loop. Although at least three components—DNA, RNA, and the protein—are required to form such a circuit, stability analysis of the fixed points of this self-regulatory circuit has been performed only after reducing the system to a two-component system, either by assuming a fast equilibration of the DNA component or by removing the RNA component. Here, stability of the fixed points of the three-component positive feedback loop is analyzed by obtaining eigenvalues of the full three-dimensional Hessian matrix. In addition to rigorously identifying the stable fixed points and saddle points, detailed information about the system can be obtained, such as the existence of complex eigenvalues near a fixed point.

  4. Acetylation Reader Proteins: Linking Acetylation Signaling to Genome Maintenance and Cancer.

    PubMed

    Gong, Fade; Chiu, Li-Ya; Miller, Kyle M

    2016-09-01

    Chromatin-based DNA damage response (DDR) pathways are fundamental for preventing genome and epigenome instability, which are prevalent in cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze the addition and removal of acetyl groups on lysine residues, a post-translational modification important for the DDR. Acetylation can alter chromatin structure as well as function by providing binding signals for reader proteins containing acetyl-lysine recognition domains, including the bromodomain (BRD). Acetylation dynamics occur upon DNA damage in part to regulate chromatin and BRD protein interactions that mediate key DDR activities. In cancer, DDR and acetylation pathways are often mutated or abnormally expressed. DNA damaging agents and drugs targeting epigenetic regulators, including HATs, HDACs, and BRD proteins, are used or are being developed to treat cancer. Here, we discuss how histone acetylation pathways, with a focus on acetylation reader proteins, promote genome stability and the DDR. We analyze how acetylation signaling impacts the DDR in the context of cancer and its treatments. Understanding the relationship between epigenetic regulators, the DDR, and chromatin is integral for obtaining a mechanistic understanding of genome and epigenome maintenance pathways, information that can be leveraged for targeting acetylation signaling, and/or the DDR to treat diseases, including cancer.

  5. Intimin-Mediated Export of Passenger Proteins Requires Maintenance of a Translocation-Competent Conformation

    PubMed Central

    Adams, Thorsten M.; Wentzel, Alexander; Kolmar, Harald

    2005-01-01

    Intimins from pathogenic bacteria promote intimate bacterial adhesion to epithelial cells. Several structurally similar domains form on the bacterial cell surface an extended rigid rod that exposes the carboxy-terminal domain, which interacts with the translocated intimin receptor. We constructed a series of intimin-derived fusion proteins consisting of carboxy-terminally truncated intimin and the immunoglobulin light-chain variable domain REIv, ubiquitin, calmodulin, β-lactamase inhibitor protein, or β-lactamase. By systematically investigating the intimin-mediated cell surface exposure of these passenger domains in the presence or absence of compounds that interfere with outer membrane stability or passenger domain folding, we acquired experimental evidence that intimin-mediated protein export across the outer membrane requires, prior to export, the maintenance of a translocation-competent conformation that may be distinct from the final protein structure. We propose that, during export, competition exists between productive translocation and folding of the passenger domain in the periplasm into a stable conformation that is not compatible with translocation through the bacterial outer membrane. These results may expand understanding of the mechanism by which intimins are inserted into the outer membrane and expose extracellular domains on the cell surface. PMID:15629924

  6. Acetylation Reader Proteins: Linking Acetylation Signaling to Genome Maintenance and Cancer

    PubMed Central

    Miller, Kyle M.

    2016-01-01

    Chromatin-based DNA damage response (DDR) pathways are fundamental for preventing genome and epigenome instability, which are prevalent in cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze the addition and removal of acetyl groups on lysine residues, a post-translational modification important for the DDR. Acetylation can alter chromatin structure as well as function by providing binding signals for reader proteins containing acetyl-lysine recognition domains, including the bromodomain (BRD). Acetylation dynamics occur upon DNA damage in part to regulate chromatin and BRD protein interactions that mediate key DDR activities. In cancer, DDR and acetylation pathways are often mutated or abnormally expressed. DNA damaging agents and drugs targeting epigenetic regulators, including HATs, HDACs, and BRD proteins, are used or are being developed to treat cancer. Here, we discuss how histone acetylation pathways, with a focus on acetylation reader proteins, promote genome stability and the DDR. We analyze how acetylation signaling impacts the DDR in the context of cancer and its treatments. Understanding the relationship between epigenetic regulators, the DDR, and chromatin is integral for obtaining a mechanistic understanding of genome and epigenome maintenance pathways, information that can be leveraged for targeting acetylation signaling, and/or the DDR to treat diseases, including cancer. PMID:27631103

  7. Invaded grassland communities have altered stability-maintenance mechanisms but equal stability compared to native communities.

    PubMed

    Wilsey, Brian J; Daneshgar, Pedram P; Hofmockel, Kirsten; Polley, H Wayne

    2014-01-01

    Theory predicts that stability should increase with diversity via several mechanisms. We tested predictions in a 5-year experiment that compared low-diversity exotic to high-diversity native plant mixtures under two irrigation treatments. The study included both wet and dry years. Variation in biomass across years (CV) was 50% lower in mixtures than monocultures of both native and exotic species. Growth among species was more asynchronous and overyielding values were greater during and after a drought in native than exotic mixtures. Mean-variance slopes indicated strong portfolio effects in both community types, but the intercept was higher for exotics than for natives, suggesting that exotics were inherently more variable than native species. However, this failed to result in higher CV's in exotic communities because species that heavily dominated plots tended to have lower than expected variance. Results indicate that diversity-stability mechanisms are altered in invaded systems compared to native ones they replaced. © 2013 John Wiley & Sons Ltd/CNRS.

  8. Evolutionary Capacitance and Control of Protein Stability in Protein-Protein Interaction Networks

    PubMed Central

    Dixit, Purushottam D.; Maslov, Sergei

    2013-01-01

    In addition to their biological function, protein complexes reduce the exposure of the constituent proteins to the risk of undesired oligomerization by reducing the concentration of the free monomeric state. We interpret this reduced risk as a stabilization of the functional state of the protein. We estimate that protein-protein interactions can account for of additional stabilization; a substantial contribution to intrinsic stability. We hypothesize that proteins in the interaction network act as evolutionary capacitors which allows their binding partners to explore regions of the sequence space which correspond to less stable proteins. In the interaction network of baker's yeast, we find that statistically proteins that receive higher energetic benefits from the interaction network are more likely to misfold. A simplified fitness landscape wherein the fitness of an organism is inversely proportional to the total concentration of unfolded proteins provides an evolutionary justification for the proposed trends. We conclude by outlining clear biophysical experiments to test our predictions. PMID:23592969

  9. Cumulative versus Stabilizing Effects of Methadone Maintenance: A Quasi-Experimental Study Using Longitudinal Self-Report Data.

    ERIC Educational Resources Information Center

    Powers, Keiko Ichikawa; Anglin, M. Douglas

    1993-01-01

    Whether methadone maintenance treatment demonstrates cumulative (rehabilitative) or stabilizing effects on behavior of narcotics addicts over multiple treatment episodes was studied involving 993 addicts in a quasi-experimental design. Observed behavioral changes and longitudinal self-reports indicate stabilizing, but not cumulative, effects. (SLD)

  10. The Intersubunit Bridge B1b of the Bacterial Ribosome Facilitates Initiation of Protein Synthesis and Maintenance of Translational Fidelity.

    PubMed

    Lilleorg, Silva; Reier, Kaspar; Remme, Jaanus; Liiv, Aivar

    2017-04-07

    In bacteria, ribosomal subunits are connected via 12 intersubunit bridges involving RNA-RNA, RNA-protein, and protein-protein interactions. The only protein-protein bridge in the ribosome is ribosomal intersubunit bridge 1b (B1b), which is mainly formed by the bacterial protein L31 (bL31) and connects the head domain of 30S subunit and the central protuberance of the 50S subunit. It is known to be the most dynamic intersubunit bridge. Here, we have evaluated the role of bL31 and thereby the bridge B1b in the working cycle of the ribosome. First, bL31-deficient ribosomes are severely compromised in their ability to ensure translational fidelity particularly in reading frame maintenance in vivo. Second, in the absence of bL31, the rate of initiation is significantly reduced both in vivo and in vitro. Third, polysome profile and subunit reassociation assays demonstrate that bL31 is important for stabilizing subunit joining in vivo and in vitro. Together, our results demonstrate that bL31 is important for determining translational fidelity and stabilizing subunit association. We conclude that the only protein-protein intersubunit bridge of the bacterial ribosome facilitates translation initiation and is essential for maintaining the reading frame of mRNA translation.

  11. Characterisation of protein stability in rod-insert vaginal rings.

    PubMed

    Pattani, Aditya; Lowry, Deborah; Curran, Rhonda M; McGrath, Stephanie; Kett, Vicky L; Andrews, Gavin P; Malcolm, R Karl

    2012-07-01

    A major goal in vaccine development is elimination of the 'cold chain', the transport and storage system for maintenance and distribution of the vaccine product. This is particularly pertinent to liquid formulation of vaccines. We have previously described the rod-insert vaginal ring (RiR) device, comprising an elastomeric body into which are inserted lyophilised, rod-shaped, solid drug dosage forms, and having potential for sustained mucosal delivery of biomacromolecules, such as HIV envelope protein-based vaccine candidates. Given the solid, lyophilised nature of these insert dosage forms, we hypothesised that antigen stability may be significantly increased compared with more conventional solubilised vaginal gel format. In this study, we prepared and tested vaginal ring devices fitted with lyophilised rod inserts containing the model antigen bovine serum albumin (BSA). Both the RiRs and the gels that were freeze-dried to prepare the inserts were evaluated for BSA stability using PAGE, turbidimetry, microbial load, MALDI-TOF and qualitative precipitate solubility measurements. When stored at 4 °C, but not when stored at 40 °C/75% RH, the RiR formulation offered protection against structural and conformational changes to BSA. The insert also retained matrix integrity and release characteristics. The results demonstrate that lypophilised gels can provide relative protection against degradation at lower temperatures compared to semi-solid gels. The major mechanism of degradation at 40 °C/75% RH was shown to be protein aggregation. Finally, in a preliminary study, we found that addition of trehalose to the formulation significantly reduces the rate of BSA degradation compared to the original formulation when stored at 40 °C/75% RH. Establishing the mechanism of degradation, and finding that degradation is decelerated in the presence of trehalose, will help inform further development of RiRs specifically and polymer based freeze-dried systems in general.

  12. Ankyrin protein networks in membrane formation and stabilization

    PubMed Central

    Cunha, Shane R; Mohler, Peter J

    2009-01-01

    In eukaryotic cells, ankyrins serve as adaptor proteins that link membrane proteins to the underlying cytoskeleton. These adaptor proteins form protein complexes consisting of integral membrane proteins, signalling molecules and cytoskeletal components. With their modular architecture and ability to interact with many proteins, ankyrins organize and stabilize these protein networks, thereby establishing the infrastructure of membrane domains with specialized functions. To this end, ankyrin collaborates with a number of proteins including cytoskeletal proteins, cell adhesion molecules and large structural proteins. This review addresses the targeting and stabilization of protein networks related to ankyrin interactions with the cytoskeletal protein β-spectrin, L1-cell adhesion molecules and the large myofibrillar protein obscurin. The significance of these interactions for differential targeting of cardiac proteins and neuronal membrane formation is also presented. Finally, this review concludes with a discussion about ankyrin dysfunction in human diseases such as haemolytic anaemia, cardiac arrhythmia and neurological disorders. PMID:19840192

  13. ATMIN Is Required for Maintenance of Genomic Stability and Suppression of B Cell Lymphoma

    PubMed Central

    Loizou, Joanna I.; Sancho, Rocio; Kanu, Nnennaya; Bolland, Daniel J.; Yang, Fengtang; Rada, Cristina; Corcoran, Anne E.; Behrens, Axel

    2011-01-01

    Summary Defective V(D)J rearrangement of immunoglobulin heavy or light chain (IgH or IgL) or class switch recombination (CSR) can initiate chromosomal translocations. The DNA-damage kinase ATM is required for the suppression of chromosomal translocations but ATM regulation is incompletely understood. Here, we show that mice lacking the ATM cofactor ATMIN in B cells (ATMINΔB/ΔB) have impaired ATM signaling and develop B cell lymphomas. Notably, ATMINΔB/ΔB cells exhibited defective peripheral V(D)J rearrangement and CSR, resulting in translocations involving the Igh and Igl loci, indicating that ATMIN is required for efficient repair of DNA breaks generated during somatic recombination. Thus, our results identify a role for ATMIN in regulating the maintenance of genomic stability and tumor suppression in B cells. PMID:21575860

  14. Road Maintenance Experience Using Polyurethane (PU) Foam Injection System and Geocrete Soil Stabilization as Ground Rehabilitation

    NASA Astrophysics Data System (ADS)

    Fakhar, A. M. M.; Asmaniza, A.

    2016-07-01

    There are many types of ground rehabilation and improvement that can be consider and implement in engineering construction works for soil improvement in order to prevent road profile deformation in later stage. However, when comes to road maintenance especially on operated expressways, not all method can be apply directly as it must comply to opreation's working window and lane closure basis. Key factors that considering ideal proposal for ground rehabilitation are time, cost, quality and most importantly practicality. It should provide long lifespan structure in order to reduce continuous cycle of maintenance. Thus, this paper will present two approaches for ground rehabilitation, namely Polyurethane (PU) Foam Injection System and Geocrete Soil Stabilization. The first approach is an injection system which consists two-parts chemical grout of Isocynate and Polyol when mixed together within soil structure through injection will polymerized with volume expansion. The strong expansion of grouting causes significant compression and compacting of the surrounding soil and subsequently improve ground properties and uplift sunken structure. The later is a cold in-place recyclying whereby mixture process that combines in-situ soil materials, cement, white powder (alkaline) additive and water to produce hard yet flexible and durable ground layer that act as solid foundation with improved bearing capacity. The improvement of the mechanical behaviour of soil through these two systems is investigated by an extensive testing programme which includes in-situ and laboratory test in determining properties such as strength, stiffness, compressibility, bearing capacity, differential settlement and etc.

  15. Replication fork stability is essential for the maintenance of centromere integrity in the absence of heterochromatin.

    PubMed

    Li, Pao-Chen; Petreaca, Ruben C; Jensen, Amanda; Yuan, Ji-Ping; Green, Marc D; Forsburg, Susan L

    2013-03-28

    The centromere of many eukaryotes contains highly repetitive sequences marked by methylation of histone H3K9 by Clr4(KMT1). This recruits multiple heterochromatin proteins, including Swi6 and Chp1, to form a rigid centromere and ensure accurate chromosome segregation. In the absence of heterochromatin, cells show an increased rate of recombination in the centromere, as well as chromosome loss. These defects are severely aggravated by loss of replication fork stability. Thus, heterochromatin proteins and replication fork protection mechanisms work in concert to prevent abnormal recombination, preserve centromere integrity, and ensure faithful chromosome segregation. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Blood profiling of proteins and steroids during weight maintenance with manipulation of dietary protein level and glycaemic index.

    PubMed

    Wang, Ping; Holst, Claus; Astrup, Arne; Bouwman, Freek G; van Otterdijk, Sanne; Wodzig, Will K W H; Andersen, Malene R; van Baak, Marleen A; Rasmussen, Lone G; Martinez, J Alfredo; Jebb, Susan A; Pfeiffer, Andreas F H; Kafatos, Anthony; Handjieva-Darlenska, Teodora; Hlavaty, Petr; Saris, Wim H M; Mariman, Edwin C M

    2012-01-01

    Weight regain after weight loss is common. In the Diogenes dietary intervention study, a high-protein and low-glycaemic index (GI) diet improved weight maintenance. The objective of the present study was to identify (1) blood profiles associated with continued weight loss and weight regain (2) blood biomarkers of dietary protein and GI levels during the weight-maintenance phase. Blood samples were collected at baseline, after 8 weeks of low-energy diet-induced weight loss and after a 6-month dietary intervention period from female continued weight losers (n 48) and weight regainers (n 48), evenly selected from four dietary groups that varied in protein and GI levels. The blood concentrations of twenty-nine proteins and three steroid hormones were measured. The changes in analytes during weight maintenance largely correlated negatively with the changes during weight loss, with some differences between continued weight losers and weight regainers. Increases in leptin (LEP) and C-reactive protein (CRP) were significantly associated with weight regain (P < 0·001 and P = 0·005, respectively), and these relationships were influenced by the diet. Consuming a high-protein and high-GI diet dissociated the positive relationship between the change in LEP concentration and weight regain. CRP increased during the weight-maintenance period only in weight regainers with a high-protein diet (P < 0·001). In addition, testosterone, luteinising hormone, angiotensinogen, plasminogen activator inhibitor-1, resistin, retinol-binding protein 4, insulin, glucagon, haptoglobin and growth hormone were also affected by the dietary intervention. The blood profile reflects not only the weight change during the maintenance period, but also the macronutrient composition of the dietary intervention, especially the protein level.

  17. SPLINTS: small-molecule protein ligand interface stabilizers.

    PubMed

    Fischer, Eric S; Park, Eunyoung; Eck, Michael J; Thomä, Nicolas H

    2016-04-01

    Regulatory protein-protein interactions are ubiquitous in biology, and small molecule protein-protein interaction inhibitors are an important focus in drug discovery. Remarkably little attention has been given to the opposite strategy-stabilization of protein-protein interactions, despite the fact that several well-known therapeutics act through this mechanism. From a structural perspective, we consider representative examples of small molecules that induce or stabilize the association of protein domains to inhibit, or alter, signaling for nuclear hormone, GTPase, kinase, phosphatase, and ubiquitin ligase pathways. These SPLINTS (small-molecule protein ligand interface stabilizers) drive interactions that are in some cases physiologically relevant, and in others entirely adventitious. The diverse structural mechanisms employed suggest approaches for a broader and systematic search for such compounds in drug discovery.

  18. Protein thermal stabilization in aqueous solutions of osmolytes.

    PubMed

    Bruździak, Piotr; Panuszko, Aneta; Jourdan, Muriel; Stangret, Janusz

    2016-01-01

    Proteins' thermal stabilization is a significant problem in various biomedical, biotechnological, and technological applications. We investigated thermal stability of hen egg white lysozyme in aqueous solutions of the following stabilizing osmolytes: Glycine (GLY), N-methylglycine (NMG), N,N-dimethylglycine (DMG), N,N,N-trimethylglycine (TMG), and trimethyl-N-oxide (TMAO). Results of CD-UV spectroscopic investigation were compared with FTIR hydration studies' results. Selected osmolytes increased lysozyme's thermal stability in the following order: Gly>NMG>TMAO≈DMG>TMG. Theoretical calculations (DFT) showed clearly that osmolytes' amino group protons and water molecules interacting with them played a distinctive role in protein thermal stabilization. The results brought us a step closer to the exact mechanism of protein stabilization by osmolytes.

  19. Protein kinesis: The dynamics of protein trafficking and stability

    SciTech Connect

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  20. Designing Whey Protein-Polysaccharide Particles for Colloidal Stability.

    PubMed

    Wagoner, Ty; Vardhanabhuti, Bongkosh; Foegeding, E Allen

    2016-01-01

    Interactions between whey proteins and polysaccharides, in particular the formation of food-grade soluble complexes, are of interest because of potential functional and health benefits. A specific application that has not received much attention is the use of complexes for enhanced colloidal stability of protein sols, such as protein-containing beverages. In beverages, the primary goal is the formation of complexes that remain dispersed after thermal processing and extended storage. This review highlights recent progress in the area of forming whey protein-polysaccharide soluble complexes that would be appropriate for beverage applications. Research in this area indicates that soluble complexes can be formed and stabilized that are reasonably small in size and possess a large surface charge that would predict colloidal stability. Selection of specific proteins and polysaccharides can be tailored to desired conditions. The principal challenges involve overcoming restrictions on protein concentration and ensuring that protein remains bioavailable.

  1. Two Putative Polysaccharide Deacetylases Are Required for Osmotic Stability and Cell Shape Maintenance in Bacillus anthracis.

    PubMed

    Arnaouteli, Sofia; Giastas, Petros; Andreou, Athina; Tzanodaskalaki, Mary; Aldridge, Christine; Tzartos, Socrates J; Vollmer, Waldemar; Eliopoulos, Elias; Bouriotis, Vassilis

    2015-05-22

    Membrane-anchored lipoproteins have a broad range of functions and play key roles in several cellular processes in Gram-positive bacteria. BA0330 and BA0331 are the only lipoproteins among the 11 known or putative polysaccharide deacetylases of Bacillus anthracis. We found that both lipoproteins exhibit unique characteristics. BA0330 and BA0331 interact with peptidoglycan, and BA0330 is important for the adaptation of the bacterium to grow in the presence of a high concentration of salt, whereas BA0331 contributes to the maintenance of a uniform cell shape. They appear not to alter the peptidoglycan structure and do not contribute to lysozyme resistance. The high resolution x-ray structure of BA0330 revealed a C-terminal domain with the typical fold of a carbohydrate esterase 4 and an N-terminal domain unique for this family, composed of a two-layered (4 + 3) β-sandwich with structural similarity to fibronectin type 3 domains. Our data suggest that BA0330 and BA0331 have a structural role in stabilizing the cell wall of B. anthracis.

  2. Two Putative Polysaccharide Deacetylases Are Required for Osmotic Stability and Cell Shape Maintenance in Bacillus anthracis*

    PubMed Central

    Arnaouteli, Sofia; Giastas, Petros; Andreou, Athina; Tzanodaskalaki, Mary; Aldridge, Christine; Tzartos, Socrates J.; Vollmer, Waldemar; Eliopoulos, Elias; Bouriotis, Vassilis

    2015-01-01

    Membrane-anchored lipoproteins have a broad range of functions and play key roles in several cellular processes in Gram-positive bacteria. BA0330 and BA0331 are the only lipoproteins among the 11 known or putative polysaccharide deacetylases of Bacillus anthracis. We found that both lipoproteins exhibit unique characteristics. BA0330 and BA0331 interact with peptidoglycan, and BA0330 is important for the adaptation of the bacterium to grow in the presence of a high concentration of salt, whereas BA0331 contributes to the maintenance of a uniform cell shape. They appear not to alter the peptidoglycan structure and do not contribute to lysozyme resistance. The high resolution x-ray structure of BA0330 revealed a C-terminal domain with the typical fold of a carbohydrate esterase 4 and an N-terminal domain unique for this family, composed of a two-layered (4 + 3) β-sandwich with structural similarity to fibronectin type 3 domains. Our data suggest that BA0330 and BA0331 have a structural role in stabilizing the cell wall of B. anthracis. PMID:25825488

  3. The role of Hklp2 in the stabilization and maintenance of spindle bipolarity.

    PubMed

    Vanneste, David; Takagi, Masatoshi; Imamoto, Naoko; Vernos, Isabelle

    2009-11-03

    Spindle bipolarity relies on a fine balance of forces exerted by various molecular motors [1-4]. In most animal cells, spindle bipolarity requires sustained outward forces to push the spindle poles apart, an activity that is provided by Eg5, a conserved homotetrameric plus-end-directed kinesin that crosslinks and slides antiparallel microtubules apart [5]. These pushing forces are balanced by inward minus-end-directed forces. Impairing both Eg5 and dynein restores the formation of functional bipolar spindles [4], although the mechanism at play is far from clear. The current model also fails to explain why in some systems Eg5 inhibition does not promote bipolar spindle collapse [6, 7] or why increasing Eg5 levels does not interfere with bipolar spindle assembly [8]. Moreover, the C. elegans Eg5 ortholog is not required for bipolar spindle formation [9]. We show here that the kinesin Hklp2 participates in the assembly and stabilization of the bipolar spindle. Hklp2 localizes to the mitotic microtubules in a TPX2-dependent manner and to the chromosomes through Ki67. Our data indicate that its mechanism of action is clearly distinct from and complementary to that of Eg5, providing an additional understanding of the mechanism driving the formation and maintenance of the bipolar spindle.

  4. Stability and the maintenance of balance following a perturbation from quiet stance

    NASA Astrophysics Data System (ADS)

    Stirling, J. R.; Zakynthinaki, M. S.

    2004-03-01

    We investigate stability and the maintenance of balance with the use of tools from dynamical systems. In particular we investigate the application of such tools to the study of the ground reaction forces resulting from an athlete being perturbed from quiet stance. We develop a nonlinear model consisting of a set of coupled vector fields for the derivative with respect to time of the angles between the resultant ground reaction forces and the vertical in the anteroposterior and mediolateral directions. This model contains a basin of attraction bound by a closed curve which we call the critical curve. It is only inside this curve that perturbations can be corrected, with the orbit spiraling onto an attractor corresponding to quiet stance. We show how the critical curve and also the strength of the attractor found in the basin of attraction can be fit to model the experimental data (time series) for an individual athlete. We also discuss how our model can be used to identify nonsymmetric behavior caused by muscle imbalances and differences in the ranges of motion on either side of the body.

  5. Protein kinase M ζ and the maintenance of long-term memory.

    PubMed

    Zhang, Yang; Zong, Wei; Zhang, Lei; Ma, Yuanye; Wang, Jianhong

    2016-10-01

    Although various molecules have been found to mediate the processes of memory acquisition and consolidation, the molecular mechanism to maintain memory still remains elusive. In recent years, a molecular pathway focusing on protein kinase Mζ (PKMζ) has become of interest to researchers because of its potential role in long-term memory maintenance. PKMζ is an isoform of protein kinase C (PKC) and has a related structure that influences its function in maintaining memory. Considerable evidence has been gathered on PKMζ activity, including loss of function studies using PKMζ inhibitors, such as PKMζ inhibitory peptide (ZIP), suggesting PKMζ plays an important role in long-term memory maintenance. This review provides an overview of the role of PKMζ in long-term memory and outlines the molecular structure of PKMζ, the molecular mechanism of PKMζ in long-term memory maintenance and future directions of PKMζ research. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. INCREASING PROTEIN STABILITY BY IMPROVING BETA-TURNS

    PubMed Central

    Fu, Hailong; Grimsley, Gerald R.; Razvi, Abbas; Scholtz, J. Martin; Pace, C. Nick

    2009-01-01

    Our goal was to gain a better understanding of how protein stability can be increased by improving β-turns. We studied 22 β-turns in nine proteins with 66 to 370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some β-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein (HPr), Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase α-subunit (TSα), and Maltose binding protein (MBP). Of the fifteen single proline mutations, 11increased stability (Average = 0.8 ± 0.3; Range = 0.3 – 1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. Based on this and our previous work, we conclude that proteins can generally be stabilized by replacing non-proline residues with proline residues at the i + 1 position of Type I and II β-turns and at the i position in Type II β-turns. Other turn positions can sometimes be used if the φ angle is near −60° for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in β-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in β-turns that could be replaced by Gly to increase protein stability. Improving β-turns by substituting Pro residues is a generally useful way of increasing protein stability. PMID:19626709

  7. Stability of domain structures in multi-domain proteins

    PubMed Central

    Bhaskara, Ramachandra M.; Srinivasan, Narayanaswamy

    2011-01-01

    Multi-domain proteins have many advantages with respect to stability and folding inside cells. Here we attempt to understand the intricate relationship between the domain-domain interactions and the stability of domains in isolation. We provide quantitative treatment and proof for prevailing intuitive ideas on the strategies employed by nature to stabilize otherwise unstable domains. We find that domains incapable of independent stability are stabilized by favourable interactions with tethered domains in the multi-domain context. Stability of such folds to exist independently is optimized by evolution. Specific residue mutations in the sites equivalent to inter-domain interface enhance the overall solvation, thereby stabilizing these domain folds independently. A few naturally occurring variants at these sites alter communication between domains and affect stability leading to disease manifestation. Our analysis provides safe guidelines for mutagenesis which have attractive applications in obtaining stable fragments and domain constructs essential for structural studies by crystallography and NMR. PMID:22355559

  8. SS-Stabilizing Proteins Rationally: Intrinsic Disorder-Based Design of Stabilizing Disulphide Bridges in GFP.

    PubMed

    Melnik, Bogdan S; Povarnitsyna, Tatiana V; Glukhov, Anatoly S; Melnik, Tatyana N; Uversky, Vladimir N; Sarma, Ramaswamy H

    2012-01-01

    Abstract The most attractive and methodologically convenient way to enhance protein stability is via the introduction of disulphide bond(s). However, the effect of the artificially introduced SS-bond on protein stability is often quite unpredictable. This raises the question of how to choose the protein sites in an intelligent manner, so that the 'fastening' of these sites by the SS-bond(s) would provide maximal protein stability. We hypothesize that the successful design of a stabilizing SS-bond requires finding highly mobile protein regions. Using GFP as an illustrative example, we demonstrate that the knowledge of the peculiarities of the intramolecular hydrophobic interactions, combined with the understanding of the local intrinsic disorder propensities (that can be evaluated by various disorder predictors, e.g., PONDRFIT), is sufficient to find the candidate sites for the introduction of stabilizing SS-bridge(s). In fact, our analysis revealed that the insertion of the engineered SS-bridge between two highly flexible regions of GFP noticeably increased the conformational stability of this protein toward the thermal and chemical unfolding. Therefore, our study represents a novel approach for the rational design of stabilizing disulphide bridges in proteins.

  9. Dispersion interactions govern the strong thermal stability of a protein.

    PubMed

    Vondrásek, Jirí; Kubar, Tomás; Jenney, Francis E; Adams, Michael W W; Kozísek, Milan; Cerný, Jirí; Sklenár, Vladimír; Hobza, Pavel

    2007-01-01

    Rubredoxin from the hyperthermophile Pyrococcus furiosus (Pf Rd) is an extremely thermostable protein, which makes it an attractive subject of protein folding and stability studies. A fundamental question arises as to what the reason for such extreme stability is and how it can be elucidated from a complex set of interatomic interactions. We addressed this issue first theoretically through a computational analysis of the hydrophobic core of the protein and its mutants, including the interactions taking place inside the core. Here we show that a single mutation of one of phenylalanine's residues inside the protein's hydrophobic core results in a dramatic decrease in its thermal stability. The calculated unfolding Gibbs energy as well as the stabilization energy differences between a few core residues follows the same trend as the melting temperature of protein variants determined experimentally by microcalorimetry measurements. NMR spectroscopy experiments have shown that the only part of the protein affected by mutation is the reasonably rearranged hydrophobic core. It is hence concluded that stabilization energies, which are dominated by London dispersion, represent the main source of stability of this protein.

  10. Mutation analysis of barley malt protein Z4 and protein Z7 on beer foam stability.

    PubMed

    Iimure, Takashi; Kimura, Tatsuji; Araki, Shigeki; Kihara, Makoto; Sato, Masahide; Yamada, Shinji; Shigyou, Tatsuro; Sato, Kazuhiro

    2012-02-15

    Beer foam stability is an important characteristic. It has been suggested that isoforms of protein Z, that is, protein Z4 and protein Z7, contribute to beer foam stability. We investigated the relationship between beer foam stability and protein Z4 and protein Z7 using their deficient mutants. As a protein Z4-deficient mutant, cv. Pirkka was used. Protein Z7 deficiency was screened in 1564 barley accessions in the world collection of Okayama University, Japan. The barley samples from normal, protein Z4-deficient, protein Z7-deficient, and double-deficient were genotyped in F(2) populations and then pooled based on the DNA marker genotypes of protein Z4 and protein Z7. For a brewing trial, F(5) pooled subpopulations were used. After malting and brewing, the foam stability was determined, and the results showed that the levels of foam stability in the four samples were comparable. Two-dimensional gel electrophoresis was used to investigate the proteome in these beer samples. The results showed that low molecular weight proteins, including lipid transfer protein (LTP2), in the deficient mutants were higher than those in the normal sample. Our results suggest that the contribution of protein Z4 and protein Z7 to beer foam stability was not greater than that of other beer proteins.

  11. Protein stability: computation, sequence statistics, and new experimental methods

    PubMed Central

    Magliery, Thomas J.

    2015-01-01

    Calculating protein stability and predicting stabilizing mutations remain exceedingly difficult tasks, largely due to the inadequacy of potential functions, the difficulty of modeling entropy and the unfolded state, and challenges of sampling, particularly of backbone conformations. Yet, computational design has produced some remarkably stable proteins in recent years, apparently owing to near ideality in structure and sequence features. With caveats, computational prediction of stability can be used to guide mutation, and mutations derived from consensus sequence analysis, especially improved by recent co-variation filters, are very likely to stabilize without sacrificing function. The combination of computational and statistical approaches with library approaches, including new technologies such as deep sequencing and high throughput stability measurements, point to a very exciting near term future for stability engineering, even with difficult computational issues remaining. PMID:26497286

  12. Post-production protein stability: trouble beyond the cell factory

    PubMed Central

    2011-01-01

    Being protein function a conformation-dependent issue, avoiding aggregation during production is a major challenge in biotechnological processes, what is often successfully addressed by convenient upstream, midstream or downstream approaches. Even when obtained in soluble forms, proteins tend to aggregate, especially if stored and manipulated at high concentrations, as is the case of protein drugs for human therapy. Post-production protein aggregation is then a major concern in the pharmaceutical industry, as protein stability, pharmacokinetics, bioavailability, immunogenicity and side effects are largely dependent on the extent of aggregates formation. Apart from acting at the formulation level, the recombinant nature of protein drugs allows intervening at upstream stages through protein engineering, to produce analogue protein versions with higher stability and enhanced therapeutic values. PMID:21806813

  13. Mechanism of Stabilization of Labile Compounds by Silk Fibroin Proteins

    DTIC Science & Technology

    2017-04-05

    to stability of labile molecules. In addition , we plan to complete efforts initiated to refine our understanding of electrogelation mechanisms of...paper approaches. Additional processing can remediate interactions with conformational structures of the silk protein to further enhance blood... additional benefits such as preventing exposure to enzymatic and UV stresses. Long-term temperature stability of dried silk formats such as films or

  14. Stabilization of supercooled fluids by thermal hysteresis proteins.

    PubMed Central

    Wilson, P W; Leader, J P

    1995-01-01

    It has been reported that thermal hysteresis proteins found in many cold-hardy, freeze-avoiding arthropods stabilize their supercooled body fluids. We give evidence that fish antifreeze proteins, which also produce thermal hysteresis, bind to and reduce the efficiency of heterogenous nucleation sites, rather than binding to embryonic ice nuclei. We discuss both possible mechanisms for stabilization of supercooled body fluids and also describe a new method for measuring and defining the supercooling point of small volumes of liquid. PMID:7612853

  15. Regulation of TET Protein Stability by Calpains

    PubMed Central

    Wang, Yu; Zhang, Yi

    2014-01-01

    SUMMARY DNA methylation at the fifth position of cytosine (5mC) is an important epigenetic modification that affects chromatin structure and gene expression. Recent studies have established a critical function of the Ten-eleven translocation (Tet) family of proteins in regulating DNA methylation dynamics. Three Tet genes have been identified in mammals, and they all encode for proteins capable of oxidizing 5mC as part of the DNA demethylation process. While regulation of Tet expression at the transcriptional level is well documented, how TET proteins are regulated at post-translational level is poorly understood. In this study, we report that all three TET proteins are direct substrates of calpains, a family of calcium-dependent proteases. Specifically, calpain1 mediates TET1 and TET2 turnover in mouse ES cells, and calpain2 regulates TET3 level during differentiation. This study provides the first evidence that TET proteins are subject to calpain-mediated degradation. PMID:24412366

  16. Applications of Protein Thermodynamic Database for Understanding Protein Mutant Stability and Designing Stable Mutants.

    PubMed

    Gromiha, M Michael; Anoosha, P; Huang, Liang-Tsung

    2016-01-01

    Protein stability is the free energy difference between unfolded and folded states of a protein, which lies in the range of 5-25 kcal/mol. Experimentally, protein stability is measured with circular dichroism, differential scanning calorimetry, and fluorescence spectroscopy using thermal and denaturant denaturation methods. These experimental data have been accumulated in the form of a database, ProTherm, thermodynamic database for proteins and mutants. It also contains sequence and structure information of a protein, experimental methods and conditions, and literature information. Different features such as search, display, and sorting options and visualization tools have been incorporated in the database. ProTherm is a valuable resource for understanding/predicting the stability of proteins and it can be accessed at http://www.abren.net/protherm/ . ProTherm has been effectively used to examine the relationship among thermodynamics, structure, and function of proteins. We describe the recent progress on the development of methods for understanding/predicting protein stability, such as (1) general trends on mutational effects on stability, (2) relationship between the stability of protein mutants and amino acid properties, (3) applications of protein three-dimensional structures for predicting their stability upon point mutations, (4) prediction of protein stability upon single mutations from amino acid sequence, and (5) prediction methods for addressing double mutants. A list of online resources for predicting has also been provided.

  17. Stability of efferent-mediated protection against acoustic overexposure with long maintenance under barbiturate anaesthesia.

    PubMed

    Rajan, R

    1996-01-01

    When anaesthetized animals are maintained over a long period, crossed-cochlear suppressive and enhancement-in-noise effects mediated by the olivocochlear bundle (OCB), as well as some OCB neuronal responses, show time-dependent variations. The present study determined if there were any such changes in OCB-mediated crossed-cochlear protection against compound action potential (CAP) threshold losses caused by a standard loud sound exposure at 11 kHz, presented under conditions either not evoking OCB-mediated protection (i.e. monaural exposure) or evoking protection (binaural exposure). Maintaining animals for periods up to approximately 30 h from initial anaesthetization resulted in non-significant changes in pre-exposure CAP thresholds. There were also only small changes over select frequency ranges in threshold losses caused by the monaural or binaural loud sound, after a single exposure as well as when the testing of OCB function was extended to examine effects after dual successive exposures, the latter result being determined by application of a previously described additivity model. The features of OCB-mediated protection also showed good stability over the long maintenance. These results are discussed as providing further circumstantial evidence that protection is mediated by a different OCB subcomponent to that/those responsible for other OCB-mediated crossed-cochlear effects. In general, the results show that the barbiturate anaesthetic used here does not significantly modulate the crossed-cochlear OCB effect of protection, even though it has been shown elsewhere to significantly depress other crossed-cochlear OCB effects.

  18. Stabilization of the Virulence Plasmid pSLT of Salmonella Typhimurium by Three Maintenance Systems and Its Evaluation by Using a New Stability Test.

    PubMed

    Lobato-Márquez, Damián; Molina-García, Laura; Moreno-Córdoba, Inma; García-Del Portillo, Francisco; Díaz-Orejas, Ramón

    2016-01-01

    Certain Salmonella enterica serovars belonging to subspecies I carry low-copy-number virulence plasmids of variable size (50-90 kb). All of these plasmids share the spv operon, which is important for systemic infection. Virulence plasmids are present at low copy numbers. Few copies reduce metabolic burden but suppose a risk of plasmid loss during bacterial division. This drawback is counterbalanced by maintenance modules that ensure plasmid stability, including partition systems and toxin-antitoxin (TA) loci. The low-copy number virulence pSLT plasmid of Salmonella enterica serovar Typhimurium encodes three auxiliary maintenance systems: one partition system (parAB) and two TA systems (ccdABST and vapBC2ST). The TA module ccdABST has previously been shown to contribute to pSLT plasmid stability and vapBC2ST to bacterial virulence. Here we describe a novel assay to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells by ParE toxin, a DNA gyrase inhibitor. Using this new maintenance assay we confirmed a crucial role of parAB in pSLT maintenance. We also showed that vapBC2ST, in addition to contribute to bacterial virulence, is important for plasmid stability. We have previously shown that ccdABST encodes an inactive CcdBST toxin. Using our new stability assay we monitored the contribution to plasmid stability of a ccdABST variant containing a single mutation (R99W) that restores the toxicity of CcdBST. The "activation" of CcdBST (R99W) did not increase pSLT stability by ccdABST. In contrast, ccdABST behaves as a canonical type II TA system in terms of transcriptional regulation. Of interest, ccdABST was shown to control the expression of a polycistronic operon in the pSLT plasmid. Collectively, these results show that the contribution of the CcdBST toxin to pSLT plasmid stability may depend on its role as a co-repressor in coordination with CcdAST antitoxin more than on its toxic activity.

  19. Stabilization of the Virulence Plasmid pSLT of Salmonella Typhimurium by Three Maintenance Systems and Its Evaluation by Using a New Stability Test

    PubMed Central

    Lobato-Márquez, Damián; Molina-García, Laura; Moreno-Córdoba, Inma; García-del Portillo, Francisco; Díaz-Orejas, Ramón

    2016-01-01

    Certain Salmonella enterica serovars belonging to subspecies I carry low-copy-number virulence plasmids of variable size (50–90 kb). All of these plasmids share the spv operon, which is important for systemic infection. Virulence plasmids are present at low copy numbers. Few copies reduce metabolic burden but suppose a risk of plasmid loss during bacterial division. This drawback is counterbalanced by maintenance modules that ensure plasmid stability, including partition systems and toxin-antitoxin (TA) loci. The low-copy number virulence pSLT plasmid of Salmonella enterica serovar Typhimurium encodes three auxiliary maintenance systems: one partition system (parAB) and two TA systems (ccdABST and vapBC2ST). The TA module ccdABST has previously been shown to contribute to pSLT plasmid stability and vapBC2ST to bacterial virulence. Here we describe a novel assay to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells by ParE toxin, a DNA gyrase inhibitor. Using this new maintenance assay we confirmed a crucial role of parAB in pSLT maintenance. We also showed that vapBC2ST, in addition to contribute to bacterial virulence, is important for plasmid stability. We have previously shown that ccdABST encodes an inactive CcdBST toxin. Using our new stability assay we monitored the contribution to plasmid stability of a ccdABST variant containing a single mutation (R99W) that restores the toxicity of CcdBST. The “activation” of CcdBST (R99W) did not increase pSLT stability by ccdABST. In contrast, ccdABST behaves as a canonical type II TA system in terms of transcriptional regulation. Of interest, ccdABST was shown to control the expression of a polycistronic operon in the pSLT plasmid. Collectively, these results show that the contribution of the CcdBST toxin to pSLT plasmid stability may depend on its role as a co-repressor in coordination with CcdAST antitoxin more than on its toxic activity. PMID

  20. Role of Atypical Protein Kinases in Maintenance of Long-Term Memory and Synaptic Plasticity.

    PubMed

    Borodinova, A A; Zuzina, A B; Balaban, P M

    2017-03-01

    Investigation of biochemical mechanisms underlying the long-term storage of information in nervous system is one of main problems of modern neurobiology. As a molecular basis of long-term memory, long-term changes in kinase activities, increase in the level and changes in the subunit composition of receptors in synaptic membranes, local activity of prion-like proteins, and epigenetic modifications of chromatin have been proposed. Perhaps a combination of all or of some of these factors underlies the storage of long-term memory in the brain. Many recent studies have shown an exclusively important role of atypical protein kinases (PKCζ, PKMζ, and PKCι/λ) in processes of learning, consolidation and maintenance of memory. The present review is devoted to consideration of mechanisms of transcriptional and translational control of atypical protein kinases and their roles in induction and maintenance of long-term synaptic plasticity and memory in vertebrates and invertebrates.

  1. Effects of Glycosylation on the Stability of Protein Pharmaceuticals

    PubMed Central

    SOLÁ, RICARDO J.; GRIEBENOW, KAI

    2008-01-01

    In recent decades, protein-based therapeutics have substantially expanded the field of molecular pharmacology due to their outstanding potential for the treatment of disease. Unfortunately, protein pharmaceuticals display a series of intrinsic physical and chemical instability problems during their production, purification, storage, and delivery that can adversely impact their final therapeutic efficacies. This has prompted an intense search for generalized strategies to engineer the long-term stability of proteins during their pharmaceutical employment. Due to the well known effect that glycans have in increasing the overall stability of glycoproteins, rational manipulation of the glycosylation parameters through glycoengineering could become a promising approach to improve both the in vitro and in vivo stability of protein pharmaceuticals. The intent of this review is therefore to further the field of protein glycoengineering by increasing the general understanding of the mechanisms by which glycosylation improves the molecular stability of protein pharmaceuticals. This is achieved by presenting a survey of the different instabilities displayed by protein pharmaceuticals, by addressing which of these instabilities can be improved by glycosylation, and by discussing the possible mechanisms by which glycans induce these stabilization effects. PMID:18661536

  2. Conformational stability of dimeric proteins: quantitative studies by equilibrium denaturation.

    PubMed Central

    Neet, K. E.; Timm, D. E.

    1994-01-01

    The conformational stability of dimeric globular proteins can be measured by equilibrium denaturation studies in solvents such as guanidine hydrochloride or urea. Many dimeric proteins denature with a 2-state equilibrium transition, whereas others have stable intermediates in the process. For those proteins showing a single transition of native dimer to denatured monomer, the conformational stabilities, delta Gu (H2O), range from 10 to 27 kcal/mol, which is significantly greater than the conformational stability found for monomeric proteins. The relative contribution of quaternary interactions to the overall stability of the dimer can be estimated by comparing delta Gu (H2O) from equilibrium denaturation studies to the free energy associated with simple dissociation in the absence of denaturant. In many cases the large stabilization energy of dimers is primarily due to the intersubunit interactions and thus gives a rationale for the formation of oligomers. The magnitude of the conformational stability is related to the size of the polypeptide in the subunit and depends upon the type of structure in the subunit interface. The practical use, interpretation, and utility of estimation of conformational stability of dimers by equilibrium denaturation methods are discussed. PMID:7756976

  3. A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function

    PubMed Central

    Araya, Carlos L.; Fowler, Douglas M.; Chen, Wentao; Muniez, Ike; Kelly, Jeffery W.; Fields, Stanley

    2012-01-01

    The ability of a protein to carry out a given function results from fundamental physicochemical properties that include the protein’s structure, mechanism of action, and thermodynamic stability. Traditional approaches to study these properties have typically required the direct measurement of the property of interest, oftentimes a laborious undertaking. Although protein properties can be probed by mutagenesis, this approach has been limited by its low throughput. Recent technological developments have enabled the rapid quantification of a protein’s function, such as binding to a ligand, for numerous variants of that protein. Here, we measure the ability of 47,000 variants of a WW domain to bind to a peptide ligand and use these functional measurements to identify stabilizing mutations without directly assaying stability. Our approach is rooted in the well-established concept that protein function is closely related to stability. Protein function is generally reduced by destabilizing mutations, but this decrease can be rescued by stabilizing mutations. Based on this observation, we introduce partner potentiation, a metric that uses this rescue ability to identify stabilizing mutations, and identify 15 candidate stabilizing mutations in the WW domain. We tested six candidates by thermal denaturation and found two highly stabilizing mutations, one more stabilizing than any previously known mutation. Thus, physicochemical properties such as stability are latent within these large-scale protein functional data and can be revealed by systematic analysis. This approach should allow other protein properties to be discovered. PMID:23035249

  4. Role of cardiolipin in stability of integral membrane proteins.

    PubMed

    Musatov, Andrej; Sedlák, Erik

    2017-08-23

    Cardiolipin (CL) is a unique phospholipid with a dimeric structure having four acyl chains and two phosphate groups found almost exclusively in certain membranes of bacteria and of mitochondria of eukaryotes. CL interacts with numerous proteins and has been implicated in function and stabilization of several integral membrane proteins (IMPs). While both functional and stabilization roles of CL in IMPs has been generally acknowledged, there are, in fact, only limited number of quantitative analysis that support this function of CL. This is likely caused by relatively complex determination of parameters characterizing stability of IMPs and particularly intricate assessment of role of specific PLs such as CL in IMPs stability. This review aims to summarize quantitative findings regarding stabilization role of CL in IMPs reported up to now. Copyright © 2017 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

  5. Energetics-Based Methods for Protein Folding and Stability Measurements

    NASA Astrophysics Data System (ADS)

    Geer, M. Ariel; Fitzgerald, Michael C.

    2014-06-01

    Over the past 15 years, a series of energetics-based techniques have been developed for the thermodynamic analysis of protein folding and stability. These techniques include Stability of Unpurified Proteins from Rates of amide H/D Exchange (SUPREX), pulse proteolysis, Stability of Proteins from Rates of Oxidation (SPROX), slow histidine H/D exchange, lysine amidination, and quantitative cysteine reactivity (QCR). The above techniques, which are the subject of this review, all utilize chemical or enzymatic modification reactions to probe the chemical denaturant- or temperature-induced equilibrium unfolding properties of proteins and protein-ligand complexes. They employ various mass spectrometry-, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-, and optical spectroscopy-based readouts that are particularly advantageous for high-throughput and in some cases multiplexed analyses. This has created the opportunity to use protein folding and stability measurements in new applications such as in high-throughput screening projects to identify novel protein ligands and in mode-of-action studies to identify protein targets of a particular ligand.

  6. Heat stability of reconstituted, protein-standardized skim milk powders.

    PubMed

    Sikand, V; Tong, P S; Walker, J

    2010-12-01

    We determined the effects of standardization material, protein content, and pH on the heat stability of reconstituted milk made from low-heat (LH) and medium-heat (MH) nonfat dry milk (NDM). Low-heat and MH NDM were standardized downward from 35.5% to 34, 32, and 30% protein by adding either edible lactose powder (ELP) or permeate powder (PP) from skim milk ultrafiltration. These powders were called standardized skim milk powders (SSMP). The LH and MH NDM and SSMP were reconstituted to 9% total solids. Furthermore, subsamples of reconstituted NDM and SSMP samples were set aside to measure heat stability at native (unadjusted) pH, and the rest were adjusted to pH 6.3 to 7.0. Heat stability is defined as heat coagulation time at 140°C of the reconstituted LH or MH NDM and SSMP samples. The entire experiment was replicated 3 times at unadjusted pH values and 2 times at adjusted pH values. At an unadjusted pH, powder type, standardization material, and protein content influenced the heat stability of the samples. Heat stability for reconstituted LH NDM and SSMP was higher than reconstituted MH NDM and SSMP. Generally, decreased heat stability was observed in reconstituted LH or MH SSMP as protein content was decreased by standardization. However, adding ELP to MH SSMP did not significantly change its heat stability. When pH was adjusted to values between 6.3 and 7.0, powder type, standardization material, and pH had a significant effect on heat stability, whereas protein content did not. Maximum heat stability was noted at pH 6.7 for both reconstituted LH NDM and SSMP samples, and at pH 6.6 for both reconstituted MH NDM and SSMP samples. Furthermore, for samples with adjusted pH, higher heat stability was observed for reconstituted LH SSMP containing PP compared with reconstituted milk from LH SSMP containing ELP. However, no statistical difference was observed in the heat stability of reconstituted milk from MH NDM and MH SSMP samples. We conclude that powder type

  7. Electrostatic Stabilization Of Growing Protein Crystals

    NASA Technical Reports Server (NTRS)

    Shlichta, Paul J.

    1991-01-01

    Proposed technique produces large crystals in compact, economical apparatus. Report presents concept for supporting protein crystals during growth in microgravity. Yields crystals larger and more-nearly perfect than those grown on Earth. Combines best features of sandwich-drop and electrostatic-levitation methods of support. Drop of protein solution inserted between pair of glass or plastic plates, as in sandwich-drop-support method. Electrostatically charged ring confines drop laterally and shapes it, as in electrostatic technique. Apparatus also made to accommodate several drops simultaneously between same pair of supporting plates. Drops can be inserted and crystals removed through ducts in plates.

  8. Stabilized helical peptides: a strategy to target protein-protein interactions.

    PubMed

    Klein, Mark A

    2014-08-14

    Protein-protein interactions are critical for cell proliferation, differentiation, and function. Peptides hold great promise for clinical applications focused on targeting protein-protein interactions. Advantages of peptides include a large chemical space and potential diversity of sequences and structures. However, peptides do present well-known challenges for drug development. Progress has been made in the development of stabilizing alpha helices for potential therapeutic applications. Advantages and disadvantages of different methods of helical peptide stabilization are discussed.

  9. Ligand binding and thermodynamic stability of a multidomain protein, calmodulin.

    PubMed Central

    Masino, L.; Martin, S. R.; Bayley, P. M.

    2000-01-01

    Chemical and thermal denaturation of calmodulin has been monitored spectroscopically to determine the stability for the intact protein and its two isolated domains as a function of binding of Ca2+ or Mg2+. The reversible urea unfolding of either isolated apo-domain follows a two-state mechanism with relatively low deltaG(o)20 values of approximately 2.7 (N-domain) and approximately 1.9 kcal/mol (C-domain). The apo-C-domain is significantly unfolded at normal temperatures (20-25 degrees C). The greater affinity of the C-domain for Ca2+ causes it to be more stable than the N-domain at [Ca2+] > or = 0.3 mM. By contrast, Mg2+ causes a greater stabilization of the N- rather than the C-domain, consistent with measured Mg2+ affinities. For the intact protein (+/-Ca2+), the bimodal denaturation profiles can be analyzed to give two deltaG(o)20 values, which differ significantly from those of the isolated domains, with one domain being less stable and one domain more stable. The observed stability of the domains is strongly dependent on solution conditions such as ionic strength, as well as specific effects due to metal ion binding. In the intact protein, different folding intermediates are observed, depending on the ionic composition. The results illustrate that a protein of low intrinsic stability is liable to major perturbation of its unfolding properties by environmental conditions and liganding processes and, by extension, mutation. Hence, the observed stability of an isolated domain may differ significantly from the stability of the same structure in a multidomain protein. These results address questions involved in manipulating the stability of a protein or its domains by site directed mutagenesis and protein engineering. PMID:10975573

  10. Separation of stem cell maintenance and transposon silencing functions of Piwi protein.

    PubMed

    Klenov, Mikhail S; Sokolova, Olesya A; Yakushev, Evgeny Y; Stolyarenko, Anastasia D; Mikhaleva, Elena A; Lavrov, Sergey A; Gvozdev, Vladimir A

    2011-11-15

    Piwi-interacting RNAs (piRNAs) and Piwi proteins have the evolutionarily conserved function of silencing of repetitive genetic elements in germ lines. The founder of the Piwi subfamily, Drosophila nuclear Piwi protein, was also shown to be required for the maintenance of germ-line stem cells (GSCs). Hence, null mutant piwi females exhibit two types of abnormalities, overexpression of transposons and severely underdeveloped ovaries. It remained unknown whether the failure of GSC maintenance is related to transposon derepression or if GSC self-renewal and piRNA silencing are two distinct functions of the Piwi protein. We have revealed a mutation, piwi(Nt), removing the nuclear localization signal of the Piwi protein. piwi(Nt) females retain the ability of GSC self-renewal and a near-normal number of egg chambers in the ovarioles but display a drastic transposable element derepression and nuclear accumulation of their transcripts in the germ line. piwi(Nt) mutants are sterile most likely because of the disturbance of piRNA-mediated transposon silencing. Analysis of chromatin modifications in the piwi(Nt) ovaries indicated that Piwi causes chromatin silencing only of certain types of transposons, whereas others are repressed in the nuclei without their chromatin modification. Thus, Piwi nuclear localization that is required for its silencing function is not essential for the maintenance of GSCs. We suggest that the Piwi function in GSC self-renewal is independent of transposon repression and is normally realized in the cytoplasm of GSC niche cells.

  11. Local hydrophobicity stabilizes secondary structures in proteins

    SciTech Connect

    Kanehisa, M.I.; Tsong, T.Y.

    1980-01-01

    The probability of occurrence of helix and ..beta..-sheet residues in 47 globular proteins was determined as a function of local hydrophobicity, which was defined by the sum of the Nozaki-Tanford transfer free energies at two nearest-neighbors on both sides of the amino acid sequence. In general, hydrophilic amino acids favor neither helix nor ..beta..-sheet formations when neighbor residues are also hydrophilic but favor helix formation at higher local hydrophobicity. On the other hand, some hydrophobic amino acids such as Met, Leu, and Ile favor helix formation when neighbor residues are hydrophilic. None of the hydrophobic amino acids favor ..beta..-sheet formation with hydrophilic neighbors, but most of them strongly favor ..beta..-sheet formation at high local hydrophobicity. When the average of 20 amino acids is taken, both helix and ..beta..-sheet residue probabilities are higher at higher local hydrophobicity, although the increase is steeper for ..beta..-sheets. Therefore, ..beta..-sheet formation is more influenced by local hydrophobicity than helix formation. Generally, helices are nearer the surface and tend to have hydrophilic and hydrophobic faces at opposite sides. The tendency of alternating regions of hydrophilic and hydrophobic residues in a helical sequence was revealed by calculating the correlation of the Nozaki-Tanford values. Such amphipathic helices may be important in protein-protein-lipid interactions and in forming hydrophilic channels in the membrane. The choice of 30 nonhomologous proteins as the data set did not alter the above results.

  12. Functional diversification of CLAVATA3-related CLE proteins in meristem maintenance in rice.

    PubMed

    Suzaki, Takuya; Yoshida, Akiko; Hirano, Hiro-Yuki

    2008-08-01

    Postembryonic development in plants depends on the activity of the shoot apical meristem (SAM) and root apical meristem (RAM). In Arabidopsis thaliana, CLAVATA signaling negatively regulates the size of the stem cell population in the SAM by repressing WUSCHEL. In other plants, however, studies of factors involved in stem cell maintenance are insufficient. Here, we report that two proteins closely related to CLAVATA3, FLORAL ORGAN NUMBER2 (FON2) and FON2-LIKE CLE PROTEIN1 (FCP1/Os CLE402), have functionally diversified to regulate the different types of meristem in rice (Oryza sativa). Unlike FON2, which regulates the maintenance of flower and inflorescence meristems, FCP1 appears to regulate the maintenance of the vegetative SAM and RAM. Constitutive expression of FCP1 results in consumption of the SAM in the vegetative phase, and application of an FCP1 CLE peptide in vitro disturbs root development by misspecification of cell fates in the RAM. FON1, a putative receptor of FON2, is likely to be unnecessary for these FCP1 functions. Furthermore, we identify a key amino acid residue that discriminates between the actions of FCP1 and FON2. Our results suggest that, although the basic framework of meristem maintenance is conserved in the angiosperms, the functions of the individual factors have diversified during evolution.

  13. The HU Protein Is Important for Apicoplast Genome Maintenance and Inheritance in Toxoplasma gondii

    PubMed Central

    Reiff, Sarah B.; Vaishnava, Shipra

    2012-01-01

    The apicoplast, a chloroplast-like organelle, is an essential cellular component of most apicomplexan parasites, including Plasmodium and Toxoplasma. The apicoplast maintains its own genome, a 35-kb DNA molecule that largely encodes proteins required for organellar transcription and translation. Interference with apicoplast genome maintenance and function is a validated target for drug therapy for malaria and toxoplasmosis. However, the many proteins required for genome maintenance and inheritance remain largely unstudied. Here we genetically characterize a nucleus-encoded homolog to the bacterial HU protein in Toxoplasma gondii. In bacteria, HU is a DNA-binding structural protein with fundamental roles in transcription, replication initiation, and DNA repair. Immunofluorescence assays reveal that in T. gondii this protein localizes to the apicoplast. We have found that the HU protein from Toxoplasma can successfully complement bacterial ΔhupA mutants, supporting a similar function. We were able to construct a genetic knockout of HU in Toxoplasma. This Δhu mutant is barely viable and exhibits significant growth retardation. Upon further analysis of the mutant phenotype, we find that this mutant has a dramatically reduced apicoplast genome copy number and, furthermore, suffers defects in the segregation of the apicoplast organelle. Our findings not only show that the HU protein is important for Toxoplasma cell biology but also demonstrate the importance of the apicoplast genome in the biogenesis of the organelle. PMID:22611021

  14. Stabilization of protein-protein interactions in drug discovery.

    PubMed

    Andrei, Sebastian A; Sijbesma, Eline; Hann, Michael; Davis, Jeremy; O'Mahony, Gavin; Perry, Matthew W D; Karawajczyk, Anna; Eickhoff, Jan; Brunsveld, Luc; Doveston, Richard G; Milroy, Lech-Gustav; Ottmann, Christian

    2017-09-01

    PPIs are involved in every disease and specific modulation of these PPIs with small molecules would significantly improve our prospects of developing therapeutic agents. Both industry and academia have engaged in the identification and use of PPI inhibitors. However in comparison, the opposite strategy of employing small-molecule stabilizers of PPIs is underrepresented in drug discovery. Areas covered: PPI stabilization has not been exploited in a systematic manner. Rather, this concept validated by a number of therapeutically used natural products like rapamycin and paclitaxel has been shown retrospectively to be the basis of the activity of synthetic molecules originating from drug discovery projects among them lenalidomide and tafamidis. Here, the authors cover the growing number of synthetic small-molecule PPI stabilizers to advocate for a stronger consideration of this as a drug discovery approach. Expert opinion: Both the natural products and the growing number of synthetic molecules show that PPI stabilization is a viable strategy for drug discovery. There is certainly a significant challenge to adapt compound libraries, screening techniques and downstream methodologies to identify, characterize and optimize PPI stabilizers, but the examples of molecules reviewed here in our opinion justify these efforts.

  15. USP7 and TDP-43: Pleiotropic Regulation of Cryptochrome Protein Stability Paces the Oscillation of the Mammalian Circadian Clock.

    PubMed

    Hirano, Arisa; Nakagawa, Tomoki; Yoshitane, Hikari; Oyama, Masaaki; Kozuka-Hata, Hiroko; Lanjakornsiripan, Darin; Fukada, Yoshitaka

    2016-01-01

    Mammalian Cryptochromes, CRY1 and CRY2, function as principal regulators of a transcription-translation-based negative feedback loop underlying the mammalian circadian clockwork. An F-box protein, FBXL3, promotes ubiquitination and degradation of CRYs, while FBXL21, the closest paralog of FBXL3, ubiquitinates CRYs but leads to stabilization of CRYs. Fbxl3 knockout extremely lengthened the circadian period, and deletion of Fbxl21 gene in Fbxl3-deficient mice partially rescued the period-lengthening phenotype, suggesting a key role of CRY protein stability for maintenance of the circadian periodicity. Here, we employed a proteomics strategy to explore regulators for the protein stability of CRYs. We found that ubiquitin-specific protease 7 (USP7 also known as HAUSP) associates with CRY1 and CRY2 and stabilizes CRYs through deubiquitination. Treatment with USP7-specific inhibitor or Usp7 knockdown shortened the circadian period of the cellular rhythm. We identified another CRYs-interacting protein, TAR DNA binding protein 43 (TDP-43), an RNA-binding protein. TDP-43 stabilized CRY1 and CRY2, and its knockdown also shortened the circadian period in cultured cells. The present study identified USP7 and TDP-43 as the regulators of CRY1 and CRY2, underscoring the significance of the stability control process of CRY proteins for period determination in the mammalian circadian clockwork.

  16. USP7 and TDP-43: Pleiotropic Regulation of Cryptochrome Protein Stability Paces the Oscillation of the Mammalian Circadian Clock

    PubMed Central

    Yoshitane, Hikari; Oyama, Masaaki; Kozuka-Hata, Hiroko; Lanjakornsiripan, Darin; Fukada, Yoshitaka

    2016-01-01

    Mammalian Cryptochromes, CRY1 and CRY2, function as principal regulators of a transcription-translation-based negative feedback loop underlying the mammalian circadian clockwork. An F-box protein, FBXL3, promotes ubiquitination and degradation of CRYs, while FBXL21, the closest paralog of FBXL3, ubiquitinates CRYs but leads to stabilization of CRYs. Fbxl3 knockout extremely lengthened the circadian period, and deletion of Fbxl21 gene in Fbxl3-deficient mice partially rescued the period-lengthening phenotype, suggesting a key role of CRY protein stability for maintenance of the circadian periodicity. Here, we employed a proteomics strategy to explore regulators for the protein stability of CRYs. We found that ubiquitin-specific protease 7 (USP7 also known as HAUSP) associates with CRY1 and CRY2 and stabilizes CRYs through deubiquitination. Treatment with USP7-specific inhibitor or Usp7 knockdown shortened the circadian period of the cellular rhythm. We identified another CRYs-interacting protein, TAR DNA binding protein 43 (TDP-43), an RNA-binding protein. TDP-43 stabilized CRY1 and CRY2, and its knockdown also shortened the circadian period in cultured cells. The present study identified USP7 and TDP-43 as the regulators of CRY1 and CRY2, underscoring the significance of the stability control process of CRY proteins for period determination in the mammalian circadian clockwork. PMID:27123980

  17. Interplay between Protein Thermal Flexibility and Kinetic Stability.

    PubMed

    Quezada, Andrea G; Díaz-Salazar, A Jessica; Cabrera, Nallely; Pérez-Montfort, Ruy; Piñeiro, Ángel; Costas, Miguel

    2017-01-03

    Kinetic stability is a key parameter to comprehend protein behavior and it plays a central role to understand how evolution has reached the balance between function and stability in cell-relevant timescales. Using an approach that includes simulations, protein engineering, and calorimetry, we show that there is a clear correlation between kinetic stability determined by differential scanning calorimetry and protein thermal flexibility obtained from a novel method based on temperature-induced unfolding molecular dynamics simulations. Thermal flexibility quantitatively measures the increment of the conformational space available to the protein when energy in provided. The (β/α)8 barrel fold of two closely related by evolution triosephosphate isomerases from two trypanosomes are used as model systems. The kinetic stability-thermal flexibility correlation has predictive power for the studied proteins, suggesting that the strategy and methodology discussed here might be applied to other proteins in biotechnological developments, evolutionary studies, and the design of protein based therapeutics.

  18. A method for direct measurement of protein stability in vivo.

    PubMed

    Ignatova, Zoya; Gierasch, Lila M

    2009-01-01

    The stability of proteins is tuned by evolution to enable them to perform their cellular functions for the success of an organism. Yet, most of the arsenal of biophysical techniques at our disposal to characterize the thermodynamic stability of proteins is limited to in vitro samples. We describe an approach that we have developed to observe a protein directly in a cell and to monitor a fluorescence signal that reports the unfolding transition of the protein, yielding quantitatively interpretable stability data in vivo. The method is based on incorporation of structurally nonperturbing, specific binding motifs for a bis-arsenical fluorescein derivative in sites that result in dye fluorescence differences between the folded and unfolded states of the protein under study. This fluorescence labeling approach makes possible the determination of thermodynamic stability by direct urea titration in Escherichia coli cells. The specific case study we describe was carried out on the predominantly beta-sheet intracellular lipid-binding protein, cellular retinoic acid-binding protein (CRABP), expressed in E. coli.

  19. Robust enzyme design: bioinformatic tools for improved protein stability.

    PubMed

    Suplatov, Dmitry; Voevodin, Vladimir; Švedas, Vytas

    2015-03-01

    The ability of proteins and enzymes to maintain a functionally active conformation under adverse environmental conditions is an important feature of biocatalysts, vaccines, and biopharmaceutical proteins. From an evolutionary perspective, robust stability of proteins improves their biological fitness and allows for further optimization. Viewed from an industrial perspective, enzyme stability is crucial for the practical application of enzymes under the required reaction conditions. In this review, we analyze bioinformatic-driven strategies that are used to predict structural changes that can be applied to wild type proteins in order to produce more stable variants. The most commonly employed techniques can be classified into stochastic approaches, empirical or systematic rational design strategies, and design of chimeric proteins. We conclude that bioinformatic analysis can be efficiently used to study large protein superfamilies systematically as well as to predict particular structural changes which increase enzyme stability. Evolution has created a diversity of protein properties that are encoded in genomic sequences and structural data. Bioinformatics has the power to uncover this evolutionary code and provide a reproducible selection of hotspots - key residues to be mutated in order to produce more stable and functionally diverse proteins and enzymes. Further development of systematic bioinformatic procedures is needed to organize and analyze sequences and structures of proteins within large superfamilies and to link them to function, as well as to provide knowledge-based predictions for experimental evaluation.

  20. White wine continuous protein stabilization by packed column.

    PubMed

    Pashova, Vesselina; Güell, Carme; López, Francisco

    2004-03-24

    Protein stabilization is an important stage in the production of white wine. This paper studies white wine protein stabilization using a continuous process with zirconium oxide (powder and pellets) packed in a column. The results show that the total proteins decrease by 50 and 70% for the pellet and powdered zirconium oxides, respectively. Treatment with all zirconium oxides improves wine stability. The effect of the heat regeneration process on both zirconium oxide forms is to increase the adsorption capacity. The wine treated with powdered zirconium oxide after the regeneration is the most effective for preventing protein haze. The protein profile of wine after treatment shows that the 20-50 kDa and 50-70 kDa fractions are the ones removed preferentially, while the 15 kDa fraction and the ones higher than 70 kDa are removed the least. The results show that the protein fraction with a molecular weight of 15 kDa does not affect the protein instability of the wines studied. The protein fraction with a molecular weight higher than 70 kDa seems to influence protein instability. The physicochemical properties of wine after treatment were not affected, and the values obtained were like those of the standardized range.

  1. Development of a simple assay system for protein-stabilizing efficiency based on hemoglobin protection against denaturation and measurement of the cooperative effect of mixing protein stabilizers.

    PubMed

    Chen, Siyu; Manabe, Yoshiyuki; Minamoto, Naoya; Saiki, Naoka; Fukase, Koichi

    2016-10-01

    We have elucidated the cooperative stabilization of proteins by sugars, amino acids, and other protein-stabilizing agents using a new and simple assay system. Our system determines the protein-stabilizing ability of various compounds by measuring their ability to protect hemoglobin from denaturation. Hemoglobin denaturation was readily measured by quantitative changes in its ultraviolet-visible absorption spectrum. The efficiency of our assay was confirmed using various sugars such as trehalose and sucrose that are known to be good protein stabilizers. We have also found that mixtures of two different types of protein stabilizers resulted in a cooperative stabilizing effect on protein.

  2. Fluorinated proteins: from design and synthesis to structure and stability.

    PubMed

    Marsh, E Neil G

    2014-10-21

    Fluorine is all but absent from biology; however, it has proved to be a remarkably useful element with which to modulate the activity of biological molecules and to study their mechanism of action. Our laboratory's interest in incorporating fluorine into proteins was stimulated by the unusual physicochemical properties exhibited by perfluorinated small molecules. These include extreme chemical inertness and thermal stability, properties that have made them valuable as nonstick coatings and fire retardants. Fluorocarbons also exhibit an unusual propensity to phase segregation. This phenomenon, which has been termed the "fluorous effect", has been effectively exploited in organic synthesis to purify compounds from reaction mixtures by extracting fluorocarbon-tagged molecules into fluorocarbon solvents. As biochemists, we were curious to explore whether the unusual physicochemical properties of perfluorocarbons could be engineered into proteins. To do this, we developed a synthesis of a highly fluorinated amino acid, hexafluoroleucine, and designed a model 4-helix bundle protein, α4H, in which the hydrophobic core was packed exclusively with leucine. We then investigated the effects of repacking the hydrophobic core of α4H with various combinations of leucine and hexafluoroleucine. These initial studies demonstrated that fluorination is a general and effective strategy for enhancing the stability of proteins against chemical and thermal denaturation and proteolytic degradation. We had originally envisaged that the "fluorous interactions", postulated from the self-segregating properties of fluorous solvents, might be used to mediate specific protein-protein interactions orthogonal to those of natural proteins. However, various lines of evidence indicate that no special, favorable fluorine-fluorine interactions occur in the core of the fluorinated α4 protein. This makes it unlikely that fluorinated amino acids can be used to direct protein-protein interactions. More

  3. Protein stability and molecular adaptation to extreme conditions.

    PubMed

    Jaenicke, R

    1991-12-18

    Proteins, due to the delicate balance of stabilizing and destabilizing interactions, are only marginally stable. Adaptation to extreme environments tends to shift the 'mesophilic' characteristics of proteins to the respective extremes of temperature, hydrostatic pressure, pH and salinity, such that, under the mutual physiological conditions, the molecular properties are similar regarding overall topology, flexibility and solvation. Enhanced intrinsic stability requires only minute local structural changes so that general strategies of stabilization cannot be established. Apart from mutative changes of amino-acid sequences, extrinsic factors (or cellular components) may be involved in 'extremophilic adaptation'. The molecular basis of acidophilic, alkalophilic and barophilic adaptation is still obscure. Mechanisms of enhanced thermal stability involve improved packing density, as well as specific local interactions. In halophiles, water and salt binding of the intrinsically stable protein inventory is accomplished by favoring acidic over basic amino acid residues and decreased hydrophobicity. General limits of viability are: (a) the susceptibility of the covalent structure of the polypeptide chain toward hydrolysis or hydrothermal degradation; (b) the competition of extreme solvent parameters with the weak electrostatic and hydrophobic interactions involved in protein stabilization; (c) perturbations of the folding and assembly of proteins; and (d) 'dislocation' of biochemical pathways due to effects of extreme conditions on the intricate network of metabolic reactions.

  4. Defining the role of salt bridges in protein stability.

    PubMed

    Jelesarov, Ilian; Karshikoff, Andrey

    2009-01-01

    Although the energetic balance of forces stabilizing proteins has been established qualitatively over the last decades, quantification of the energetic contribution of particular interactions still poses serious problems. The reasons are the strong cooperativity and the interdependence ofnoncovalent interactions. Salt bridges are a typical example. One expects that ionizable side chains frequently form ion pairs in innumerable crystal structures. Since electrostatic attraction between opposite charges is strong per se, salt bridges can intuitively be regarded as an important factor stabilizing the native structure. Is that really so? In this chapter we critically reassess the available methods to delineate the role ofelectrostatic interactions and salt bridges to protein stability, and discuss the progress and the obstacles in this endeavor. The basic problem is that formation of salt bridges depends on the ionization properties of the participating groups, which is significantly influenced by the protein environment. Furthermore, salt bridges experience thermal fluctuations, continuously break and re-form, and their lifespan in solution is governed by the flexibility of the protein. Finally, electrostatic interactions are long-range and might be significant in the unfolded state, thus seriously influencing the energetic profile. Elimination of salt bridges by protonation/deprotonation at extreme pH or by mutation provides only rough energetic estimates, since there is no way to account for the nonadditive response of the protein moiety. From what we know so far, the strength of electrostatic interactions is strongly context-dependent, yet it is unlikely that salt bridges are dominant factors governing protein stability. Nevertheless, proteins from thermophiles and hyperthermophiles exhibit more, and frequently networked, salt bridges than proteins from the mesophilic counterparts. Increasing the thermal (not the thermodynamic) stability of proteins by optimization

  5. An Entropic Perspective of Protein Stability on Surfaces

    PubMed Central

    Knotts, Thomas A.; Rathore, Nitin; Pablo, Juan J. de

    2008-01-01

    The interaction of proteins with surfaces regulates numerous processes in nature, science, and technology. In many applications, it is desirable to place proteins on surfaces in an active state, and tethering represents one manner in which to accomplish this. However, a clear understanding of how tether placement and design affects protein activity is lacking. Available theoretical models predict that proteins will be stabilized when tethered to substrates. Such models suggest that the surface reduces the number of states accessible to the unfolded state of the protein, thereby reducing the entropic cost of folding on the surface compared to the bulk case. Recent studies, however, have shown that this stabilization is not always seen. The purpose of this article is to determine the validity of the theory with a thorough thermodynamic analysis of the folding of peptides attached to surfaces. Configuration-temperature-density-of-states Monte Carlo simulations are used to examine the behavior of four different peptides of different secondary and tertiary structure. It is found that the surface does reduce the entropic cost of folding for tethered peptides, as the theory suggests. This effect, however, does not always translate into improved stability because the surface may also have a destabilizing enthalpic effect. The theory neglects this effect and assumes that the enthalpy of folding is the same on and off the surface. Both the enthalpic and entropic contributions to the stability are found to be topology- and tether-placement-specific; we show that stability cannot be predicted a priori. A detailed analysis of the folding of protein A shows how the same protein can be both stabilized and destabilized on a surface depending upon how the tethering enhances or hinders the ability of the peptide to form correct tertiary structures. PMID:18326646

  6. An entropic perspective of protein stability on surfaces.

    PubMed

    Knotts, Thomas A; Rathore, Nitin; de Pablo, Juan J

    2008-06-01

    The interaction of proteins with surfaces regulates numerous processes in nature, science, and technology. In many applications, it is desirable to place proteins on surfaces in an active state, and tethering represents one manner in which to accomplish this. However, a clear understanding of how tether placement and design affects protein activity is lacking. Available theoretical models predict that proteins will be stabilized when tethered to substrates. Such models suggest that the surface reduces the number of states accessible to the unfolded state of the protein, thereby reducing the entropic cost of folding on the surface compared to the bulk case. Recent studies, however, have shown that this stabilization is not always seen. The purpose of this article is to determine the validity of the theory with a thorough thermodynamic analysis of the folding of peptides attached to surfaces. Configuration-temperature-density-of-states Monte Carlo simulations are used to examine the behavior of four different peptides of different secondary and tertiary structure. It is found that the surface does reduce the entropic cost of folding for tethered peptides, as the theory suggests. This effect, however, does not always translate into improved stability because the surface may also have a destabilizing enthalpic effect. The theory neglects this effect and assumes that the enthalpy of folding is the same on and off the surface. Both the enthalpic and entropic contributions to the stability are found to be topology- and tether-placement-specific; we show that stability cannot be predicted a priori. A detailed analysis of the folding of protein A shows how the same protein can be both stabilized and destabilized on a surface depending upon how the tethering enhances or hinders the ability of the peptide to form correct tertiary structures.

  7. Mechanisms of protein stabilization and prevention of protein aggregation by glycerol.

    PubMed

    Vagenende, Vincent; Yap, Miranda G S; Trout, Bernhardt L

    2009-11-24

    The stability of proteins in aqueous solution is routinely enhanced by cosolvents such as glycerol. Glycerol is known to shift the native protein ensemble to more compact states. Glycerol also inhibits protein aggregation during the refolding of many proteins. However, mechanistic insight into protein stabilization and prevention of protein aggregation by glycerol is still lacking. In this study, we derive mechanisms of glycerol-induced protein stabilization by combining the thermodynamic framework of preferential interactions with molecular-level insight into solvent-protein interactions gained from molecular simulations. Contrary to the common conception that preferential hydration of proteins in polyol/water mixtures is determined by the molecular size of the polyol and the surface area of the protein, we present evidence that preferential hydration of proteins in glycerol/water mixtures mainly originates from electrostatic interactions that induce orientations of glycerol molecules at the protein surface such that glycerol is further excluded. These interactions shift the native protein toward more compact conformations. Moreover, glycerol preferentially interacts with large patches of contiguous hydrophobicity where glycerol acts as an amphiphilic interface between the hydrophobic surface and the polar solvent. Accordingly, we propose that glycerol prevents protein aggregation by inhibiting protein unfolding and by stabilizing aggregation-prone intermediates through preferential interactions with hydrophobic surface regions that favor amphiphilic interface orientations of glycerol. These mechanisms agree well with experimental data available in the literature, and we discuss the extent to which these mechanisms apply to other cosolvents, including polyols, arginine, and urea.

  8. Role of arginine in the stabilization of proteins against aggregation.

    PubMed

    Baynes, Brian M; Wang, Daniel I C; Trout, Bernhardt L

    2005-03-29

    The amino acid arginine is frequently used as a solution additive to stabilize proteins against aggregation, especially in the process of protein refolding. Despite arginine's prevalence, the mechanism by which it stabilizes proteins is not presently understood. We propose that arginine deters aggregation by slowing protein-protein association reactions, with only a small concomitant effect on protein folding. The associated rate effect was observed experimentally in association of globular proteins (insulin and a monoclonal anti-insulin) and in refolding of carbonic anhydrase. We suggest that this effect arises because arginine is preferentially excluded from protein-protein encounter complexes but not from dissociated protein molecules. Such an effect is predicted by our gap effect theory [Baynes and Trout (2004) Biophys. J. 87, 1631] for "neutral crowder" additives such as arginine which are significantly larger than water but have only a small effect on the free energies of isolated protein molecules. The effect of arginine on refolding of carbonic anhydrase was also shown to be consistent with this hypothesis.

  9. Protein stability and enzyme activity at extreme biological temperatures.

    PubMed

    Feller, Georges

    2010-08-18

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 °C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins.

  10. Effects of sugars on the thermal stability of a protein

    NASA Astrophysics Data System (ADS)

    Oshima, Hiraku; Kinoshita, Masahiro

    2013-06-01

    It is experimentally known that the heat-denaturation temperature of a protein is raised (i.e., its thermal stability is enhanced) by sugar addition. In earlier works, we proposed a physical picture of thermal denaturation of proteins in which the measure of the thermal stability is defined as the solvent-entropy gain upon protein folding at 298 K normalized by the number of residues. A multipolar-model water was adopted as the solvent. The polyatomic structures of the folded and unfolded states of a protein were taken into account in the atomic detail. A larger value of the measure implies higher thermal stability. First, we show that the measure remains effective even when the model water is replaced by the hard-sphere solvent whose number density and molecular diameter are set at those of real water. The physical picture is then adapted to the elucidation of the effects of sugar addition on the thermal stability of a protein. The water-sugar solution is modeled as a binary mixture of hard spheres. The thermal stability is determined by a complex interplay of the diameter of sugar molecules dC and the total packing fraction of the solution η: dC is estimated from the volume per molecule in the sugar crystal and η is calculated using the experimental data of the solution density. We find that the protein is more stabilized as the sucrose or glucose concentration becomes higher and the stabilization effect is stronger for sucrose than for glucose. These results are in accord with the experimental observations. Using a radial-symmetric integral equation theory and the morphometric approach, we decompose the change in the measure upon sugar addition into two components originating from the protein-solvent pair and protein-solvent many-body correlations, respectively. Each component is further decomposed into the excluded-volume and solvent-accessible-surface terms. These decompositions give physical insights into the microscopic origin of the thermal-stability

  11. Stability of ALS-related Superoxide Dismutase Protein variants

    NASA Astrophysics Data System (ADS)

    Lusebrink, Daniel; Plotkin, Steven

    Superoxide dismutase (SOD1) is a metal binding, homodimeric protein, whose misfolding is implicated in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Monomerization is believed to be a key step in the propagation of the disease. The dimer stability is often difficult to measure experimentally however, because it is entangled with protein unfolding and metal loss. We thus computationally investigate the dimer stability of mutants of SOD1 known to be associated with ALS. We report on systematic trends in dimer stability, as well as intriguing allosteric communication between mutations and the dimer interface. We study the dimer stabilities in molecular dynamics simulations and obtain the binding free energies of the dimers from pulling essays. Mutations are applied in silicoand we compare the differences of binding free energies compared to the wild type.

  12. Rational stabilization of complex proteins: a divide and combine approach

    PubMed Central

    Lamazares, Emilio; Clemente, Isabel; Bueno, Marta; Velázquez-Campoy, Adrián; Sancho, Javier

    2015-01-01

    Increasing the thermostability of proteins is often crucial for their successful use as analytic, synthetic or therapeutic tools. Most rational thermostabilization strategies were developed on small two-state proteins and, unsurprisingly, they tend to fail when applied to the much more abundant, larger, non-fully cooperative proteins. We show that the key to stabilize the latter is to know the regions of lower stability. To prove it, we have engineered apoflavodoxin, a non-fully cooperative protein on which previous thermostabilizing attempts had failed. We use a step-wise combination of structure-based, rationally-designed, stabilizing mutations confined to the less stable structural region, and obtain variants that, according to their van't Hoff to calorimetric enthalpy ratios, exhibit fully-cooperative thermal unfolding with a melting temperature of 75°C, 32 degrees above the lower melting temperature of the non-cooperative wild type protein. The ideas introduced here may also be useful for the thermostabilization of complex proteins through formulation or using specific stabilizing ligands (e.g. pharmacological chaperones). PMID:25774740

  13. Conformational stability of adrenodoxin mutant proteins.

    PubMed Central

    Burova, T. V.; Beckert, V.; Uhlmann, H.; Ristau, O.; Bernhardt, R.; Pfeil, W.

    1996-01-01

    Adrenodoxin and the mutants at the positions T54, H56, D76, Y82, and C95, as well as the deletion mutants 4-114 and 4-108, were studied by high-sensitivity scanning microcalorimetry, limited proteolysis, and absorption spectroscopy. The mutants show thermal transition temperatures ranging from 46 to 56 degrees C, enthalpy changes from 250 to 370 kJ/mol, and heat capacity change delta Cp = 7.28 +/- 0.67 kJ/mol/K, except H56R. The amino acid replacement H56R produces substantial local changes in the region around positions 56 and Y82, as indicated by reduced heat capacity change (delta Cp = 4.29 +/- 0.37 kJ/mol/K) and enhanced fluorescence. Deletion mutant 4-108 is apparently more stable than the wild type, as judged by higher specific denaturation enthalpy and resistance toward proteolytic degradation. No simple correlation between conformational stability and functional properties could be found. PMID:8880913

  14. Interactions of phospholipase D and cytochrome P450 protein stability

    SciTech Connect

    Zangar, Richard C.; Fan, Yang-Yi; Chapkin, Robert S.

    2004-08-01

    Previous studies have suggested a relationship between cytochrome P450 (P450) 3A (CYP3A) conformation and the phospholipid composition of the associated membrane. In this study, we utilized a novel microsomal incubation system that mimics many of the characteristics of CYP3A degradation pathway that have been observed in vivo and in cultured cells to study the effects of phospholipid composition on protein stability. We found that addition of phosphatidylcholine-specific phospholipase D (PLD) stabilized CYP3A in this system, but that phosphatidylinositol-specific phospholipase C (PLC) was without effect. Addition of phosphatidic acid also stabilized CYP3A protein in the microsomes. The use of 1,10-phenanthroline (phenanthroline), an inhibitor of PLD activity, decreased CYP3A stability in incubated microsomes. Similarly, 6-h treatment of primary cultures of rat hepatocytes with phenanthroline resulted in nearly complete loss of CYP3A protein. Treatment of rats with nicardipine or dimethylsulfoxide (DMSO), which have been shown to affect CYP3A stability, altered the phospholipid composition of hepatic microsomes. It did not appear, though, that the changes in phospholipid composition that resulted from these in vivo treatments accounted for the change in CYP3A stability observed in hepatic microsomes from these animals.

  15. The role of interfacial lipids in stabilizing membrane protein oligomers.

    PubMed

    Gupta, Kallol; Donlan, Joseph A C; Hopper, Jonathan T S; Uzdavinys, Povilas; Landreh, Michael; Struwe, Weston B; Drew, David; Baldwin, Andrew J; Stansfeld, Phillip J; Robinson, Carol V

    2017-01-19

    Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways but is often difficult to define or predict. Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT, one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET, another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na(+)/H(+) antiporter NhaA from E. coli, which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors.

  16. Conformational stability as a design target to control protein aggregation.

    PubMed

    Costanzo, Joseph A; O'Brien, Christopher J; Tiller, Kathryn; Tamargo, Erin; Robinson, Anne Skaja; Roberts, Christopher J; Fernandez, Erik J

    2014-05-01

    Non-native protein aggregation is a prevalent problem occurring in many biotechnological manufacturing processes and can compromise the biological activity of the target molecule or induce an undesired immune response. Additionally, some non-native aggregation mechanisms lead to amyloid fibril formation, which can be associated with debilitating diseases. For natively folded proteins, partial or complete unfolding is often required to populate aggregation-prone conformational states, and therefore one proposed strategy to mitigate aggregation is to increase the free energy for unfolding (ΔGunf) prior to aggregation. A computational design approach was tested using human γD crystallin (γD-crys) as a model multi-domain protein. Two mutational strategies were tested for their ability to reduce/increase aggregation rates by increasing/decreasing ΔGunf: stabilizing the less stable domain and stabilizing the domain-domain interface. The computational protein design algorithm, RosettaDesign, was implemented to identify point variants. The results showed that although the predicted free energies were only weakly correlated with the experimental ΔGunf values, increased/decreased aggregation rates for γD-crys correlated reasonably well with decreases/increases in experimental ΔGunf, illustrating improved conformational stability as a possible design target to mitigate aggregation. However, the results also illustrate that conformational stability is not the sole design factor controlling aggregation rates of natively folded proteins.

  17. Temperature compensation via cooperative stability in protein degradation

    NASA Astrophysics Data System (ADS)

    Peng, Yuanyuan; Hasegawa, Yoshihiko; Noman, Nasimul; Iba, Hitoshi

    2015-08-01

    Temperature compensation is a notable property of circadian oscillators that indicates the insensitivity of the oscillator system's period to temperature changes; the underlying mechanism, however, is still unclear. We investigated the influence of protein dimerization and cooperative stability in protein degradation on the temperature compensation ability of two oscillators. Here, cooperative stability means that high-order oligomers are more stable than their monomeric counterparts. The period of an oscillator is affected by the parameters of the dynamic system, which in turn are influenced by temperature. We adopted the Repressilator and the Atkinson oscillator to analyze the temperature sensitivity of their periods. Phase sensitivity analysis was employed to evaluate the period variations of different models induced by perturbations to the parameters. Furthermore, we used experimental data provided by other studies to determine the reasonable range of parameter temperature sensitivity. We then applied the linear programming method to the oscillatory systems to analyze the effects of protein dimerization and cooperative stability on the temperature sensitivity of their periods, which reflects the ability of temperature compensation in circadian rhythms. Our study explains the temperature compensation mechanism for circadian clocks. Compared with the no-dimer mathematical model and linear model for protein degradation, our theoretical results show that the nonlinear protein degradation caused by cooperative stability is more beneficial for realizing temperature compensation of the circadian clock.

  18. Electrostatic stabilization of a thermophilic cold shock protein.

    PubMed

    Perl, D; Schmid, F X

    2001-10-19

    The cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus differs from its mesophilic homolog Bs-CspB from Bacillus subtilis by 15.8 kJ mol(-1) in the Gibbs free energy of denaturation (DeltaG(D)). The two proteins vary in sequence at 12 positions but only two of them, Arg3 and Leu66 of Bc-Csp, which replace Glu3 and Glu66 of Bs-CspB, are responsible for the additional stability of Bc-Csp. These two positions are near the ends of the protein chain, but close to each other in the three-dimensional structure. The Glu3Arg exchange alone changed the stability by more than 11 kJ mol(-1). Here, we elucidated the molecular origins of the stability difference between the two proteins by a mutational analysis. Electrostatic contributions to stability were characterized by measuring the thermodynamic stabilities of many variants as a function of salt concentration. Double and triple mutant analyses indicate that the stabilization by the Glu3Arg exchange originates from three sources. Improved hydrophobic interactions of the aliphatic moiety of Arg3 contribute about 4 kJ mol(-1). Another 4 kJ mol(-1) is gained from the relief of a pairwise electrostatic repulsion between Glu3 and Glu66, as in the mesophilic protein, and 3 kJ mol(-1) originate from a general electrostatic stabilization by the positive charge of Arg3, which is not caused by a pairwise interaction. Mutations of all potential partners for an ion pair within a radius of 10 A around Arg3 had only marginal effects on stability. The Glu3-->Arg3 charge reversal thus optimizes ionic interactions at the protein surface by both local and global effects. However, it cannot convert the coulombic repulsion with another Glu residue into a corresponding attraction. Avoidance of unfavorable coulombic repulsions is probably a much simpler route to thermostability than the creation of stabilizing surface ion pairs, which can form only at the expense of conformational entropy. Copyright 2001 Academic Press.

  19. Storage Stability of Food Protein Hydrolysates-A Review.

    PubMed

    Rao, Qinchun; Klaassen Kamdar, Andre; Labuza, Theodore P

    2016-05-18

    In recent years, mainly due to the specific health benefits associated with (1) the discovery of bioactive peptides in protein hydrolysates, (2) the reduction of protein allergenicity by protein hydrolysis, and (3) the improved protein digestibility and absorption of protein hydrolysates, the utilization of protein hydrolysates in functional foods and beverages has significantly increased. Although the specific health benefits from different hydrolysates are somewhat proven, the delivery and/or stability of these benefits is debatable during distribution, storage, and consumption. In this review, we discuss (1) the quality changes in different food protein hydrolysates during storage; (2) the resulting changes in the structure and texture of three food matrices, i.e., low moisture foods (LMF, aw < 0.6), intermediate moisture foods (IMF, 0.6 ≤ aw < 0.85), and high moisture foods (HMF, aw ≥ 0.85); and (3) the potential solutions to improve storage stability of food protein hydrolysates. In addition, we note there is a great need for evaluation of biofunction availability of bioactive peptides in food protein hydrolysates during storage.

  20. Thermodynamic stability and folding of proteins from hyperthermophilic organisms.

    PubMed

    Luke, Kathryn A; Higgins, Catherine L; Wittung-Stafshede, Pernilla

    2007-08-01

    Life grows almost everywhere on earth, including in extreme environments and under harsh conditions. Organisms adapted to high temperatures are called thermophiles (growth temperature 45-75 degrees C) and hyperthermophiles (growth temperature >or= 80 degrees C). Proteins from such organisms usually show extreme thermal stability, despite having folded structures very similar to their mesostable counterparts. Here, we summarize the current data on thermodynamic and kinetic folding/unfolding behaviors of proteins from hyperthermophilic microorganisms. In contrast to thermostable proteins, rather few (i.e. less than 20) hyperthermostable proteins have been thoroughly characterized in terms of their in vitro folding processes and their thermodynamic stability profiles. Examples that will be discussed include co-chaperonin proteins, iron-sulfur-cluster proteins, and DNA-binding proteins from hyperthermophilic bacteria (i.e. Aquifex and Theromotoga) and archea (e.g. Pyrococcus, Thermococcus, Methanothermus and Sulfolobus). Despite the small set of studied systems, it is clear that super-slow protein unfolding is a dominant strategy to allow these proteins to function at extreme temperatures.

  1. Protein turnover forms one of the highest maintenance costs in Lactococcus lactis.

    PubMed

    Lahtvee, Petri-Jaan; Seiman, Andrus; Arike, Liisa; Adamberg, Kaarel; Vilu, Raivo

    2014-07-01

    Protein turnover plays an important role in cell metabolism by regulating metabolic fluxes. Furthermore, the energy costs for protein turnover have been estimated to account for up to a third of the total energy production during cell replication and hence may represent a major limiting factor in achieving either higher biomass or production yields. This work aimed to measure the specific growth rate (μ)-dependent abundance and turnover rate of individual proteins, estimate the ATP cost for protein production and turnover, and compare this with the total energy balance and other maintenance costs. The lactic acid bacteria model organism Lactococcus lactis was used to measure protein turnover rates at μ = 0.1 and 0.5 h(-1) in chemostat experiments. Individual turnover rates were measured for ~75% of the total proteome. On average, protein turnover increased by sevenfold with a fivefold increase in growth rate, whilst biomass yield increased by 35%. The median turnover rates found were higher than the specific growth rate of the bacterium, which suggests relatively high energy consumption for protein turnover. We found that protein turnover costs alone account for 38 and 47% of the total energy produced at μ = 0.1 and 0.5 h(-1), respectively, and gene ontology groups Energy metabolism and Translation dominated synthesis costs at both growth rates studied. These results reflect the complexity of metabolic changes that occur in response to changes in environmental conditions, and signify the trade-off between biomass yield and the need to produce ATP for maintenance processes.

  2. Flocculation of protein-stabilized oil-in-water emulsions.

    PubMed

    Dickinson, Eric

    2010-11-01

    The flocculation properties of oil-in-water emulsions stabilized by proteins are reviewed from the colloid science perspective. Emphasis is placed on insight from systematic studies of the stability of emulsions prepared with a milk protein ingredient as the sole emulsifying agent. The main factors considered are pH, ionic strength, calcium ion concentration, thermal processing, and the presence of cosolutes (alcohol, sugars). Contrasting dependences of the flocculation behaviour on these factors are observed for the pH-sensitive disordered caseins (alpha(s1)-casein or beta-casein) and the heat-sensitive globular proteins (especially beta-lactoglobulin). In comparing characteristic emulsion properties obtained with different proteins, we consider the relative importance of the different kinds of molecular and colloidal interactions-electrostatic, steric, hydrophobic and covalent.

  3. Biglycan is an extracellular MuSK binding protein important for synapse stability

    PubMed Central

    Amenta, A.R.; Creely, H.E.; Mercado, M.L.; Hagiwara, H.; McKechnie, B. A.; Lechner, B.E.; Rossi, S. G.; Wang, Q.; Owens, R. T.; Marrero, E.; Mei, L.; Hoch, W.; Young, M. F.; McQuillan, D. J.; Rotundo, R. L.; Fallon, J.R.

    2012-01-01

    The receptor tyrosine kinase MuSK is indispensable for nerve-muscle synapse formation and maintenance. MuSK is necessary for pre-patterning of the endplate zone anlage and as a signaling receptor for agrin-mediated postsynaptic differentiation. MuSK-associated proteins such as Dok7, LRP4, and Wnt11r are involved in these early events in neuromuscular junction formation. However, the mechanisms regulating synapse stability are poorly understood. Here we examine a novel role for the extracellular matrix protein biglycan in synapse stability. Synaptic development in fetal and early postnatal biglycan null (bgn-/o) muscle is indistinguishable from wild type controls. However, by 5 wks after birth nerve-muscle synapses in bgn-/o mice are abnormal as judged by the presence of perijunctional folds, increased segmentation and focal misalignment of acetylcholinesterase and AChRs. These observations indicate that previously occupied pre- and post- synaptic territory has been vacated. Biglycan binds MuSK and the levels of this receptor tyrosine kinase are selectively reduced at bgn-/o synapses. In bgn-/o myotubes, the initial stages of agrin-induced MuSK phosphorylation and AChR clustering are normal, but the AChR clusters are unstable. This stability defect can be substantially rescued by the addition of purified biglycan. Together, these results indicate that biglycan is an extracellular ligand for MuSK that is important for synapse stability. PMID:22396407

  4. Positively selected sites in cetacean myoglobins contribute to protein stability.

    PubMed

    Dasmeh, Pouria; Serohijos, Adrian W R; Kepp, Kasper P; Shakhnovich, Eugene I

    2013-01-01

    Since divergence ∼50 Ma ago from their terrestrial ancestors, cetaceans underwent a series of adaptations such as a ∼10-20 fold increase in myoglobin (Mb) concentration in skeletal muscle, critical for increasing oxygen storage capacity and prolonging dive time. Whereas the O2-binding affinity of Mbs is not significantly different among mammals (with typical oxygenation constants of ∼0.8-1.2 µM(-1)), folding stabilities of cetacean Mbs are ∼2-4 kcal/mol higher than for terrestrial Mbs. Using ancestral sequence reconstruction, maximum likelihood and bayesian tests to describe the evolution of cetacean Mbs, and experimentally calibrated computation of stability effects of mutations, we observe accelerated evolution in cetaceans and identify seven positively selected sites in Mb. Overall, these sites contribute to Mb stabilization with a conditional probability of 0.8. We observe a correlation between Mb folding stability and protein abundance, suggesting that a selection pressure for stability acts proportionally to higher expression. We also identify a major divergence event leading to the common ancestor of whales, during which major stabilization occurred. Most of the positively selected sites that occur later act against other destabilizing mutations to maintain stability across the clade, except for the shallow divers, where late stability relaxation occurs, probably due to the shorter aerobic dive limits of these species. The three main positively selected sites 66, 5, and 35 undergo changes that favor hydrophobic folding, structural integrity, and intra-helical hydrogen bonds.

  5. Evidence for a Role of Arabidopsis CDT1 Proteins in Gametophyte Development and Maintenance of Genome Integrity[C][W

    PubMed Central

    Domenichini, Séverine; Benhamed, Moussa; De Jaeger, Geert; Van De Slijke, Eveline; Blanchet, Sophie; Bourge, Mickaël; De Veylder, Lieven; Bergounioux, Catherine; Raynaud, Cécile

    2012-01-01

    Meristems retain the ability to divide throughout the life cycle of plants, which can last for over 1000 years in some species. Furthermore, the germline is not laid down early during embryogenesis but originates from the meristematic cells relatively late during development. Thus, accurate cell cycle regulation is of utmost importance to avoid the accumulation of mutations during vegetative growth and reproduction. The Arabidopsis thaliana genome encodes two homologs of the replication licensing factor CDC10 Target1 (CDT1), and overexpression of CDT1a stimulates DNA replication. Here, we have investigated the respective functions of Arabidopsis CDT1a and CDT1b. We show that CDT1 proteins have partially redundant functions during gametophyte development and are required for the maintenance of genome integrity. Furthermore, CDT1-RNAi plants show endogenous DNA stress, are more tolerant than the wild type to DNA-damaging agents, and show constitutive induction of genes involved in DNA repair. This DNA stress response may be a direct consequence of reduced CDT1 accumulation on DNA repair or may relate to the ability of CDT1 proteins to form complexes with DNA polymerase ε, which functions in DNA replication and in DNA stress checkpoint activation. Taken together, our results provide evidence for a crucial role of Arabidopsis CDT1 proteins in genome stability. PMID:22773747

  6. Evidence for a role of Arabidopsis CDT1 proteins in gametophyte development and maintenance of genome integrity.

    PubMed

    Domenichini, Séverine; Benhamed, Moussa; De Jaeger, Geert; Van De Slijke, Eveline; Blanchet, Sophie; Bourge, Mickaël; De Veylder, Lieven; Bergounioux, Catherine; Raynaud, Cécile

    2012-07-01

    Meristems retain the ability to divide throughout the life cycle of plants, which can last for over 1000 years in some species. Furthermore, the germline is not laid down early during embryogenesis but originates from the meristematic cells relatively late during development. Thus, accurate cell cycle regulation is of utmost importance to avoid the accumulation of mutations during vegetative growth and reproduction. The Arabidopsis thaliana genome encodes two homologs of the replication licensing factor CDC10 Target1 (CDT1), and overexpression of CDT1a stimulates DNA replication. Here, we have investigated the respective functions of Arabidopsis CDT1a and CDT1b. We show that CDT1 proteins have partially redundant functions during gametophyte development and are required for the maintenance of genome integrity. Furthermore, CDT1-RNAi plants show endogenous DNA stress, are more tolerant than the wild type to DNA-damaging agents, and show constitutive induction of genes involved in DNA repair. This DNA stress response may be a direct consequence of reduced CDT1 accumulation on DNA repair or may relate to the ability of CDT1 proteins to form complexes with DNA polymerase ε, which functions in DNA replication and in DNA stress checkpoint activation. Taken together, our results provide evidence for a crucial role of Arabidopsis CDT1 proteins in genome stability.

  7. Development of dextran nanoparticles for stabilizing delicate proteins

    NASA Astrophysics Data System (ADS)

    Wu, Fei; Zhou, Zhihua; Su, Jing; Wei, Liangming; Yuan, Weien; Jin, Tuo

    2013-04-01

    One of the most challenging problems in the development of protein pharmaceuticals is to deal with stabilities of proteins due to its complicated structures. This study aims to develop a novel approach to stabilize and encapsulate proteins into dextran nanoparticles without contacting the interface between the aqueous phase and the organic phase. The bovine serum albumin, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), β-galactosidase, and myoglobin were selected as model proteins. The proteins were added into an aqueous solution containing the dextran and polyethylene glycol, and then encapsulated into dextran nanoparticles by aqueous-aqueous freezing-induced phase separation. The encapsulation efficiency and recovery of dextran nanoparticles were determined. The dextran nanoparticles loaded with proteins were characterized by scanning electron microscopy and particle size analysis. The protein aggregation was determined by size-exclusion chromatography-high-performance chromatography, and the bioactivity of proteins recovered during formulation steps was determined. The bioactivity of GM-CSF, G-CSF, and β-galactosidase were examined by the proliferation of TF-1 cell, NSF-60 cell, and ortho-nitrophenyl- β-galactoside assay, respectively. The results of bioactivity recovered show that this novel dextran nanoparticle can preserve the protein's bioactivity during the preparation process. LysoSensor™ Yellow/Blue dextran, a pH-sensitive indicator with fluorescence excited at two channels, was encapsulated into dextran nanoparticles to investigate the ability of dextran nanoparticles to resist the acidic microenvironment (pH < 2.5). The result shows that the dextran nanoparticles attenuate the acidic microenvironment in the poly (lactic-co-glycolic acid) microsphere by means of the dilution effect. These novel dextran nanoparticles provided an appealing approach to stabilize the delicate proteins for

  8. Rheological properties of highly concentrated protein-stabilized emulsions.

    PubMed

    Dimitrova, Tatiana D; Leal-Calderon, Fernando

    2004-05-20

    We prepared concentrated quasi monodisperse hexadecane-in-water emulsions stabilized by various proteins and investigated their rheological properties. Some protein-stabilized emulsions possess remarkably high elasticity and at the same time they are considerably fragile--they exhibit coalescence at yield strain and practically do not flow. The elastic storage modulus G' and the loss modulus G" of the emulsions were determined for different oil volume fractions above the random close packing. Surprisingly, the dimensionless elastic moduli G'/(sigma/a), sigma being the interfacial tension, and a being the mean drop radius, obtained for emulsions stabilized by different proteins do not collapse on a single master curve. They are almost always substantially higher than the corresponding values obtained for equivalent Sodium Dodecyl Sulfate (SDS)-stabilized emulsions. The unusually high elasticity cannot be attributed to a specificity of the continuous phase, because the osmotic equation of state of our emulsions is found identical to the one obtained for samples stabilized by classical surfactants. In parallel, we mimicked the thin films that separate the droplets in the concentrated emulsion and found that the protein adsorption layers contain a substantial number of sticky surface aggregates. These severely obstruct local rearrangements of individual drops in respect to their neighbors which leads to coalescence at yield strain. Furthermore, we found that G'/(sigma/a) is correlated (for a given oil volume fraction) to the dilatational elastic modulus, of the protein layer adsorbed on the droplets. The intrinsic elasticity of the protein layers, together with the blocked local rearrangements are considered as the main factors determining the unusual bulk elasticity of the studied emulsions.

  9. Role of cholecystokinin in induction and maintenance of dietary protein-stimulated pancreatic growth.

    PubMed

    Green, G M; Jurkowska, G; Berube, F L; Rivard, N; Guan, D; Morisset, J

    1992-04-01

    The role of cholecystokinin (CCK) in induction and maintenance of pancreatic growth stimulated by a high-protein diet was investigated. Rats adapted to 5% casein diet were switched to 70% casein for 21 days. MK-329, a CCK receptor antagonist, was administered at 2.5 mg.kg-1.day-1 ip, beginning on day zero (day zero treatment) or day 7 (midcourse treatment) of feeding 70% casein and thereafter. Another group was returned to 5% casein after 7 days of feeding 70% casein. Feeding 70% casein significantly stimulated increases of 32, 87, 74, 216, and 1,450% in pancreatic DNA, RNA, wet weight, protein content, and chymotrypsin content, respectively. Midcourse treatment with MK-329 was more effective than day zero treatment, and it completely reversed increases in pancreatic weight and RNA content, partially reversed increases in protein and chymotrypsin content, and had no effect on DNA content. Return to 5% casein rapidly reversed increases in pancreatic parameters, except for DNA. The results indicate that CCK is essential for induction and maintenance of dietary protein-stimulated pancreatic hypertrophy.

  10. Polyethylene glycol on stability of chitosan microparticulate carrier for protein.

    PubMed

    Luangtana-Anan, Manee; Limmatvapirat, Sontaya; Nunthanid, Jurairat; Chalongsuk, Rapeepun; Yamamoto, Keiji

    2010-09-01

    Stability enhancement of protein-loaded chitosan microparticles under storage was investigated. Chitosan glutamate at 35 kDa and bovine serum albumin as model protein drug were used in this study. The chitosan microparticles were prepared by ionotropic gelation, and polyethylene glycol 200 (PEG 200) was applied after the formation of the particles. All chitosan microparticles were kept at 25°C for 28 days. A comparison was made between those preparations with PEG 200 and without PEG 200. The changes in the physicochemical properties of the microparticles such as size, zeta potential, pH, and percent loading capacity were investigated after 0, 3, 7, 14, and 28 days of storage. It was found that the stability decreased upon storage and the aggregation of microparticles could be observed for both preparations. The reduction in the zeta potential and the increase in the pH, size, and loading capacity were observed when they were kept at a longer period. The significant change of those preparations without PEG 200 was evident after 7 days of storage whereas those with PEG 200 underwent smaller changes with enhanced stability after 28 days of storage. Therefore, this investigation gave valuable information on the stability enhancement of the microparticles. Hence, enhanced stability of chitosan glutamate microparticles for the delivery of protein could be achieved by the application of PEG 200.

  11. Sde2: A novel nuclear protein essential for telomeric silencing and genomic stability in Schizosaccharomyces pombe

    SciTech Connect

    Sugioka-Sugiyama, Rie; Sugiyama, Tomoyasu

    2011-03-18

    Research highlights: {yields} Sde2 is essential for telomere silencing. {yields} Sde2 is involved in the maintenance of genomic stability. {yields} Sde2 promotes the recruitment of SHREC, a histone deacetylase complex, to telomeres. -- Abstract: Telomeres, specialized domains assembled at the ends of linear chromosomes, are essential for genomic stability in eukaryotes. The formation and maintenance of telomeres are governed by numerous factors such as telomeric repeats, telomere-binding proteins, heterochromatin proteins, and telomerase. Here, we report Sde2, a novel nuclear protein essential for telomeric silencing and genomic stability in the fission yeast Schizosaccharomyces pombe. A deficiency in sde2 results in the derepression of the ura4{sup +} gene inserted near telomeric repeats, and the noncoding transcripts from telomeric regions accumulate in sde2{Delta} cells. The loss of Sde2 function compromises transcriptional silencing at telomeres, and this silencing defect is accompanied by increased levels of acetylated histone H3K14 and RNA polymerase II occupancy at telomeres as well as reduced recruitment of the SNF2 ATPase/histone deacetylase-containing complex SHREC to telomeres. Deletion of sde2 also leads to a higher frequency of mitotic minichromosome loss, and sde2{Delta} cells often form asci that contain spores in abnormal numbers, shapes, or both. In addition, sde2{Delta} cells are highly sensitive to several stresses, including high/low temperatures, bleomycin, which induces DNA damage, and thiabendazole, a microtubule-destabilizing agent. Furthermore, Sde2 genetically interacts with the telomere regulators Taz1, Pof3, and Ccq1. These findings demonstrate that Sde2 cooperates with other telomere regulators to maintain functional telomeres, thereby preventing genomic instability.

  12. Separation of stem cell maintenance and transposon silencing functions of Piwi protein

    PubMed Central

    Klenov, Mikhail S.; Sokolova, Olesya A.; Yakushev, Evgeny Y.; Stolyarenko, Anastasia D.; Mikhaleva, Elena A.; Lavrov, Sergey A.; Gvozdev, Vladimir A.

    2011-01-01

    Piwi-interacting RNAs (piRNAs) and Piwi proteins have the evolutionarily conserved function of silencing of repetitive genetic elements in germ lines. The founder of the Piwi subfamily, Drosophila nuclear Piwi protein, was also shown to be required for the maintenance of germ-line stem cells (GSCs). Hence, null mutant piwi females exhibit two types of abnormalities, overexpression of transposons and severely underdeveloped ovaries. It remained unknown whether the failure of GSC maintenance is related to transposon derepression or if GSC self-renewal and piRNA silencing are two distinct functions of the Piwi protein. We have revealed a mutation, piwiNt, removing the nuclear localization signal of the Piwi protein. piwiNt females retain the ability of GSC self-renewal and a near-normal number of egg chambers in the ovarioles but display a drastic transposable element derepression and nuclear accumulation of their transcripts in the germ line. piwiNt mutants are sterile most likely because of the disturbance of piRNA-mediated transposon silencing. Analysis of chromatin modifications in the piwiNt ovaries indicated that Piwi causes chromatin silencing only of certain types of transposons, whereas others are repressed in the nuclei without their chromatin modification. Thus, Piwi nuclear localization that is required for its silencing function is not essential for the maintenance of GSCs. We suggest that the Piwi function in GSC self-renewal is independent of transposon repression and is normally realized in the cytoplasm of GSC niche cells. PMID:22065765

  13. Yeast hnRNP-related proteins contribute to the maintenance of telomeres

    SciTech Connect

    Lee-Soety, Julia Y.; Jones, Jennifer; MacGibeny, Margaret A.; Remaly, Erin C.; Daniels, Lynsey; Ito, Andrea; Jean, Jessica; Radecki, Hannah; Spencer, Shannon

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Yeast hnRNP-related proteins are able to prevent faster senescence in telomerase-null cells. Black-Right-Pointing-Pointer The conserved RRMs in Npl3 are important for telomere maintenance. Black-Right-Pointing-Pointer Human hnRNP A1 is unable to complement the lack of NPL3 in yeast. Black-Right-Pointing-Pointer Npl3 and Cbc2 may work as telomere capping proteins. -- Abstract: Telomeres protect the ends of linear chromosomes, which if eroded to a critical length can become uncapped and lead to replicative senescence. Telomerase maintains telomere length in some cells, but inappropriate expression facilitates the immortality of cancer cells. Recently, proteins involved in RNA processing and ribosome assembly, such as hnRNP (heterogeneous nuclear ribonucleoprotein) A1, have been found to participate in telomere maintenance in mammals. The Saccharomyces cerevisiae protein Npl3 shares significant amino acid sequence similarities with hnRNP A1. We found that deleting NPL3 accelerated the senescence of telomerase null cells. The highly conserved RNA recognition motifs (RRM) in Npl3 appear to be important for preventing faster senescence. Npl3 preferentially binds telomere sequences in vitro, suggesting that Npl3 may affect telomeres directly. Despite similarities between the two proteins, human hnRNP A1 is unable to complement the lack of Npl3 to rescue accelerated senescence in tlc1 npl3 cells. Deletion of CBC2, which encodes another hnRNP-related protein that associates with Npl3, also accelerates senescence. Potential mechanisms by which hnRNP-related proteins maintain telomeres are discussed.

  14. Communication: Using multiple tethers to stabilize proteins on surfaces

    NASA Astrophysics Data System (ADS)

    Loong, Brandon K.; Knotts, Thomas A.

    2014-08-01

    Protein surface interactions are important in many applications in biotechnology including protein arrays, but these technologies have not lived up to their transformative potential because it is difficult to attach proteins to surfaces in a manner that preserves function and theoretical understanding of the relevant phenomena remains limited. Here is reported the effect of using multiple tethers to attach a protein (lysozyme) to a surface and the effects on the structure and stability of the molecule. The simulations show how using two tethers can drastically change the folding mechanism such that a protein that is initially unstable and inactive when attached using a single tether can become more stable and functional when two tethers are used. The results offer hope that the rational design of protein arrays is possible.

  15. Communication: Using multiple tethers to stabilize proteins on surfaces.

    PubMed

    Loong, Brandon K; Knotts, Thomas A

    2014-08-07

    Protein surface interactions are important in many applications in biotechnology including protein arrays, but these technologies have not lived up to their transformative potential because it is difficult to attach proteins to surfaces in a manner that preserves function and theoretical understanding of the relevant phenomena remains limited. Here is reported the effect of using multiple tethers to attach a protein (lysozyme) to a surface and the effects on the structure and stability of the molecule. The simulations show how using two tethers can drastically change the folding mechanism such that a protein that is initially unstable and inactive when attached using a single tether can become more stable and functional when two tethers are used. The results offer hope that the rational design of protein arrays is possible.

  16. Sample preservation through heat stabilization of proteins: principles and examples.

    PubMed

    Borén, Mats

    2015-01-01

    Due to post-sampling changes, caused by residual enzyme activity in the sample, levels of analytes can change from their in vivo levels so that they no longer accurately reflect conditions in the living system. The Stabilizor(™) system accomplishes elimination of enzyme activity through heat-induced denaturation of enzymes by permanently altering the 3D protein structure of the enzymes. Heat stabilization can be introduced in the workflow either directly after sampling, with the instrument just next to where the sample is taken, or prior to sample homogenization and extraction, when samples are heat denatured directly from a frozen state. Initially, heat stabilization was developed to enable mass spectrometric analysis of neuropeptides. Heat stabilization has since been further developed and applied to a range of samples and downstream protein analysis techniques such as western blot, 2D gels and phosphorylation analysis with LC-MS.

  17. Some implications of colloid stability theory for protein crystallization

    NASA Technical Reports Server (NTRS)

    Young, C. C.; De Mattei, R. C.; Feigelson, R. S.; Tiller, W. A.

    1988-01-01

    Colloid stability theory has been applied to protein crystallization and predicts a narrow range of conditions under which crystals can be grown without the agglomeration of protein molecules (colloids) in the bulk solution. It also predicts a critical electrolyte concentration above which agglomeration will always occur. Using this theory, the rapid protein agglomeration occurring during Schlieren experiments as well as a terminal crystal size effect in a fixed container were explained. Following this concept, the supposed 'terminal' crystal size has been at least doubled.

  18. Some implications of colloid stability theory for protein crystallization

    NASA Technical Reports Server (NTRS)

    Young, C. C.; De Mattei, R. C.; Feigelson, R. S.; Tiller, W. A.

    1988-01-01

    Colloid stability theory has been applied to protein crystallization and predicts a narrow range of conditions under which crystals can be grown without the agglomeration of protein molecules (colloids) in the bulk solution. It also predicts a critical electrolyte concentration above which agglomeration will always occur. Using this theory, the rapid protein agglomeration occurring during Schlieren experiments as well as a terminal crystal size effect in a fixed container were explained. Following this concept, the supposed 'terminal' crystal size has been at least doubled.

  19. Large scale analysis of protein stability in OMIM disease related human protein variants.

    PubMed

    Martelli, Pier Luigi; Fariselli, Piero; Savojardo, Castrense; Babbi, Giulia; Aggazio, Francesco; Casadio, Rita

    2016-06-23

    Modern genomic techniques allow to associate several Mendelian human diseases to single residue variations in different proteins. Molecular mechanisms explaining the relationship among genotype and phenotype are still under debate. Change of protein stability upon variation appears to assume a particular relevance in annotating whether a single residue substitution can or cannot be associated to a given disease. Thermodynamic properties of human proteins and of their disease related variants are lacking. In the present work, we take advantage of the available three dimensional structure of human proteins for predicting the role of disease related variations on the perturbation of protein stability. We develop INPS3D, a new predictor based on protein structure for computing the effect of single residue variations on protein stability (ΔΔG), scoring at the state-of-the-art (Pearson's correlation value of the regression is equal to 0.72 with mean standard error of 1.15 kcal/mol on a blind test set comprising 351 variations in 60 proteins). We then filter 368 OMIM disease related proteins known with atomic resolution (where the three dimensional structure covers at least 70 % of the sequence) with 4717 disease related single residue variations and 685 polymorphisms without clinical consequence. We find that the effect on protein stability of disease related variations is larger than the effect of polymorphisms: in particular, by setting to |1 kcal/mol| the threshold between perturbing and not perturbing variations of the protein stability, about 44 % of disease related variations and 20 % of polymorphisms are predicted with |ΔΔG| > 1 kcal/mol, respectively. A consistent fraction of OMIM disease related variations is however predicted to promote |ΔΔG| ≤ 1 kcal/mol and we focus here on detecting features that can be associated to the thermodynamic property of the protein variant. Our analysis reveals that some 47 % of disease related variations

  20. Designed protein reveals structural determinants of extreme kinetic stability

    PubMed Central

    Broom, Aron; Ma, S. Martha; Xia, Ke; Rafalia, Hitesh; Trainor, Kyle; Colón, Wilfredo; Gosavi, Shachi; Meiering, Elizabeth M.

    2015-01-01

    The design of stable, functional proteins is difficult. Improved design requires a deeper knowledge of the molecular basis for design outcomes and properties. We previously used a bioinformatics and energy function method to design a symmetric superfold protein composed of repeating structural elements with multivalent carbohydrate-binding function, called ThreeFoil. This and similar methods have produced a notably high yield of stable proteins. Using a battery of experimental and computational analyses we show that despite its small size and lack of disulfide bonds, ThreeFoil has remarkably high kinetic stability and its folding is specifically chaperoned by carbohydrate binding. It is also extremely stable against thermal and chemical denaturation and proteolytic degradation. We demonstrate that the kinetic stability can be predicted and modeled using absolute contact order (ACO) and long-range order (LRO), as well as coarse-grained simulations; the stability arises from a topology that includes many long-range contacts which create a large and highly cooperative energy barrier for unfolding and folding. Extensive data from proteomic screens and other experiments reveal that a high ACO/LRO is a general feature of proteins with strong resistances to denaturation and degradation. These results provide tractable approaches for predicting resistance and designing proteins with sufficient topological complexity and long-range interactions to accommodate destabilizing functional features as well as withstand chemical and proteolytic challenge. PMID:26554002

  1. Capture-stabilize approach for membrane protein SPR assays.

    PubMed

    Chu, Ruiyin; Reczek, David; Brondyk, William

    2014-12-08

    Measuring the binding kinetics of antibodies to intact membrane proteins by surface plasmon resonance has been challenging largely because of the inherent difficulties in capturing membrane proteins on chip surfaces while retaining their native conformation. Here we describe a method in which His-tagged CXCR5, a GPCR, was purified and captured on a Biacore chip surface via the affinity tag. The captured receptor protein was then stabilized on the chip surface by limited cross-linking. The resulting chip surface retained ligand binding activity and was used for monoclonal antibody kinetics assays by a standard Biacore kinetics assay method with a simple low pH regeneration step. We demonstrate the advantages of this whole receptor assay when compared to available peptide-based binding assays. We further extended the application of the capture-stabilize approach to virus-like particles and demonstrated its utility analyzing antibodies against CD52, a GPI-anchored protein, in its native membrane environment. The results are the first demonstration of chemically stabilized chip surfaces for membrane protein SPR assays.

  2. Designed protein reveals structural determinants of extreme kinetic stability.

    PubMed

    Broom, Aron; Ma, S Martha; Xia, Ke; Rafalia, Hitesh; Trainor, Kyle; Colón, Wilfredo; Gosavi, Shachi; Meiering, Elizabeth M

    2015-11-24

    The design of stable, functional proteins is difficult. Improved design requires a deeper knowledge of the molecular basis for design outcomes and properties. We previously used a bioinformatics and energy function method to design a symmetric superfold protein composed of repeating structural elements with multivalent carbohydrate-binding function, called ThreeFoil. This and similar methods have produced a notably high yield of stable proteins. Using a battery of experimental and computational analyses we show that despite its small size and lack of disulfide bonds, ThreeFoil has remarkably high kinetic stability and its folding is specifically chaperoned by carbohydrate binding. It is also extremely stable against thermal and chemical denaturation and proteolytic degradation. We demonstrate that the kinetic stability can be predicted and modeled using absolute contact order (ACO) and long-range order (LRO), as well as coarse-grained simulations; the stability arises from a topology that includes many long-range contacts which create a large and highly cooperative energy barrier for unfolding and folding. Extensive data from proteomic screens and other experiments reveal that a high ACO/LRO is a general feature of proteins with strong resistances to denaturation and degradation. These results provide tractable approaches for predicting resistance and designing proteins with sufficient topological complexity and long-range interactions to accommodate destabilizing functional features as well as withstand chemical and proteolytic challenge.

  3. Designing an extracellular matrix protein with enhanced mechanical stability

    PubMed Central

    Ng, Sean P.; Billings, Kate S.; Ohashi, Tomoo; Allen, Mark D.; Best, Robert B.; Randles, Lucy G.; Erickson, Harold P.; Clarke, Jane

    2007-01-01

    The extracellular matrix proteins tenascin and fibronectin experience significant mechanical forces in vivo. Both contain a number of tandem repeating homologous fibronectin type III (fnIII) domains, and atomic force microscopy experiments have demonstrated that the mechanical strength of these domains can vary significantly. Previous work has shown that mutations in the core of an fnIII domain from human tenascin (TNfn3) reduce the unfolding force of that domain significantly: The composition of the core is apparently crucial to the mechanical stability of these proteins. Based on these results, we have used rational redesign to increase the mechanical stability of the 10th fnIII domain of human fibronectin, FNfn10, which is directly involved in integrin binding. The hydrophobic core of FNfn10 was replaced with that of the homologous, mechanically stronger TNfn3 domain. Despite the extensive substitution, FNoTNc retains both the three-dimensional structure and the cell adhesion activity of FNfn10. Atomic force microscopy experiments reveal that the unfolding forces of the engineered protein FNoTNc increase by ≈20% to match those of TNfn3. Thus, we have specifically designed a protein with increased mechanical stability. Our results demonstrate that core engineering can be used to change the mechanical strength of proteins while retaining functional surface interactions. PMID:17535921

  4. Acute myeloid leukemia fusion proteins deregulate genes involved in stem cell maintenance and DNA repair

    PubMed Central

    Alcalay, Myriam; Meani, Natalia; Gelmetti, Vania; Fantozzi, Anna; Fagioli, Marta; Orleth, Annette; Riganelli, Daniela; Sebastiani, Carla; Cappelli, Enrico; Casciari, Cristina; Sciurpi, Maria Teresa; Mariano, Angela Rosa; Minardi, Simone Paolo; Luzi, Lucilla; Muller, Heiko; Di Fiore, Pier Paolo; Frosina, Guido; Pelicci, Pier Giuseppe

    2003-01-01

    Acute myelogenous leukemias (AMLs) are genetically heterogeneous and characterized by chromosomal rearrangements that produce fusion proteins with aberrant transcriptional regulatory activities. Expression of AML fusion proteins in transgenic mice increases the risk of myeloid leukemias, suggesting that they induce a preleukemic state. The underlying molecular and biological mechanisms are, however, unknown. To address this issue, we performed a systematic analysis of fusion protein transcriptional targets. We expressed AML1/ETO, PML/RAR, and PLZF/RAR in U937 hemopoietic precursor cells and measured global gene expression using oligonucleotide chips. We identified 1,555 genes regulated concordantly by at least two fusion proteins that were further validated in patient samples and finally classified according to available functional information. Strikingly, we found that AML fusion proteins induce genes involved in the maintenance of the stem cell phenotype and repress DNA repair genes, mainly of the base excision repair pathway. Functional studies confirmed that ectopic expression of fusion proteins constitutively activates pathways leading to increased stem cell renewal (e.g., the Jagged1/Notch pathway) and provokes accumulation of DNA damage. We propose that expansion of the stem cell compartment and induction of a mutator phenotype are relevant features underlying the leukemic potential of AML-associated fusion proteins. PMID:14660751

  5. Genome-health nutrigenomics and nutrigenetics: nutritional requirements or 'nutriomes' for chromosomal stability and telomere maintenance at the individual level.

    PubMed

    Bull, Caroline; Fenech, Michael

    2008-05-01

    It is becoming increasingly evident that (a) risk for developmental and degenerative disease increases with more DNA damage, which in turn is dependent on nutritional status, and (b) the optimal concentration of micronutrients for prevention of genome damage is also dependent on genetic polymorphisms that alter the function of genes involved directly or indirectly in the uptake and metabolism of micronutrients required for DNA repair and DNA replication. The development of dietary patterns, functional foods and supplements that are designed to improve genome-health maintenance in individuals with specific genetic backgrounds may provide an important contribution to an optimum health strategy based on the diagnosis and individualised nutritional prevention of genome damage, i.e. genome health clinics. The present review summarises some of the recent knowledge relating to micronutrients that are associated with chromosomal stability and provides some initial insights into the likely nutritional factors that may be expected to have an impact on the maintenance of telomeres. It is evident that developing effective strategies for defining nutrient doses and combinations or 'nutriomes' for genome-health maintenance at the individual level is essential for further progress in this research field.

  6. A simple protein-energy wasting score predicts survival in maintenance hemodialysis patients.

    PubMed

    Moreau-Gaudry, Xavier; Jean, Guillaume; Genet, Leslie; Lataillade, Dominique; Legrand, Eric; Kuentz, François; Fouque, Denis

    2014-11-01

    Nutritional status is a powerful predictor of survival in maintenance hemodialysis patients but remains challenging to assess. We defined a new Protein Energy Wasting (PEW) score based on the nomenclature proposed by the International Society of Renal Nutrition and Metabolism in 2008. This score, graded from 0 (worse) to 4 (best) was derived from 4 body nutrition compartments: serum albumin, body mass index, a normalized serum creatinine value, and protein intake as assessed by nPNA. We applied this score to 1443 patients from the ARNOS prospective dialysis cohort and provide survival data from 2005 until 2008. Patients survival at 3.5 year. Survival ranged from 84%-69% according to the protein-energy wasting score. There was a clear-cut reduction in survival (5%-7%; P < 0.01) for each unit decrement in the score grade. There was a 99% survival at 1 year for patients with the score of 4. In addition, the 6-month variation of this PEW score also strongly predicted patients' survival (P < 0.01). A new simple and easy-to-get PEW score predicts survival in maintenance hemodialysis patients. Furthermore, increase of this nutritional score over time also indicates survival improvement, and may help to better identify subgroups of patients with a high mortality rate, in which nutrition support should be enforced. Copyright © 2014 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

  7. Evaluation of protein stability and in vitro permeation of lyophilized polysaccharides-based microparticles for intranasal protein delivery.

    PubMed

    Cho, Hyun-Jong; Balakrishnan, Prabagar; Chung, Suk-Jae; Shim, Chang-Koo; Kim, Dae-Duk

    2011-09-15

    Biocompatible microparticles prepared by lyophilization were developed for intranasal protein delivery. To test for the feasibility of this formulation, stability of the incorporated protein and enhancement of in vitro permeation across the nasal epithelium were evaluated. Lyophilization was processed with hydroxypropylmethylcellulose (HPMC) or water soluble chitosan (WCS) as biocompatible polymers, hydroxypropyl-β-cyclodextrin (HP-β-CD) and d-alpha-tocopheryl poly(ethylene glycol 1000) succinate (TPGS 1000) as permeation enhancers, sugars as cryoprotectants and lysozyme as the model protein. As a result, microparticles ranging from 6 to 12μm were developed where the maintenance of the protein conformation was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism and fluorescence intensity detection. Moreover, in vitro bioassay showed that the lysozyme activity was preserved during the preparation process while exhibiting less cytotoxicity in primary human nasal epithelial (HNE) cells. Results of the in vitro release study revealed slower release rate in these microparticles compared to that of the lysozyme itself. On the other hand, the in vitro permeation study exhibited a 9-fold increase in absorption of lysozyme when prepared in lyophilized microparticles with HPMC, HP-β-CD and TPGS 1000 (F4-2). These microparticles could serve as efficient intranasal delivery systems for therapeutic proteins. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Protein stabilization by introduction of cross-strand disulfides.

    PubMed

    Chakraborty, Kausik; Thakurela, Sudhir; Prajapati, Ravindra Singh; Indu, S; Ali, P Shaik Syed; Ramakrishnan, C; Varadarajan, Raghavan

    2005-11-08

    Disulfides cross-link residues in a protein that are separated in primary sequence and stabilize the protein through entropic destabilization of the unfolded state. While the removal of naturally occurring disulfides leads to protein destabilization, introduction of engineered disulfides does not always lead to significant stabilization of a protein. We have analyzed naturally occurring disulfides that span adjacent antiparallel strands of beta sheets (cross-strand disulfides). Cross-strand disulfides have recently been implicated as redox-based conformational switches in proteins such as gp120 and CD4. The propensity of these disulfides to act as conformational switches was postulated on the basis of the hypothesis that this class of disulfide is conformationally strained. In the present analysis, there was no evidence to suggest that cross-strand disulfides are more strained compared to other disulfides as assessed by their torsional energy. It was also observed that these disulfides occur solely at non-hydrogen-bonded (NHB) registered pairs of adjacent antiparallel strands and not at hydrogen-bonded (HB) positions as suggested previously. One of the half-cystines involved in cross-strand disulfide formation often occurs at an edge strand. Experimental confirmation of the stabilizing effects of such disulfides was carried out in Escherichia coli thioredoxin. Four pairs of cross-strand cysteines were introduced, two at HB and two at NHB pairs. Disulfides were formed in all four cases. However, as predicted from our analysis, disulfides at NHB positions resulted in an increase in melting temperature of 7-10 degrees C, while at HB positions there was a corresponding decrease of -7 degrees C. The reduced state of all proteins had similar stability.

  9. Detergent Stabilized Nanopore Formation Kinetics of an Anthrax Protein

    NASA Astrophysics Data System (ADS)

    Peterson, Kelby

    2015-03-01

    This summer research project funded through the Society of Physics Students Internship Program and The National Institute of Standards and Technology focused on optimization of pore formation of Protective Antigen protein secreted by Bacillus Anthraces. This experiment analyzes the use of N-tetradecylphosphocholine (FOS-14 Detergent) to stabilize the water soluble protein, protective antigen protein (PA63) to regulate the kinetics of pore formation in a model bilayer lipid membrane. The FOS-14 Detergent was tested under various conditions to understand its impact on the protein pore formation. The optimization of this channel insertion is critical in preparing samples of oriented for neutron reflectometry that provide new data to increase the understanding of the protein's structure.

  10. RNA stabilizing proteins as molecular targets in cardiovascular pathologies

    PubMed Central

    Babu, Sahana Suresh; Joladarashi, Darukeshwara; Jeyabal, Prince; Thandavarayan, Rajarajan Amirthalingam; Krishnamurthy, Prasanna

    2015-01-01

    The stability of mRNA has emerged as a key step in the regulation of eukaryotic gene expression and function. RNA stabilizing proteins (RSPs) contain several RNA recognition motifs, and selectively bind to Adenylate- and uridylate- Rich Elements in the 3′ untranslated region of several mRNAs leading to altered processing, stability and translation. These post-transcriptional gene regulations play a critical role in cellular homeostasis; therefore act as molecular switch between ‘normal cell’ and ‘disease state’. Many mRNA binding proteins have been discovered to date, which either stabilize (HuR/HuA, HuB, HuC, HuD) or destabilize (AUF1, Tristetraprolin, KSRP) the target transcripts. Although the function of RSPs has been widely studied in cancer biology, its role in cardiovascular pathologies is only beginning to evolve. The current review provides an overall understanding of the potential role of RSP, specifically HuR-mediated mRNA stability in myocardial infarction, hypertension and hypertrophy. Also, the effect of RSPs on various cellular processes including inflammation, fibrosis, angiogenesis, cell-death and proliferation and its relevance to cardiovascular pathophysiological processes is presented. We also discuss the potential clinical implications of RSPs as therapeutic targets in cardiovascular diseases. PMID:25801788

  11. Protein stabilization by macromolecular crowding through enthalpy rather than entropy.

    PubMed

    Senske, Michael; Törk, Lisa; Born, Benjamin; Havenith, Martina; Herrmann, Christian; Ebbinghaus, Simon

    2014-06-25

    The interior of the cell is a densely crowded environment in which protein stability is affected differently than in dilute solution. Macromolecular crowding is commonly understood in terms of an entropic volume exclusion effect based on hardcore repulsions among the macromolecules. We studied the thermal unfolding of ubiquitin in the presence of different cosolutes (glucose, dextran, poly(ethylene glycol), KCl, urea). Our results show that for a correct dissection of the cosolute-induced changes of the free energy into its enthalpic and entropic contributions, the temperature dependence of the heat capacity change needs to be explicitly taken into account. In contrast to the prediction by the excluded volume theory, we observed an enthalpic stabilization and an entropic destabilization for glucose, dextran, and poly(ethylene glycol). The enthalpic stabilization mechanism induced by the macromolecular crowder dextran was similar to the enthalpic stabilization mechanism of its monomeric building block glucose. In the case of poly(ethylene glycol), entropy is dominating over enthalpy leading to an overall destabilization. We propose a new model to classify cosolute effects in terms of their enthalpic contributions to protein stability.

  12. Sgs1, a Homologue of the Bloom's and Werner's Syndrome Genes, Is Required for Maintenance of Genome Stability in Saccharomyces Cerevisiae

    PubMed Central

    Watt, P. M.; Hickson, I. D.; Borts, R. H.; Louis, E. J.

    1996-01-01

    The Saccharomyces cerevisiae SGS1 gene is homologous to Escherichia coli RecQ and the human BLM and WRN proteins that are defective in the cancer-prone disorder Bloom's syndrome and the premature aging disorder Werner's syndrome, respectively. While recQ mutants are deficient in conjugational recombination and DNA repair, Bloom's syndrome cell lines show hyperrecombination. Bloom's and Werner's syndrome cell lines both exhibit chromosomal instability. sgs1Δ strains show mitotic hyperrecombination, as do Bloom's cells. This was manifested as an increase in the frequency of interchromosomal homologous recombination, intrachromosomal excision recombination, and ectopic recombination. Hyperrecombination was partially independent of both RAD52 and RAD1. Meiotic recombination was not increased in sgs1Δ mutants, although meiosis I chromosome missegregation has been shown to be elevated. sgs1Δ suppresses the slow growth of a top3Δ strain lacking topoisomerase III. Although there was an increase in subtelomeric Y' instability in sgs1Δ strains due to hyperrecombination, no evidence was found for an increase in the instability of terminal telomeric sequences in a top3Δ or a sgs1Δ strain. This contrasts with the telomere maintenance defects of Werner's patients. We conclude that the SGS1 gene product is involved in the maintenance of genome stability in S. cerevisiae. PMID:8913739

  13. Potential role of minichromosome maintenance protein 2 as a screening biomarker in esophageal cancer high-risk population in China.

    PubMed

    Huang, Bo; Hu, Bin; Su, Min; Tian, Dongping; Guo, Yu; Lian, Shiyong; Liu, Zhicai; Wu, Xianying; Li, Qiaoshan; Zheng, Ruiming; Gao, Yuxia

    2011-06-01

    Minichromosome maintenance proteins are novel proliferative markers that have been proposed as diagnostic markers in many cancers. We evaluated the potential role of minichromosome maintenance protein 2 as a screening biomarker and compared it with proliferating cell nuclear antigen and Ki67 in a population survey of esophageal squamous cell carcinoma. A total of 299 esophageal samples from a high-risk region in China, including 171 from an endoscopy population survey, 30 from brushing cytology, and 98 from surgery and autopsy, underwent immunostaining with minichromosome maintenance protein 2, proliferating cell nuclear antigen, and Ki67 antibodies. Minichromosome maintenance protein 2 expression was confined to the proliferative compartment of normal and abnormal esophageal epithelium and particularly manifested in the surface layer of dysplasia and carcinoma in situ. The expression of proliferating cell nuclear antigen and Ki67 was positively correlated with that of minichromosome maintenance protein 2 (r(s) >0.39, P < .01); but their positive nuclei seldom reached the surface layer, and the labeling indices were significantly lower than those for minichromosome maintenance protein 2 in dysplasia (P < .05) and carcinoma in situ (P < .001). The sensitivity and specificity of minichromosome maintenance protein 2 in diagnosing dysplasia were 91.3% and 61.8%, respectively, higher than those for proliferating cell nuclear antigen (88.4% and 47.1%) and Ki67 (78.3% and 57.8%). Nine of 10 cancer and paracancerous surface-brushing samples expressed minichromosome maintenance protein 2, and the detection was higher than that for proliferating cell nuclear antigen (8/10 and 7/10) and Ki67 (7/10 and 7/10). However, none of 10 normal surface-brushing samples expressed the 3 markers. Minichromosome maintenance protein 2 is more sensitive and specific than proliferating cell nuclear antigen and Ki67 in indicating esophageal dysplasia. Minichromosome maintenance protein 2

  14. Expression, function, and targeting of the nuclear exporter chromosome region maintenance 1 (CRM1) protein.

    PubMed

    Ishizawa, Jo; Kojima, Kensuke; Hail, Numsen; Tabe, Yoko; Andreeff, Michael

    2015-09-01

    Nucleocytoplasmic trafficking of proteins/RNAs is essential to normal cellular function. Indeed, accumulating evidence suggests that cancer cells escape anti-neoplastic mechanisms and benefit from pro-survival signals via the dysregulation of this system. The nuclear exporter chromosome region maintenance 1 (CRM1) protein is the only protein in the karyopherin-β protein family that contributes to the trafficking of numerous proteins and RNAs from the nucleus. It is considered to be an oncogenic, anti-apoptotic protein in transformed cells, since it reportedly functions as a gatekeeper for cell survival, including affecting p53 function, and ribosomal biogenesis. Furthermore, abnormally high expression of CRM1 is correlated with poor patient prognosis in various malignancies. Therapeutic targeting of CRM1 has emerged as a novel cancer treatment strategy, starting with a clinical trial with leptomycin B, the original specific inhibitor of CRM1, followed by development of several next-generation small molecules. KPT-330, a novel member of the CRM1-selective inhibitors of nuclear export (SINE) class of compounds, is currently undergoing clinical evaluation for the therapy of various malignancies. Results from these trials suggest that SINE compounds may be particularly useful against hematological malignancies, which often become refractory to standard chemotherapeutic agents. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Expression, function, and targeting of the nuclear exporter chromosome region maintenance 1 (CRM1) protein

    PubMed Central

    Ishizawa, Jo; Kojima, Kensuke; Hail, Numsen; Tabe, Yoko; Andreeff, Michael

    2015-01-01

    Nucleocytoplasmic trafficking of proteins/RNAs is essential to normal cellular function. Indeed, accumulating evidence suggests that cancer cells escape anti-neoplastic mechanisms and benefit from pro-survival signals via the dysregulation of this system. The nuclear exporter chromosome region maintenance 1 (CRM1) protein is the only protein in the karyopherin-β protein family that contributes to the trafficking of numerous proteins and RNAs from the nucleus. It is considered to be an oncogenic, anti-apoptotic protein in transformed cells, since it reportedly functions as a gatekeeper for cell survival, including affecting p53 function, and ribosomal biogenesis. Furthermore, abnormally high expression of CRM1 is correlated with poor patient prognosis in various malignancies. Therapeutic targeting of CRM1 has emerged as a novel cancer treatment strategy, starting with a clinical trial with leptomycin B, the original specific inhibitor of CRM1, followed by development of several next-generation small molecules. KPT-330, a novel member of the CRM1-selective inhibitors of nuclear export (SINE) class of compounds, is currently undergoing clinical evaluation for the therapy of various malignancies. Results from these trials suggest that SINE compounds may be particularly useful against hematological malignancies, which often become refractory to standard chemotherapeutic agents. PMID:26048327

  16. Beer consumption and changes in stability of human serum proteins.

    PubMed

    Gorinstein, S; Caspi, A; Goshev, I; Moncheva, S; Zemser, M; Weisz, M; Libman, I; Lerner, H T; Trakhtenberg, S; Martín-Belloso, O

    2001-03-01

    The aim of this study was to evaluate the influence of beer consumption (BC) on the functional and structural properties of human serum proteins (HSP). Thirty-eight volunteers (after coronary bypass) were divided into two groups: experimental (EG) and control (CG). Nineteen volunteers of the EG consumed 330 mL per day of beer (about 20 g of alcohol) for 30 consecutive days. The CG volunteers consumed mineral water instead of beer. Blood samples were collected from EG and CG patients before and after the experiment. Albumin (Alb), globulin (Glo), and methanol-precipitable proteins (MPP) from human serum were denatured with 8 M urea. Fluorescence and electrophoresis were employed in order to elucidate urea-induced conformational changes and structural behavior of proteins. The measured fluorescence emission spectra were used to estimate the stability of native and denatured protein fractions before and after BC. It was found that before BC the fractions most stable to urea denaturation were Glo, Alb, and MPP fractions. After BC in most of the beer-consuming patients (EG) some changes in native and denatured protein fractions were detected: a tendency to lower stability and minor structural deviations. These qualitative changes were more profound in MPP than in Alb and Glo. Thus, Glo is more resistible to alcohol influence than Alb, which in turn is more resistible than MPP. No serum protein changes were detected in patients of CG.

  17. SUMO modification regulates the protein stability of NDRG1.

    PubMed

    Lee, Jae Eun; Kim, Jung Hwa

    2015-03-27

    N-myc Downstream Regulated Gene 1 (NDRG1) is a metastasis suppressor protein which suppresses metastasis without affecting primary tumorigenesis. There have been many reports about the anti-metastatic function of NDRG1 in various cancers. However, the regulatory mechanism of NDRG1 at the protein level has not been studied widely. Here, we found that NDRG1 is posttranslationally modified by Small Ubiquitin-like Modifier (SUMO), preferentially by SUMO-2, and the major SUMO acceptor site of NDRG1 is Lys 14. Using various SUMO-2 modification status mimicking NDRG1 mutants, we characterized the role of SUMO-2 modification on NDRG1. SUMO-2 modification does not affect the subcellular distribution of NDRG1. However, the protein stability of NDRG1 is influenced by SUMO-2 modification. We found that both the wildtype and the SUMO modification site mutant form of the NDRG1 protein were very stable but the protein stability of SUMO-2 fused NDRG1 K14R had dramatically decreased. In addition, the expression of p21 is downregulated by overexpression of SUMO-2 fused NDRG1 K14R mutants. These results indicate that SUMO-2 modification is implicated in the modulation of NDRG1 protein level and function. This novel link between SUMO modification and regulation of NDRG1 could be a therapeutic target for treatment of various metastatic cancers.

  18. Plk phosphorylation regulates the microtubule-stabilizing protein TCTP.

    PubMed

    Yarm, Frederic R

    2002-09-01

    The mitotic polo-like kinases have been implicated in the formation and function of bipolar spindles on the basis of their respective localizations and mutant phenotypes. To date, this putative regulation has been limited to a kinesin-like motor protein, a centrosomal structural protein, and two microtubule-associated proteins (MAPs). In this study, another spindle-regulating protein, the mammalian non-MAP microtubule-binding and -stabilizing protein, the translationally controlled tumor protein (TCTP), was identified as a putative Plk-interacting clone by a two-hybrid screen. Plk phosphorylates TCTP on two serine residues in vitro and cofractionates with the majority of kinase activity toward TCTP in mitotic cell lysates. In addition, these sites were demonstrated to be phosphorylated in vivo. Overexpression of a Plk phosphorylation site-deficient mutant of TCTP induced a dramatic increase in the number of multinucleate cells, rounded cells with condensed ball-like nuclei, and cells undergoing cell death, similar to both the reported anti-Plk antibody microinjection and the low-concentration taxol treatment phenotypes. These results suggest that phosphorylation decreases the microtubule-stabilizing activity of TCTP and promotes the increase in microtubule dynamics that occurs after metaphase.

  19. High Dose Omega-3 Fatty Acid Administration and Skeletal Muscle Protein Turnover in Maintenance Hemodialysis Patients.

    PubMed

    Deger, Serpil Muge; Hung, Adriana M; Ellis, Charles D; Booker, Cindy; Bian, Aihua; Chen, Guanhua; Abumrad, Naji N; Ikizler, T Alp

    2016-07-07

    Protein energy wasting and systemic inflammation are prevalent in maintenance hemodialysis (MHD) patients. Omega-3 (ω-3) fatty acids have anti-inflammatory properties and have been shown to improve protein homeostasis. We hypothesized that administration of high-dose (2.9 g/d) ω-3 would be associated with decreased muscle protein breakdown in MHD patients with systemic inflammation. This is a substudy from a randomized, placebo-controlled study (NCT00655525). Patients were recruited between September 2008 and June 2011. Primary inclusion criteria included signs of chronic inflammation (average C-reactive protein of ≥5 mg/L over three consecutive measurements), lack of active infectious or inflammatory disease, no hospitalization within 1 month prior to the study, and not receiving steroids (>5 mg/d) and/or immunosuppressive agents. The primary outcomes were forearm muscle and whole body protein breakdown and synthesis before and after the intervention. The patients received ω-3 (n=11) versus placebo (n=9) for 12 weeks. Analysis of covariance was used to compare outcome variables at 12 weeks. Models were adjusted for a propensity score that was derived from age, sex, race, baseline high sensitivity C-reactive protein, diabetes mellitus, and fat mass because the groups were not balanced for several characteristics. Compared with placebo, ω-3 supplementation was significantly associated with decreased muscle protein breakdown at 12 weeks (-31, [interquartile range, -98--13] versus 26 [interquartile range, 13-87] µg/100 ml per min; P=0.01), which remained significant after multivariate adjustment (-46, [95% confidence interval, -102 to -1] µg/100 ml per min). ω-3 Supplementation resulted in decreased forearm muscle protein synthesis while the rate in the placebo group increased; however, there is no longer a statistically significant difference in skeletal muscle protein synthesis or in net protein balance after multivariate adjustment. There was no

  20. Translocation and Stability of Replicative DNA Helicases upon Encountering DNA-Protein Cross-links*

    PubMed Central

    Nakano, Toshiaki; Miyamoto-Matsubara, Mayumi; Shoulkamy, Mahmoud I.; Salem, Amir M. H.; Pack, Seung Pil; Ishimi, Yukio; Ide, Hiroshi

    2013-01-01

    DNA-protein cross-links (DPCs) are formed when cells are exposed to various DNA-damaging agents. Because DPCs are extremely large, steric hindrance conferred by DPCs is likely to affect many aspects of DNA transactions. In DNA replication, DPCs are first encountered by the replicative helicase that moves at the head of the replisome. However, little is known about how replicative helicases respond to covalently immobilized protein roadblocks. In the present study we elucidated the effect of DPCs on the DNA unwinding reaction of hexameric replicative helicases in vitro using defined DPC substrates. DPCs on the translocating strand but not on the nontranslocating strand impeded the progression of the helicases including the phage T7 gene 4 protein, simian virus 40 large T antigen, Escherichia coli DnaB protein, and human minichromosome maintenance Mcm467 subcomplex. The impediment varied with the size of the cross-linked proteins, with a threshold size for clearance of 5.0–14.1 kDa. These results indicate that the central channel of the dynamically translocating hexameric ring helicases can accommodate only small proteins and that all of the helicases tested use the steric exclusion mechanism to unwind duplex DNA. These results further suggest that DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. The helicases stalled by DPC had limited stability and dissociated from DNA with a half-life of 15–36 min. The implications of the results are discussed in relation to the distinct stabilities of replisomes that encounter tight but reversible DNA-protein complexes and irreversible DPC roadblocks. PMID:23283980

  1. Diagnosis of pancreaticobiliary malignancy by detection of minichromosome maintenance protein 5 in biliary brush cytology

    PubMed Central

    Keane, Margaret G; Huggett, Matthew T; Chapman, Michael H; Johnson, Gavin J; Webster, George J; Thorburn, Douglas; Mackay, James; Pereira, Stephen P

    2017-01-01

    Background: Biliary brush cytology is the standard method of evaluating biliary strictures, but is insensitive at detecting malignancy. In pancreaticobiliary cancer minichromosome maintenance replication proteins (MCM 2–7) are dysregulated in the biliary epithelium and MCM5 levels are elevated in bile samples. This study aimed to validate an immunocolorimetric ELISA assay for MCM5 as a pancreaticobiliary cancer biomarker in biliary brush samples. Methods: Biliary brush specimens were collected prospectively at ERCP from patients with a biliary stricture. Collected samples were frozen at −80 °C. The supernatant was washed and lysed cells incubated with HRP-labelled anti-MCM5 mouse monoclonal antibody. Test positivity was determined by optical density absorbance. Patients underwent biliary brush cytology or additional investigations as per clinical routine. Results: Ninety-seven patients were included in the study; 50 had malignant strictures. Median age was 65 years (range 21–94) and 51 were male. Compared with final diagnosis the MCM5 assay had a sensitivity for malignancy of 65.4% compared with 25.0% for cytology. In the 72 patients with paired MCM5 assay and biliary brush cytology, MCM5 demonstrated an improved sensitivity (55.6% vs 25.0% P=0.0002) for the detection of malignancy. Conclusions: Minichromosome maintenance replication protein5 is a more sensitive indicator of pancreaticobiliary malignancy than standard biliary brush cytology. PMID:28081547

  2. Probing protein mechanical stability with controlled shear flows

    NASA Astrophysics Data System (ADS)

    Dusting, Jonathan; Ashton, Lorna; Leontini, Justin; Blanch, Ewan; Balabani, Stavroula

    2009-11-01

    Understanding and controlling protein aggregation or misfolding is of both fundamental and medical interest. The structural changes experienced by proteins in response to forces such as those generated within flows have not been well characterised, despite the importance of mechanics in many biological processes. By monitoring the structural conformation of proteins in different concentric cylinder flows using Raman Spectroscopy we have quantified the relative stability of β-sheet dominated proteins compared with those containing a greater proportion of α-helix. To ensure that the fluid stresses are quantified accurately, a combined DNS and PIV approach has been undertaken for flow cell characterisation across the full range of operating Re. This is important for practical concentric cylinder geometries where the shear components are non-zero and spatially dependent, with the peak stresses located near the endwalls. Furthermore, recirculation regions appear well below the crtical Reynolds number for Taylor vortex formation.

  3. Nanobody stabilization of G protein coupled receptor conformational states

    PubMed Central

    Steyaert, Jan; K Kobilka, Brian

    2011-01-01

    Remarkable progress has been made in the field of G protein coupled receptor (GPCR) structural biology during the past four years. Several obstacles to generating diffraction quality crystals of GPCRs have been overcome by combining innovative methods ranging from protein engineering to lipid-based screens and microdiffraction technology. The initial GPCR structures represent energetically stable inactive-state conformations. However, GPCRs signal through different G protein isoforms or G protein-independent effectors upon ligand binding suggesting the existence of multiple ligand-specific active states. These active-state conformations are unstable in the absence of specific cytosolic signaling partners representing new challenges for structural biology. Camelid single chain antibody fragments (nanobodies) show promise for stabilizing active GPCR conformations and as chaperones for crystallogenesis. PMID:21782416

  4. Physical and oxidative stability of fish oil-in-water emulsions stabilized with fish protein hydrolysates.

    PubMed

    García-Moreno, Pedro J; Guadix, Antonio; Guadix, Emilia M; Jacobsen, Charlotte

    2016-07-15

    The emulsifying and antioxidant properties of fish protein hydrolysates (FPH) for the physical and oxidative stabilization of 5% (by weight) fish oil-in-water emulsions were investigated. Muscle proteins from sardine (Sardina pilchardus) and small-spotted catshark (Scyliorhinus canicula) were hydrolyzed to degrees of hydrolysis (DH) of 3-4-5-6% with subtilisin. Sardine hydrolysates with low DH, 3% and 4%, presented the most effective peptides to physically stabilize emulsions with smaller droplet size. This implied more protein adsorbed at the interface to act as physical barrier against prooxidants. This fact might also be responsible for the higher oxidative stability of these emulsions, as shown by their lowest peroxide value and concentration of volatiles such as 1-penten-3-one and 1-penten-3-ol. Among the hydrolysates prepared from small-spotted catshark only the hydrolysate with DH 3% yielded a physically stable emulsion with low concentration of unsaturated aldehydes. These results show the potential of FPH as alternative protein emulsifiers for the production of oxidatively stable fish oil-in-water emulsions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. A Critical Balance: dNTPs and the Maintenance of Genome Stability

    PubMed Central

    Pai, Chen-Chun; Kearsey, Stephen E.

    2017-01-01

    A crucial factor in maintaining genome stability is establishing deoxynucleoside triphosphate (dNTP) levels within a range that is optimal for chromosomal replication. Since DNA replication is relevant to a wide range of other chromosomal activities, these may all be directly or indirectly affected when dNTP concentrations deviate from a physiologically normal range. The importance of understanding these consequences is relevant to genetic disorders that disturb dNTP levels, and strategies that inhibit dNTP synthesis in cancer chemotherapy and for treatment of other disorders. We review here how abnormal dNTP levels affect DNA replication and discuss the consequences for genome stability. PMID:28146119

  6. BDNF Facilitates L-LTP Maintenance in the Absence of Protein Synthesis through PKMζ

    PubMed Central

    Mei, Fan; Nagappan, Guhan; Ke, Yang; Sacktor, Todd C.; Lu, Bai

    2011-01-01

    Late-phase long term potentiation (L-LTP) is thought to be the cellular basis for long-term memory (LTM). While LTM as well as L-LTP is known to depend on transcription and translation, it is unclear why brain-derived neurotrophic factor (BDNF) could sustain L-LTP when protein synthesis is inhibited. The persistently active protein kinase ζ (PKMζ) is the only molecule implicated in perpetuating L-LTP maintenance. Here, in mouse acute brain slices, we show that inhibition of PKMζ reversed BDNF-dependent form of L-LTP. While BDNF did not alter the steady-state level of PKMζ, BDNF together with the L-LTP inducing theta-burst stimulation (TBS) increased PKMζ level even without protein synthesis. Finally, in the absence of de novo protein synthesis, BDNF maintained TBS-induced PKMζ at a sufficient level. These results suggest that BDNF sustains L-LTP through PKMζ in a protein synthesis-independent manner, revealing an unexpected link between BDNF and PKMζ. PMID:21747912

  7. Protein Folding, Stability, and Solvation Structure in Osmolyte Solutions

    PubMed Central

    Rösgen, Jörg; Pettitt, B. Montgomery; Bolen, David Wayne

    2005-01-01

    An understanding of the impact of the crowded conditions in the cytoplasm on its biomolecules is of clear importance to biochemical, medical, and pharmaceutical science. Our previous work on the use of small biochemical compounds to crowd protein solutions indicates that a quantitative description of their nonideal behavior is possible and straightforward. Here, we show the structural origin of the nonideal solution behavior. We discuss the consequences of these findings regarding protein folding stability and solvation in crowded solutions through a structural analysis of the m-value or the change in free-energy difference of a macromolecule in solution with respect to the concentration of a third component. PMID:16113118

  8. Cellular Proteomes Have Broad Distributions of Protein Stability

    PubMed Central

    Ghosh, Kingshuk; Dill, Ken

    2010-01-01

    Biological cells are extremely sensitive to temperature. What is the mechanism? We compute the thermal stabilities of the whole proteomes of Escherichia coli, yeast, and Caenorhabditis elegans using an analytical model and an extensive database of stabilities of individual proteins. Our results support the hypothesis that a cell's thermal sensitivities arise from the collective instability of its proteins. This model shows a denaturation catastrophe at temperatures of 49–55°C, roughly the thermal death point of mesophiles. Cells live on the edge of a proteostasis catastrophe. According to the model, it is not that the average protein is problematic; it is the tail of the distribution. About 650 of E. coli's 4300 proteins are less than 4 kcal mol−1 stable to denaturation. And upshifting by only 4° from 37° to 41°C is estimated to destabilize an average protein by nearly 20%. This model also treats effects of denaturants, osmolytes, and other physical stressors. In addition, it predicts the dependence of cellular growth rates on temperature. This approach may be useful for studying physical forces in biological evolution and the role of climate change on biology. PMID:21156142

  9. Protein stability in Artemia embryos during prolonged anoxia.

    PubMed

    Clegg, James S

    2007-02-01

    Encysted embryos (cysts) of the brine shrimp, Artemia franciscana, are arguably the most stress-resistant of all animal life-history stages. One of their many adaptations is the ability to tolerate anoxia for periods of years, while fully hydrated and at physiological temperatures. Previous work indicated that the overall metabolism of anoxic embryos is brought to a reversible standstill, including the transduction of free energy and the turnover of macromolecules. But the issue of protein stability at the level of tertiary and quaternary structure was not examined. Here I provide evidence that the great majority of proteins do not irreversibly lose their native conformation during years of anoxia, despite the absence of detectable protein turnover. Although a modest degree of protein denaturation and aggregation occurs, that is quickly reversed by a brief post-anoxic aerobic incubation. I consider how such extraordinary stability is achieved and suggest that at least part of the answer involves massive amounts of a small heat shock protein (p26) that acts as a molecular chaperone, the function of which does not appear to require ribonucleoside di- or tri-phosphates.

  10. CAPNS1 Regulates USP1 Stability and Maintenance of Genome Integrity

    PubMed Central

    Cataldo, Francesca; Peche, Leticia Y.; Klaric, Enio; Brancolini, Claudio; Myers, Michael P.

    2013-01-01

    Calpains regulate a wide spectrum of biological functions, including migration, adhesion, apoptosis, secretion, and autophagy, through the modulating cleavage of specific substrates. Ubiquitous microcalpain (μ-calpain) and millicalpain (m-calpain) are heterodimers composed of catalytic subunits encoded, respectively, by CAPN1 and CAPN2 and a regulatory subunit encoded by CAPNS1. Here we show that calpain is required for the stability of the deubiquitinating enzyme USP1 in several cell lines. USP1 modulates DNA replication polymerase choice and repair by deubiquitinating PCNA. The ubiquitinated form of the USP1 substrate PCNA is stabilized in CAPNS1-depleted U2OS cells and mouse embryonic fibroblasts (MEFs), favoring polymerase-η loading on chromatin and increased mutagenesis. USP1 degradation directed by the cell cycle regulator APC/Ccdh1, which marks USP1 for destruction in the G1 phase, is upregulated in CAPNS1-depleted cells. USP1 stability can be rescued upon forced expression of calpain-activated Cdk5/p25, previously reported as a cdh1 repressor. These data suggest that calpain stabilizes USP1 by activating Cdk5, which in turn inhibits cdh1 and, consequently, USP1 degradation. Altogether these findings point to a connection between the calpain system and the ubiquitin pathway in the regulation of DNA damage response and place calpain at the interface between cell cycle modulation and DNA repair. PMID:23589330

  11. The emulsion flocculation stability of protein-carbohydrate diblock copolymers.

    PubMed

    Wooster, Tim J; Augustin, Mary Ann

    2007-09-15

    The effect of the steric layer thickness on the flocculation stability of beta-lactoglobulin-carbohydrate diblock copolymers was assessed. The diblock copolymers were created by conjugating beta-lactoglobulin to maltose or a series of different M(n) maltodextrins using the Maillard reaction. The thickness and spatial arrangement of the interfacial layers were assessed via latex adsorption and selective enzymatic digestion studies. An increase in the molecular weight of the maltodextrin (900, 1900 and 3800 Da) increased the interfacial thickness (1.1, 2.5 and 7.3 nm, respectively). No detectable change to interfacial thickness was observed upon the attachment of maltose. The increase in the interfacial layer thickness scaled with the hydrodynamic size of the carbohydrate. The beta-lactoglobulin-maltodextrin conjugates were found to have a diblock architecture, with the protein anchored at the surface and the carbohydrate protruding into the aqueous continuous phase. The stability of oil-in-water emulsions formed using the conjugates was assessed by exposing them to salt (150 mM NaCl or 0-20 mM CaCl(2)), heat alone or heat in the presence of 150 mM NaCl. Conjugation of a 900 Da maltodextrin provided sufficient steric stabilization to prevent flocculation in high salt environments. The effect of the (number) density of the steric layer was also assessed by controlling the average number of maltodextrins attached per beta-lactoglobulin molecule. The steric layer density at which emulsions became unstable was a function of carbohydrate M(n). Emulsions made from the 900 Da maltodextrin conjugate became unstable below a steric layer density of one tail per 7.5 nm(2), whilst emulsions made from the 1900 Da maltodextrin were unstable below a steric layer density of one tail per 9.5 nm(2). This trend was expected and can be explained by the stronger van der Waals attraction that arises from the closer interdroplet separations that are permissible with the shorter

  12. Stabilization and inhibition of protein-protein interactions: the 14-3-3 case study.

    PubMed

    Milroy, Lech-Gustav; Brunsveld, Luc; Ottmann, Christian

    2013-01-18

    Small-molecule modulation of protein-protein interactions (PPIs) is one of the most exciting but also difficult fields in chemical biology and drug development. As one of the most important "hub" proteins with at least 200-300 interaction partners, the 14-3-3 proteins are an especially fruitful case for PPI intervention. Here, we summarize recent success stories in small-molecule modulation, both inhibition and stabilization, of 14-3-3 PPIs. The chemical breath of modulators includes natural products such as fusicoccin A and derivatives but also compounds identified via high-throughput and in silico screening, which has yielded a toolbox of useful inhibitors and stabilizers for this interesting class of adapter proteins. Protein-protein interactions (PPIs) are involved in almost all biological processes, with any given protein typically engaged in complexes with other proteins for the majority of its lifetime. Hence, proteins function not simply as single, isolated entities but display their roles by interacting with other cellular components. These different interaction patterns are presumably as important as the intrinsic biochemical activity status of the protein itself. The biological role of a protein is therefore decisively dependent on the underlying PPI network that furthermore can show great spatial and temporal variations. A thorough appreciation and understanding of this concept and its regulation mechanisms could help to develop new therapeutic agents and concepts.

  13. The Effect of Chemical Chaperones on the Assembly and Stability of HIV-1 Capsid Protein

    PubMed Central

    Lampel, Ayala; Bram, Yaron; Levy-Sakin, Michal; Bacharach, Eran; Gazit, Ehud

    2013-01-01

    Chemical chaperones are small organic molecules which accumulate in a broad range of organisms in various tissues under different stress conditions and assist in the maintenance of a correct proteostasis under denaturating environments. The effect of chemical chaperones on protein folding and aggregation has been extensively studied and is generally considered to be mediated through non-specific interactions. However, the precise mechanism of action remains elusive. Protein self-assembly is a key event in both native and pathological states, ranging from microtubules and actin filaments formation to toxic amyloids appearance in degenerative disorders, such as Alzheimer's and Parkinson's diseases. Another pathological event, in which protein assembly cascade is a fundamental process, is the formation of virus particles. In the late stage of the virus life cycle, capsid proteins self-assemble into highly-ordered cores, which encapsulate the viral genome, consequently protect genome integrity and mediate infectivity. In this study, we examined the effect of different groups of chemical chaperones on viral capsid assembly in vitro, focusing on HIV-1 capsid protein as a system model. We found that while polyols and sugars markedly inhibited capsid assembly, methylamines dramatically enhanced the assembly rate. Moreover, chemical chaperones that inhibited capsid core formation, also stabilized capsid structure under thermal denaturation. Correspondingly, trimethylamine N-oxide, which facilitated formation of high-order assemblies, clearly destabilized capsid structure under similar conditions. In contrast to the prevailing hypothesis suggesting that chemical chaperones affect proteins through preferential exclusion, the observed dual effects imply that different chaperones modify capsid assembly and stability through different mechanisms. Furthermore, our results indicate a correlation between the folding state of capsid to its tendency to assemble into highly

  14. The effect of chemical chaperones on the assembly and stability of HIV-1 capsid protein.

    PubMed

    Lampel, Ayala; Bram, Yaron; Levy-Sakin, Michal; Bacharach, Eran; Gazit, Ehud

    2013-01-01

    Chemical chaperones are small organic molecules which accumulate in a broad range of organisms in various tissues under different stress conditions and assist in the maintenance of a correct proteostasis under denaturating environments. The effect of chemical chaperones on protein folding and aggregation has been extensively studied and is generally considered to be mediated through non-specific interactions. However, the precise mechanism of action remains elusive. Protein self-assembly is a key event in both native and pathological states, ranging from microtubules and actin filaments formation to toxic amyloids appearance in degenerative disorders, such as Alzheimer's and Parkinson's diseases. Another pathological event, in which protein assembly cascade is a fundamental process, is the formation of virus particles. In the late stage of the virus life cycle, capsid proteins self-assemble into highly-ordered cores, which encapsulate the viral genome, consequently protect genome integrity and mediate infectivity. In this study, we examined the effect of different groups of chemical chaperones on viral capsid assembly in vitro, focusing on HIV-1 capsid protein as a system model. We found that while polyols and sugars markedly inhibited capsid assembly, methylamines dramatically enhanced the assembly rate. Moreover, chemical chaperones that inhibited capsid core formation, also stabilized capsid structure under thermal denaturation. Correspondingly, trimethylamine N-oxide, which facilitated formation of high-order assemblies, clearly destabilized capsid structure under similar conditions. In contrast to the prevailing hypothesis suggesting that chemical chaperones affect proteins through preferential exclusion, the observed dual effects imply that different chaperones modify capsid assembly and stability through different mechanisms. Furthermore, our results indicate a correlation between the folding state of capsid to its tendency to assemble into highly

  15. Bromodomain Proteins Contribute to Maintenance of Bloodstream Form Stage Identity in the African Trypanosome

    PubMed Central

    Schulz, Danae; Mugnier, Monica R.; Paulsen, Eda-Margaret; Kim, Hee-Sook; Chung, Chun-wa W.; Tough, David F.; Rioja, Inmaculada; Prinjha, Rab K.; Papavasiliou, F. Nina; Debler, Erik W.

    2015-01-01

    Trypanosoma brucei, the causative agent of African sleeping sickness, is transmitted to its mammalian host by the tsetse. In the fly, the parasite’s surface is covered with invariant procyclin, while in the mammal it resides extracellularly in its bloodstream form (BF) and is densely covered with highly immunogenic Variant Surface Glycoprotein (VSG). In the BF, the parasite varies this highly immunogenic surface VSG using a repertoire of ~2500 distinct VSG genes. Recent reports in mammalian systems point to a role for histone acetyl-lysine recognizing bromodomain proteins in the maintenance of stem cell fate, leading us to hypothesize that bromodomain proteins may maintain the BF cell fate in trypanosomes. Using small-molecule inhibitors and genetic mutants for individual bromodomain proteins, we performed RNA-seq experiments that revealed changes in the transcriptome similar to those seen in cells differentiating from the BF to the insect stage. This was recapitulated at the protein level by the appearance of insect-stage proteins on the cell surface. Furthermore, bromodomain inhibition disrupts two major BF-specific immune evasion mechanisms that trypanosomes harness to evade mammalian host antibody responses. First, monoallelic expression of the antigenically varied VSG is disrupted. Second, rapid internalization of antibodies bound to VSG on the surface of the trypanosome is blocked. Thus, our studies reveal a role for trypanosome bromodomain proteins in maintaining bloodstream stage identity and immune evasion. Importantly, bromodomain inhibition leads to a decrease in virulence in a mouse model of infection, establishing these proteins as potential therapeutic drug targets for trypanosomiasis. Our 1.25Å resolution crystal structure of a trypanosome bromodomain in complex with I-BET151 reveals a novel binding mode of the inhibitor, which serves as a promising starting point for rational drug design. PMID:26646171

  16. Maintenance of Positive Diversity-Stability Relations along a Gradient of Environmental Stress

    PubMed Central

    Romanuk, Tamara N.; Vogt, Richard J.; Young, Angela; Tuck, Constance; Carscallen, Mather W.

    2010-01-01

    Background Environmental stress is widely considered to be an important factor in regulating whether changes in diversity will affect the functioning and stability of ecological communities. Methodology/Principal Findings We investigated the effects of a major environmental stressor (a decrease in water volume) on diversity-abundance and diversity-stability relations in laboratory microcosms composed of temperate multi-trophic rock pool communities to identify differences in community and functional group responses to increasing functional group richness along a gradient of environmental stress (low, medium, and high water volume). When a greater number of functional groups were present, communities were less temporally variable and achieved higher abundances. The stabilizing effect of increased functional group richness was observed regardless of the level of environmental stress the community was subjected too. Despite the strong consistent stabilizing effect of increased functional group richness on abundance, the way that individual functional groups were affected by functional group richness differed along the stress gradient. Under low stress, communities with more functional groups present were more productive and showed evidence of strong facilitative interactions. As stress increased, the positive effect of functional group richness on community abundance was no longer observed and compensatory responses became more common. Responses of individual functional groups to functional group richness became increasing heterogeneous are stress increased, prompting shifts from linear diversity-variability/abundance relations under low stress to a mix of linear and non-linear responses under medium and high stress. The strength of relations between functional group richness and both the abundances and temporal variability of functional groups also increased as stress increased. Conclusions/Significance While stress did not affect the relation between functional

  17. Nucleic acid aptamers as stabilizers of proteins: the stability of tetanus toxoid.

    PubMed

    Jain, Nishant Kumar; Jetani, Hardik C; Roy, Ipsita

    2013-07-01

    Exposure of tetanus toxoid to moisture leads to its aggregation and reduction of potency. The aim of this work was to use SELEX (systematic evolution of ligands by exponential enrichment) protocol and select aptamers which recognize tetanus toxoid (Mr ~150 kDa) with high affinity. Colyophilized preparations of tetanus toxoid and specific aptamers were encapsulated in PLGA microspheres and sustained release of the antigen was observed up to 55 days using different techniques. The total protein released was between 40-55% (24-45% residual antigenicity) in the presence of the aptamers as compared to 25% (11% residual antigenicity) for the antigen alone. We show that instead of inhibiting absorption of moisture, the aptamers blocked the protein unfolding upon absorption of moisture, inhibiting the initiation of aggregation. When exposed to accelerated storage conditions, some of the RNA sequences were able to inhibit moisture-induced aggregation in vitro and retain antigenicity of tetanus toxoid. Nucleic acid aptamers represent a novel class of protein stabilizers which stabilize the protein by interacting directly with it. This mechanism is unlike that of small molecules which alter the medium properties and hence depend on the stress condition a protein is exposed to.

  18. Intercellular chaperone transmission via exosomes contributes to maintenance of protein homeostasis at the organismal level.

    PubMed

    Takeuchi, Toshihide; Suzuki, Mari; Fujikake, Nobuhiro; Popiel, H Akiko; Kikuchi, Hisae; Futaki, Shiroh; Wada, Keiji; Nagai, Yoshitaka

    2015-05-12

    The heat shock response (HSR), a transcriptional response that up-regulates molecular chaperones upon heat shock, is necessary for cell survival in a stressful environment to maintain protein homeostasis (proteostasis). However, there is accumulating evidence that the HSR does not ubiquitously occur under stress conditions, but largely depends on the cell types. Despite such imbalanced HSR among different cells and tissues, molecular mechanisms by which multicellular organisms maintain their global proteostasis have remained poorly understood. Here, we report that proteostasis can be maintained by molecular chaperones not only in a cell-autonomous manner but also in a non-cell-autonomous manner. We found that elevated expression of molecular chaperones, such as Hsp40 and Hsp70, in a group of cells improves proteostasis in other groups of cells, both in cultured cells and in Drosophila expressing aggregation-prone polyglutamine proteins. We also found that Hsp40, as well as Hsp70 and Hsp90, is physiologically secreted from cells via exosomes, and that the J domain at the N terminus is responsible for its exosome-mediated secretion. Addition of Hsp40/Hsp70-containing exosomes to the culture medium of the polyglutamine-expressing cells results in efficient suppression of inclusion body formation, indicating that molecular chaperones non-cell autonomously improve the protein-folding environment via exosome-mediated transmission. Our study reveals that intercellular chaperone transmission mediated by exosomes is a novel molecular mechanism for non-cell-autonomous maintenance of organismal proteostasis that could functionally compensate for the imbalanced state of the HSR among different cells, and also provides a novel physiological role of exosomes that contributes to maintenance of organismal proteostasis.

  19. Properties and stability of oil-in-water emulsions stabilized by coconut skim milk proteins.

    PubMed

    Onsaard, Ekasit; Vittayanont, Manee; Srigam, Sukoncheun; McClements, D Julian

    2005-07-13

    Protein fractions were isolated from coconut: coconut skim milk protein isolate (CSPI) and coconut skim milk protein concentrate (CSPC). The ability of these proteins to form and stabilize oil-in-water emulsions was compared with that of whey protein isolate (WPI). The solubility of the proteins in CSPI, CSPC, and WPI was determined in aqueous solutions containing 0, 100, and 200 mM NaCl from pH 3 to 8. In the absence of salt, the minimum protein solubility occurred between pH 4 and 5 for CSPI and CSPC and around pH 5 for WPI. In the presence of salt (100 and 200 mM NaCl), all proteins had a higher solubility than in distilled water. Corn oil-in-water emulsions (10 wt %) with relatively small droplet diameters (d32 approximately 0.46, 1.0, and 0.5 mum for CSPI, CSPC, and WPI, respectively) could be produced using 0.2 wt % protein fraction. Emulsions were prepared with different pH values (3-8), salt concentrations (0-500 mM NaCl), and thermal treatments (30-90 degrees C for 30 min), and the mean particle diameter, particle size distribution, zeta-potential, and creaming stability were measured. Considerable droplet flocculation occurred in the emulsions near the isoelectric point of the proteins: CSPI, pH approximately 4.0; CSPC, pH approximately 4.5; WPI, pH approximately 4.8. Emulsions with monomodal particle size distributions, small mean droplet diameters, and good creaming stability could be produced at pH 7 for CSPI and WPI, whereas CSPC produced bimodal distributions. The CSPI and WPI emulsions remained relatively stable to droplet aggregation and creaming at NaCl concentrations of < or =50 and < or =100 mM, respectively. In the absence salt, the CSPI and WPI emulsions were also stable to thermal treatments at < or =80 and < or =90 degrees C for 30 min, respectively. These results suggest that CSPI may be suitable for use as an emulsifier in the food industry.

  20. Small-molecule tools for dissecting the roles of SSB/protein interactions in genome maintenance

    SciTech Connect

    Lu, Duo; Bernstein, Douglas A.; Satyshur, Kenneth A.; Keck, James L.

    2010-09-03

    Bacterial single-stranded DNA-binding proteins (SSBs) help to recruit a diverse array of genome maintenance enzymes to their sites of action through direct protein interactions. For all cases examined to date, these interactions are mediated by the evolutionarily conserved C terminus of SSB (SSB-Ct). The essential nature of SSB protein interactions makes inhibitors that block SSB complex formation valuable biochemical tools and attractive potential antibacterial agents. Here, we identify four small molecules that disrupt complexes formed between Escherichia coli SSB and Exonuclease I (ExoI), a well-studied SSB-interacting enzyme. Each compound disrupts ExoI/SSB-Ct peptide complexes and abrogates SSB stimulation of ExoI nuclease activity. Structural and biochemical studies support a model for three of the compounds in which they compete with SSB for binding to ExoI. The fourth appears to rely on an allosteric mechanism to disrupt ExoI/SSB complexes. Subsets of the inhibitors block SSB-Ct complex formation with two other SSB-interaction partners as well, which highlights their utility as reagents for investigating the roles of SSB/protein interactions in diverse DNA replication, recombination, and repair reactions.

  1. Altered Dimer Interface Decreases Stability in an Amyloidogenic Protein

    SciTech Connect

    Baden, Elizabeth M.; Owen, Barbara A.L.; Peterson, Francis C.; Volkman, Brian F.; Ramirez-Alvarado, Marina; Thompson, James R.

    2008-07-21

    Amyloidoses are devastating and currently incurable diseases in which the process of amyloid formation causes fatal cellular and organ damage. The molecular mechanisms underlying amyloidoses are not well known. In this study, we address the structural basis of immunoglobulin light chain amyloidosis, which results from deposition of light chains produced by clonal plasma cells. We compare light chain amyloidosis protein AL-09 to its wild-type counterpart, the kl O18/O8 light chain germline. Crystallographic studies indicate that both proteins form dimers. However, AL-09 has an altered dimer interface that is rotated 90 degrees from the kl O18/O8 dimer interface. The three non-conservative mutations in AL-09 are located within the dimer interface, consistent with their role in the decreased stability of this amyloidogenic protein. Moreover, AL-09 forms amyloid fibrils more quickly than kl O18/O8 in vitro. These results support the notion that the increased stability of the monomer and delayed fibril formation, together with a properly formed dimer, may be protective against amyloidogenesis. This could open a new direction into rational drug design for amyloidogenic proteins.

  2. Structuring detergents for extracting and stabilizing functional membrane proteins.

    PubMed

    Matar-Merheb, Rima; Rhimi, Moez; Leydier, Antoine; Huché, Frédéric; Galián, Carmen; Desuzinges-Mandon, Elodie; Ficheux, Damien; Flot, David; Aghajari, Nushin; Kahn, Richard; Di Pietro, Attilio; Jault, Jean-Michel; Coleman, Anthony W; Falson, Pierre

    2011-03-31

    Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. Anionic calix[4]arene based detergents (C4Cn, n=1-12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may

  3. Structuring Detergents for Extracting and Stabilizing Functional Membrane Proteins

    PubMed Central

    Matar-Merheb, Rima; Galián, Carmen; Desuzinges-Mandon, Elodie; Ficheux, Damien; Flot, David; Aghajari, Nushin; Kahn, Richard; Di Pietro, Attilio; Jault, Jean-Michel; Coleman, Anthony W.; Falson, Pierre

    2011-01-01

    Background Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. Methodology/Principal Findings Anionic calix[4]arene based detergents (C4Cn, n = 1–12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5–24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. Conclusion/Significance These compounds seem promising to extract in a functional state

  4. Exploring the Metabolic Stability of Engineered Hairy Roots after 16 Years Maintenance.

    PubMed

    Häkkinen, Suvi T; Moyano, Elisabeth; Cusidó, Rosa M; Oksman-Caldentey, Kirsi-Marja

    2016-01-01

    Plants remain a major source of new drugs, leads and fine chemicals. Cell cultures deriving from plants offer a fascinating tool to study plant metabolic pathways and offer large scale production systems for valuable compounds - commercial examples include compounds such as paclitaxel. The major constraint with undifferentiated cell cultures is that they are generally considered to be genetically unstable and cultured cells tend to produce low yields of secondary metabolites especially over time. Hairy roots, a tumor tissue caused by infection of Agrobacterium rhizogenes is a relevant alternative for plant secondary metabolite production for being fast growing, able to grow without phytohormones, and displaying higher stability than undifferentiated cells. Although genetic and metabolic stability has often been connected to transgenic hairy roots, there are only few reports on how a very long-term subculturing effects on the production capacity of hairy roots. In this study, hairy roots producing high tropane alkaloid levels were subjected to 16-year follow-up in relation to genetic and metabolic stability. Cryopreservation method for hairy roots of Hyoscyamus muticus was developed to replace laborious subculturing, and although the post-thaw recovery rates remained low, the expression of transgene remained unaltered in cryopreserved roots. It was shown that although displaying some fluctuation in the metabolite yields, even an exceedingly long-term subculturing was successfully applied without significant loss of metabolic activity.

  5. Exploring the Metabolic Stability of Engineered Hairy Roots after 16 Years Maintenance

    PubMed Central

    Häkkinen, Suvi T.; Moyano, Elisabeth; Cusidó, Rosa M.; Oksman-Caldentey, Kirsi-Marja

    2016-01-01

    Plants remain a major source of new drugs, leads and fine chemicals. Cell cultures deriving from plants offer a fascinating tool to study plant metabolic pathways and offer large scale production systems for valuable compounds – commercial examples include compounds such as paclitaxel. The major constraint with undifferentiated cell cultures is that they are generally considered to be genetically unstable and cultured cells tend to produce low yields of secondary metabolites especially over time. Hairy roots, a tumor tissue caused by infection of Agrobacterium rhizogenes is a relevant alternative for plant secondary metabolite production for being fast growing, able to grow without phytohormones, and displaying higher stability than undifferentiated cells. Although genetic and metabolic stability has often been connected to transgenic hairy roots, there are only few reports on how a very long-term subculturing effects on the production capacity of hairy roots. In this study, hairy roots producing high tropane alkaloid levels were subjected to 16-year follow-up in relation to genetic and metabolic stability. Cryopreservation method for hairy roots of Hyoscyamus muticus was developed to replace laborious subculturing, and although the post-thaw recovery rates remained low, the expression of transgene remained unaltered in cryopreserved roots. It was shown that although displaying some fluctuation in the metabolite yields, even an exceedingly long-term subculturing was successfully applied without significant loss of metabolic activity. PMID:27746806

  6. Human CDC45 protein binds to minichromosome maintenance 7 protein and the p70 subunit of DNA polymerase alpha.

    PubMed

    Kukimoto, I; Igaki, H; Kanda, T

    1999-11-01

    Budding yeast CDC45 encodes Cdc45p, an essential protein required to trigger initiation of DNA replication in late G1 phase. We cloned four and one species of the human Cdc45p homolog cDNA, resulting from different splicing patterns, from HeLa cell and human placenta cDNA libraries, respectively. A comparison of the cDNAs and the genomic sequence showed that the longest encoding a 610-amino acid protein was comprised of 20 exons. One species, which lacks exon 7 and contains the shorter of two exons 18, was identical with the previously reported CDC45L cDNA and constituted 24 out of 28 clones from HeLa cells. Splicing was different in HeLa cells and TIG-1 cells, a human diploid cell line. Human CDC45 protein was found to bind directly in vitro to human minichromosome maintenance 7 protein (hMCM7) and to the p70 subunit of DNA polymerase alpha. The data support a thesis that human CDC45 acts as a molecular tether to mediate loading of the DNA polymerase alpha on to the DNA replication complex through binding to hMCM7.

  7. Protein folding, stability, and solvation structure in osmolyte solutions hydrophobicity

    NASA Astrophysics Data System (ADS)

    Montgomery Pettitt, B.

    2008-03-01

    The hydrophobic effect between solutes in aqueous solutions plays a central role in our understanding of recognition and folding of proteins and self assembly of lipids. Hydrophobicity induces nonideal solution behavior which plays a role in many aspects of biophysics. Work on the use of small biochemical compounds to crowd protein solutions indicates that a quantitative description of their non-ideal behavior is possible and straightforward. Here, we will show what the structural origin of this non-ideal solution behavior is from expression derived from a semi grand ensemble approach. We discuss the consequences of these findings regarding protein folding stability and solvation in crowded solutions through a structural analysis of the m-value or the change in free energy difference of a macromolecule in solution with respect to the concentration of a third component. This effect has recently been restudied and new mechanisms proposed for its origins in terms of transfer free energies and hydrophobicity.

  8. Liquid drop stability for protein crystal growth in microgravity

    NASA Technical Reports Server (NTRS)

    Owen, Robert B.; Broom, Beth H.; Snyder, Robert S.; Daniel, Ron

    1987-01-01

    It is possible to grow protein crystals for biomedical research in microgravity by deploying a protein-rich solution from a syringe, forming a drop in which crystallization can occur with the proper degree of supersaturation. Drop stability is critical to the success of this research, due to the large drop sizes which can be achieved in space. In order to determine the type of syringe tips most suitable to support these large drops, tests were performed during brief periods of weightlessness onboard the NASA KC-135 low-gravity simulation aircraft. The drops were analyzed using three simple models in which the samples were approximated by modified pendulum and spring systems. It was concluded that the higher frequency systems were the most stable, indicating that of the syringes utilized, a disk-shaped configuration provided the most stable environment of low-gravity protein crystal growth.

  9. Protein stability regulators screening assay (Pro-SRSA): protein degradation meets the CRISPR-Cas9 library.

    PubMed

    Wu, Yuanzhong; Kang, Tiebang

    2016-06-29

    The regulation of protein stability is a fundamental issue for biophysical processes, but there has not previously been a convenient and unbiased method of identifying regulators of protein stability. However, as reported in the article entitled "A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A," recently published in Cell Discovery, our team developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9 (CRISPR-Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Based on our findings, we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability.

  10. Effects of phosphatidylethanolamine glycation on lipid-protein interactions and membrane protein thermal stability.

    PubMed

    Levi, Valeria; Villamil Giraldo, Ana M; Castello, Pablo R; Rossi, Juan P F C; González Flecha, F Luis

    2008-11-15

    Non-enzymatic glycation of biomolecules has been implicated in the pathophysiology of aging and diabetes. Among the potential targets for glycation are biological membranes, characterized by a complex organization of lipids and proteins interacting and forming domains of different size and stability. In the present study, we analyse the effects of glycation on the interactions between membrane proteins and lipids. The phospholipid affinity for the transmembrane surface of the PMCA (plasma-membrane Ca(2+)-ATPase) was determined after incubating the protein or the phospholipids with glucose. Results show that the affinity between PMCA and the surrounding phospholipids decreases significantly after phosphospholipid glycation, but remains unmodified after glycation of the protein. Furthermore, phosphatidylethanolamine glycation decreases by approximately 30% the stability of PMCA against thermal denaturation, suggesting that glycated aminophospholipids induce a structural rearrangement in the protein that makes it more sensitive to thermal unfolding. We also verified that lipid glycation decreases the affinity of lipids for two other membrane proteins, suggesting that this effect might be common to membrane proteins. Extending these results to the in vivo situation, we can hypothesize that, under hyperglycaemic conditions, glycation of membrane lipids may cause a significant change in the structure and stability of membrane proteins, which may affect the normal functioning of membranes and therefore of cells.

  11. Role of platelets in maintenance of pulmonary vascular permeability to protein

    SciTech Connect

    Lo, S.K.; Burhop, K.E.; Kaplan, J.E.; Malik, A.B. )

    1988-04-01

    The authors examined the role of platelets in maintenance of pulmonary vascular integrity by inducing thrombocytopenia in sheep using antiplatelet serum (APS). A causal relationship between thrombocytopenia and increase in pulmonary vascular permeability was established by platelet repletion using platelet-rich plasma (PRP). Sheep were chronically instrumented and lung lymph fistulas prepared to monitor pulmonary lymph flow (Q{sub lym}). A balloon catheter was positioned in the left atrium to assess pulmonary vascular permeability to protein after raising the left atrial pressure (P{sub la}). Thrombocytopenia was maintained for 3 days by daily intramuscular APS injections. In studies using cultured bovine pulmonary artery endothelial monolayers, transendothelia permeability of {sup 125}I-labeled albumin was reduced 50 and 95%, respectively, when 2.5 {times} 10{sup 7} or 5 {times} 10{sup 7} platelets were added onto endothelial monolayers. However, addition of 5 {times} 10{sup 6} platelets or 5 {times} 10{sup 7} red blood cells did not reduce endothelial monolayer albumin permeability. Results indicate that platelets are required for the maintenance of pulmonary vascular permeability. Reduction in permeability appears to involve an interaction of platelets with the endothelium.

  12. USP7 controls Chk1 protein stability by direct deubiquitination.

    PubMed

    Alonso-de Vega, Ignacio; Martín, Yusé; Smits, Veronique A J

    2014-01-01

    Chk1, an essential checkpoint kinase in the DNA damage response pathway (DDR), is tightly regulated by both ATR-dependent phosphorylation and proteasome-mediated degradation. Here we identify ubiquitin hydrolase USP7 as a novel regulator of Chk1 protein stability. USP7 was shown before to regulate other DDR proteins such as p53, Hdm2 and Claspin, an adaptor protein in the ATR-Chk1 pathway required for Chk1 activation. Depletion or inhibition of USP7 leads to lower Chk1 levels. The decreased Chk1 protein after USP7 knock down cannot be rescued by simultaneously elevating Claspin levels, demonstrating that the effect of USP7 on Chk1 is independent of its known effect on Claspin. Conversely, overexpression of USP7 wild type, but not a catalytic mutant version, elevates Chk1 levels and increases the half-life of Chk1 protein. Importantly, wild type, but not catalytic mutant USP7 can deubiquitinate Chk1 in vivo and in vitro, confirming that USP7 directly regulates Chk1 protein levels. Finally we show that USP7 catalytic mutant is (mono-)ubiquitinated, which suggests auto-deubiquitination by this ubiquitin hydrolase, possibly important for its regulation.

  13. Small-molecule stabilization of the p53 - 14-3-3 protein-protein interaction.

    PubMed

    Doveston, Richard G; Kuusk, Ave; Andrei, Sebastian A; Leysen, Seppe; Cao, Qing; Castaldi, Maria P; Hendricks, Adam; Brunsveld, Luc; Chen, Hongming; Boyd, Helen; Ottmann, Christian

    2017-08-01

    14-3-3 proteins are positive regulators of the tumor suppressor p53, the mutation of which is implicated in many human cancers. Current strategies for targeting of p53 involve restoration of wild-type function or inhibition of the interaction with MDM2, its key negative regulator. Despite the efficacy of these strategies, the alternate approach of stabilizing the interaction of p53 with positive regulators and, thus, enhancing tumor suppressor activity, has not been explored. Here, we report the first example of small-molecule stabilization of the 14-3-3 - p53 protein-protein interaction (PPI) and demonstrate the potential of this approach as a therapeutic modality. We also observed a disconnect between biophysical and crystallographic data in the presence of a stabilizing molecule, which is unusual in 14-3-3 PPIs. © 2017 Federation of European Biochemical Societies.

  14. Positive and negative roles of homologous recombination in the maintenance of genome stability in Saccharomyces cerevisiae.

    PubMed Central

    Yoshida, Jumpei; Umezu, Keiko; Maki, Hisaji

    2003-01-01

    In previous studies of the loss of heterozygosity (LOH), we analyzed a hemizygous URA3 marker on chromosome III in S. cerevisiae and showed that homologous recombination is involved in processes that lead to LOH in multiple ways, including allelic recombination, chromosome size alterations, and chromosome loss. To investigate the role of homologous recombination more precisely, we examined LOH events in rad50 Delta, rad51 Delta, rad52 Delta, rad50 Delta rad52 Delta, and rad51 Delta rad52 Delta mutants. As compared to Rad(+) cells, the frequency of LOH was significantly increased in all mutants, and most events were chromosome loss. Other LOH events were differentially affected in each mutant: the frequencies of all types of recombination were decreased in rad52 mutants and enhanced in rad50 mutants. The rad51 mutation increased the frequency of ectopic but not allelic recombination. Both the rad52 and rad51 mutations increased the frequency of intragenic point mutations approximately 25-fold, suggesting that alternative mutagenic pathways partially substitute for homologous recombination. Overall, these results indicate that all of the genes are required for chromosome maintenance and that they most likely function in homologous recombination between sister chromatids. In contrast, other recombination pathways can occur at a substantial level even in the absence of one of the genes and contribute to generating various chromosome rearrangements. PMID:12750319

  15. Monoclonal antibody probe for assessing beer foam stabilizing proteins.

    PubMed

    Onishi, A; Proudlove, M O; Dickie, K; Mills, E N; Kauffman, J A; Morgan, M R

    1999-08-01

    A monoclonal antibody (Mab; IFRN 1625) has been produced, which is specific for the most hydrophobic polypeptides responsible for foam stabilization. The binding characteristics of the Mab suggest that it is the conformation of certain hydrophobic polypeptides which is important for foam stabilization. An enzyme-linked immunosorbent assay (ELISA) for assessing the foam-positive form of the foam-stabilizing polypeptides in beer was developed using IFRN 1625. A good correlation was obtained between ELISA determination of foam-stabilizing polypeptides and an empirical means of determining foaming, that is, the Rudin head retention values, for a collection of beers of various foam qualities. Application of the ELISA to different stages of the brewing process showed that the amounts of foam-positive polypeptides increased during barley germination. During the brewing process the proportion of foam-positive polypeptides present after fermentation increased slightly, although a large amount was lost along with other beer proteins during subsequent steps, such as filtering. The present study demonstrates that the amounts of beer polypeptide present in a foam-positive form have a direct relationship with the foaming potential of beer, that their levels are altered by processing, and that there is potential for greater quality control.

  16. Structure of an octameric form of the minichromosome maintenance protein from the archaeon Pyrococcus abyssi

    PubMed Central

    Cannone, Giuseppe; Visentin, Silvia; Palud, Adeline; Henneke, Ghislaine; Spagnolo, Laura

    2017-01-01

    Cell division is a complex process that requires precise duplication of genetic material. Duplication is concerted by replisomes. The Minichromosome Maintenance (MCM) replicative helicase is a crucial component of replisomes. Eukaryotic and archaeal MCM proteins are highly conserved. In fact, archaeal MCMs are powerful tools for elucidating essential features of MCM function. However, while eukaryotic MCM2-7 is a heterocomplex made of different polypeptide chains, the MCM complexes of many Archaea form homohexamers from a single gene product. Moreover, some archaeal MCMs are polymorphic, and both hexameric and heptameric architectures have been reported for the same polypeptide. Here, we present the structure of the archaeal MCM helicase from Pyrococcus abyssi in its single octameric ring assembly. To our knowledge, this is the first report of a full-length octameric MCM helicase. PMID:28176822

  17. Effective stabilization of CLA by microencapsulation in pea protein.

    PubMed

    Costa, A M M; Nunes, J C; Lima, B N B; Pedrosa, C; Calado, V; Torres, A G; Pierucci, A P T R

    2015-02-01

    CLA was microencapsulated by spray drying in ten varied wall systems (WS) consisting of pea protein isolate or pea protein concentrate (PPC) alone at varied core:WS ratios (1:2; 1:3 and 1:4), or blended with maltodextrin (M) and carboxymethylcellulose at a pea protein:carbohydrate ratio of 3:1. The physical-chemical properties of the CLA microparticles were characterised by core retention, microencapsulation efficiency (ME), particle size and moisture. CLA:M:PPC (1:1:3) showed the most promising results, thus we evaluated the effect of M addition in the WS on other physical-chemical characteristics and oxidative stability (CLA isomer profile, quantification of CLA and volatile compounds by SPME coupled with CG-MS) during two months of storage at room temperature, CLA:PPC (1:4) was selected for comparisons. CLA:M:PPC (1:1:3) microparticles demonstrated better morphology, solubility, dispersibility and higher glass-transition temperature values. M addition did not influence the oxidative stability of CLA, however its presence improved physical-chemical characteristics necessary for food applications.

  18. Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability.

    PubMed

    Webb, Stacy; Nagy, Tamas; Moseley, Hunter; Fried, Michael; Dutch, Rebecca

    2017-04-07

    Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726-35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Stabilization of Protein-Protein Interactions in chemical biology and drug discovery.

    PubMed

    Bier, David; Thiel, Philipp; Briels, Jeroen; Ottmann, Christian

    2015-10-01

    More than 300,000 Protein-Protein Interactions (PPIs) can be found in human cells. This number is significantly larger than the number of single proteins, which are the classical targets for pharmacological intervention. Hence, specific and potent modulation of PPIs by small, drug-like molecules would tremendously enlarge the "druggable genome" enabling novel ways of drug discovery for essentially every human disease. This strategy is especially promising in diseases with difficult targets like intrinsically disordered proteins or transcription factors, for example neurodegeneration or metabolic diseases. Whereas the potential of PPI modulation has been recognized in terms of the development of inhibitors that disrupt or prevent a binary protein complex, the opposite (or complementary) strategy to stabilize PPIs has not yet been realized in a systematic manner. This fact is rather surprising given the number of impressive natural product examples that confer their activity by stabilizing specific PPIs. In addition, in recent years more and more examples of synthetic molecules are being published that work as PPI stabilizers, despite the fact that in the majority they initially have not been designed as such. Here, we describe examples from both the natural products as well as the synthetic molecules advocating for a stronger consideration of the PPI stabilization approach in chemical biology and drug discovery. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Protein-stabilized nanoemulsions and emulsions: comparison of physicochemical stability, lipid oxidation, and lipase digestibility.

    PubMed

    Lee, Sung Je; Choi, Seung Jun; Li, Yan; Decker, Eric Andrew; McClements, David Julian

    2011-01-12

    The properties of whey protein isolate (WPI) stabilized oil-in-water (O/W) nanoemulsions (d(43) ≈ 66 nm; 0.5% oil, 0.9% WPI) and emulsions (d(43) ≈ 325 nm; 0.5% oil, 0.045% WPI) were compared. Emulsions were prepared by high-pressure homogenization, while nanoemulsions were prepared by high-pressure homogenization and solvent (ethyl acetate) evaporation. The effects of pH, ionic strength (0-500 mM NaCl), thermal treatment (30-90 °C), and freezing/thawing on the stability and properties of the nanoemulsions and emulsions were compared. In general, nanoemulsions had better stability to droplet aggregation and creaming than emulsions. The nanoemulsions were unstable to droplet flocculation near the isoelectric point of WPI but remained stable at higher or lower pH values. In addition, the nanoemulsions were stable to salt addition, thermal treatment, and freezing/thawing (pH 7). Lipid oxidation was faster in nanoemulsions than emulsions, which was attributed to the increased surface area. Lipase digestibility of lipids was slower in nanoemulsions than emulsions, which was attributed to changes in interfacial structure and protein content. These results have important consequences for the design and utilization of food-grade nanoemulsions.

  1. Diets with High or Low Protein Content and Glycemic Index for Weight-Loss Maintenance

    PubMed Central

    Larsen, Thomas Meinert; Dalskov, Stine-Mathilde; van Baak, Marleen; Jebb, Susan A.; Papadaki, Angeliki; Pfeiffer, Andreas F.H.; Martinez, J. Alfredo; Handjieva-Darlenska, Teodora; Kunešová, Marie; Pihlsgård, Mats; Stender, Steen; Holst, Claus; Saris, Wim H.M.; Astrup, Arne

    2012-01-01

    Background Studies of weight-control diets that are high in protein or low in glycemic index have reached varied conclusions, probably owing to the fact that the studies had insufficient power. Methods We enrolled overweight adults from eight European countries who had lost at least 8% of their initial body weight with a 3.3-MJ (800-kcal) low-calorie diet. Participants were randomly assigned, in a two-by-two factorial design, to one of five ad libitum diets to prevent weight regain over a 26-week period: a low-protein and low-glycemic-index diet, a low-protein and high-glycemic-index diet, a high-protein and low-glycemic-index diet, a high-protein and high-glycemic-index diet, or a control diet. Results A total of 1209 adults were screened (mean age, 41 years; body-mass index [the weight in kilograms divided by the square of the height in meters], 34), of whom 938 entered the low-calorie-diet phase of the study. A total of 773 participants who completed that phase were randomly assigned to one of the five maintenance diets; 548 completed the intervention (71%). Fewer participants in the high-protein and the low-glycemic-index groups than in the low-protein–high-glycemic-index group dropped out of the study (26.4% and 25.6%, respectively, vs. 37.4%; P = 0.02 and P = 0.01 for the respective comparisons). The mean initial weight loss with the low-calorie diet was 11.0 kg. In the analysis of participants who completed the study, only the low-protein–high-glycemic-index diet was associated with subsequent significant weight regain (1.67 kg; 95% confidence interval [CI], 0.48 to 2.87). In an intention-to-treat analysis, the weight regain was 0.93 kg less (95% CI, 0.31 to 1.55) in the groups assigned to a high-protein diet than in those assigned to a low-protein diet (P = 0.003) and 0.95 kg less (95% CI, 0.33 to 1.57) in the groups assigned to a low-glycemic-index diet than in those assigned to a high-glycemic-index diet (P = 0.003). The analysis involving

  2. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins

    PubMed Central

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-01-01

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions. PMID:28266541

  3. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins.

    PubMed

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-03-07

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions.

  4. What makes a protein a protein? Hydrophobic core designs that specify stability and structural properties.

    PubMed Central

    Munson, M.; Balasubramanian, S.; Fleming, K. G.; Nagi, A. D.; O'Brien, R.; Sturtevant, J. M.; Regan, L.

    1996-01-01

    Here we describe how the systematic redesign of a protein's hydrophobic core alters its structure and stability. We have repacked the hydrophobic core of the four-helix-bundle protein, Rop, with altered packing patterns and various side chain shapes and sizes. Several designs reproduce the structure and native-like properties of the wild-type, while increasing the thermal stability. Other designs, either with similar sizes but different shapes, or with decreased sizes of the packing residues, destabilize the protein. Finally, overpacking the core with the larger side chains causes a loss of native-like structure. These results allow us to further define the roles of tight residue packing and the burial of hydrophobic surface area in the construction of native-like proteins. PMID:8844848

  5. Inulin glasses for the stabilization of therapeutic proteins.

    PubMed

    Hinrichs, W L; Prinsen, M G; Frijlink, H W

    2001-03-14

    Sugar glasses are widely used to stabilize proteins during drying and subsequent storage. To act successfully as a protectant, the sugars should have a high glass transition temperature (Tg), a poor hygroscopicity, a low crystallization rate, and contain no reducing groups. When freeze drying is envisaged as method of drying, a relatively high Tg of the freeze concentrated fraction (Tg') is preferrable. In this study, whether inulins meet these requirements was investigated. Inulins of various degrees of polymerisation (DP) were evaluated. Trehalose glass was used as a positive control. It was found that the Tg and the Tg' of inulins with a number/weight average DP (DP(n)/DP(w)) higher than 5.5/6.0 were higher than those of trehalose glass. Furthermore, inulin glasses showed a similar hygroscopicity to that of trehalose glass but crystallized less rapidly. Less than 6% of the sugar units of inulins with a DP(n)/DP(w) higher than 5.5/6.0 contained reducing groups. Trehalose contained no reducing groups. Freeze drying of an alkaline phosphatase solution without protectant induced an almost complete loss of the activity of the protein. In contrast, when inulins with a DP(n)/DP(w) higher than 5.5/6.0 or trehalose were used as stabilizer, the activity was fully maintained, also after subsequent storage for 4 weeks at 20 degrees C and 0, 45, or 60% RH, respectively. The stabilizing capacities of inulin with a lower DP and glucose were substantially less pronounced. After storage at 60 degrees C for 6 days, the activity of freeze dried samples containing inulins with a DP(n)/DP(w) higher than 5.5/6.0 was still about 50% whereas the activity of samples containing inulin with a lower DP, glucose, or trehalose was completely lost. It is concluded that inulins with a DP(n)/DP(w) higher than 5.5/6.0 meet the physico-chemical characteristics to successfully act as protectants for proteins. The stabilizing potential of these inulins was clearly shown using alkaline phosphatase

  6. On the Stability of Parainfluenza Virus 5 F Proteins

    PubMed Central

    Poor, Taylor A.; Song, Albert S.; Welch, Brett D.; Kors, Christopher A.; Jardetzky, Theodore S.

    2015-01-01

    The crystal structure of the F protein (prefusion form) of the paramyxovirus parainfluenza virus 5 (PIV5) WR isolate was determined. We investigated the basis by which point mutations affect fusion in PIV5 isolates W3A and WR, which differ by two residues in the F ectodomain. The P22 stabilizing site acts through a local conformational change and a hydrophobic pocket interaction, whereas the S443 destabilizing site appears sensitive to both conformational effects and amino acid charge/polarity changes. PMID:25589638

  7. Early and Late Loss of the Cytoskeletal Scaffolding Protein, Ankyrin G Reveals its Role in Maturation and Maintenance of Nodes of Ranvier in Myelinated Axons.

    PubMed

    Saifetiarova, Julia; Taylor, Anna M; Bhat, Manzoor A

    2017-02-01

    The mechanisms that govern node of Ranvier organization, stability and long-term maintenance remain to be fully elucidated. One of the molecular components of the node is the cytoskeletal scaffolding protein, Ankyrin G (AnkG), which interacts with multiple members of the nodal complex. The role of AnkG in nodal organization and maintenance is still not clearly defined, as to whether AnkG functions as an initial nodal organizer or whether it functions as a nodal stabilizer after the nodal complex has been assembled. Using a mouse model system, we report here that perinatal and juvenile neuronal ablation of AnkG has differential consequences on nodal stability. Early loss of AnkG creates immature nodes with abnormal morphology, which undergo accelerated destabilization within a month, resulting in rapid NaV channel and βIV Spectrin loss with reduced effects on Neurofascin 186. On the other hand, late ablation of AnkG from established nodal complexes leads to slow but progressive nodal destabilization over 10 months, primarily affecting βIV Spectrin, followed by NaV channels, with modest impact on Neurofascin 186. We also show that Ankyrin R and βI Spectrin are not sufficient to prevent nodal disorganization after AnkG ablation. Additionally, nodal disorganization in both early and late AnkG mutants is accompanied by axonal pathology and neurological dysfunction. Together, our results suggest that AnkG plays an indispensable role in maturation and long-term stabilization of the newly assembled nodal complex, and that loss of AnkG after nodal stabilization does not lead to rapid nodal disassembly but loss of specific nodal components in a time-dependent manner.

  8. The Drosophila Microtubule-Associated Protein Mars Stabilizes Mitotic Spindles by Crosslinking Microtubules through Its N-Terminal Region

    PubMed Central

    Zhang, Gang; Beati, Hamze; Nilsson, Jakob; Wodarz, Andreas

    2013-01-01

    Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs) are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs) have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs. PMID:23593258

  9. Contribution of surface salt bridges to protein stability: guidelines for protein engineering.

    PubMed

    Makhatadze, George I; Loladze, Vakhtang V; Ermolenko, Dmitri N; Chen, XiaoFen; Thomas, Susan T

    2003-04-11

    The small globular protein, ubiquitin, contains a pair of oppositely charged residues, K11 and E34, that according to the three-dimensional structure are located on the surface of this protein with a spatial orientation characteristic of a salt bridge. We investigated the strength of this salt bridge and its contribution to the global stability of the ubiquitin molecule. Using the "double mutant cycle" analysis, the strength of the pairwise interactions between K11 and E34 was estimated to be favorable by 3.6kJ/mol. Further, the salt bridge of the reverse orientation, i.e. E11/K34, can be formed and is found to have a strength (3.8kJ/mol) similar to that of the K11/E34 pair. However, the global stability of the K11/E34 variant of ubiquitin is 2.2kJ/mol higher than that of the E11/K34 variant. The difference in the contribution of the opposing salt bridge orientations to the overall stability of the ubiquitin molecule is attributed to the difference in the charge-charge interactions between residues forming the salt bridge and the rest of the ionizable groups in this protein. On the basis of these results, we concluded that surface salt bridges are stabilizing, but their contribution to the overall protein stability is strongly context-dependent, with charge-charge interactions being the largest determinant. Analysis of 16 salt bridges from six different proteins, for which detailed experimental data on energetics have been reported, support the conclusions made from the analysis of the salt bridge in ubiquitin. Implications of these findings for engineering proteins with enhanced thermostability are discussed.

  10. Stabilization of collagen through bioconversion: An insight in protein-protein interaction.

    PubMed

    Usharani, Nagarajan; Jayakumar, Gladstone Christopher; Kanth, Swarna Vinodh; Rao, Jonnalagadda Raghava

    2014-08-01

    Collagen is a natural protein, which is used as a vital biomaterial in tissue engineering. The major concern about native collagen is lack of its thermal stability and weak resistance to proteolytic degradation. In this scenario, the crosslinking compounds used for stabilization of collagen are mostly of chemical nature and exhibit toxicity. The enzyme mediated crosslinking of collagen provides a novel alternative, nontoxic method for stabilization. In this study, aldehyde forming enzyme (AFE) is used in the bioconversion of hydroxylmethyl groups of collagen to formyl groups that results in the formation of peptidyl aldehyde. The resulted peptidyl aldehyde interacts with bipolar ions of basic amino acid residues of collagen. Further interaction leads to the formation of conjugated double bonds (aldol condensation involving the aldehyde group of peptidyl aldehyde) within the collagen. The enzyme modified collagen matrices have shown an increase in the denaturation temperature, when compared with native collagen. Enzyme modified collagen membranes exhibit resistance toward collagenolytic activity. Moreover, they exhibited a nontoxic nature. The catalytic activity of AFE on collagen as a substrate establishes an efficient modification, which enhances the structural stability of collagen. This finds new avenues in the context of protein-protein stabilization and discovers paramount application in tissue engineering.

  11. Hdac3 is essential for the maintenance of chromatin structure and genome stability.

    PubMed

    Bhaskara, Srividya; Knutson, Sarah K; Jiang, Guochun; Chandrasekharan, Mahesh B; Wilson, Andrew J; Zheng, Siyuan; Yenamandra, Ashwini; Locke, Kimberly; Yuan, Jia-Ling; Bonine-Summers, Alyssa R; Wells, Christina E; Kaiser, Jonathan F; Washington, M Kay; Zhao, Zhongming; Wagner, Florence F; Sun, Zu-Wen; Xia, Fen; Holson, Edward B; Khabele, Dineo; Hiebert, Scott W

    2010-11-16

    Hdac3 is essential for efficient DNA replication and DNA damage control. Deletion of Hdac3 impaired DNA repair and greatly reduced chromatin compaction and heterochromatin content. These defects corresponded to increases in histone H3K9,K14ac; H4K5ac; and H4K12ac in late S phase of the cell cycle, and histone deposition marks were retained in quiescent Hdac3-null cells. Liver-specific deletion of Hdac3 culminated in hepatocellular carcinoma. Whereas HDAC3 expression was downregulated in only a small number of human liver cancers, the mRNA levels of the HDAC3 cofactor NCOR1 were reduced in one-third of these cases. siRNA targeting of NCOR1 and SMRT (NCOR2) increased H4K5ac and caused DNA damage, indicating that the HDAC3/NCOR/SMRT axis is critical for maintaining chromatin structure and genomic stability. Copyright © 2010 Elsevier Inc. All rights reserved.

  12. Potential Benefit of the Charge-Stabilized Nanostructure Saline RNS60 for Myelin Maintenance and Repair.

    PubMed

    Rao, Vijayaraghava T S; Khan, Damla; Jones, Russell G; Nakamura, Diane S; Kennedy, Timothy E; Cui, Qiao-Ling; Rone, Malena B; Healy, Luke M; Watson, Richard; Ghosh, Supurna; Antel, Jack P

    2016-07-25

    Myelin injury in multiple sclerosis (MS) has been attributed both to "outside-in" primary immune mediated and "inside-out" metabolic stress of oligodendrocyte (OL) related mechanisms. Subsequent remyelination is dependent on recruitment and differentiation of oligodendrocyte progenitor cells (OPCs). RNS60 is a physically-modified saline containing charge-stabilized nanobubbles generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. Administration of RNS60 has been shown to reduce the severity of EAE by dampening the immune response and myelin loss. Additionally, RNS60 has been demonstrated to enhance mitochondrial ATP synthesis in neurons. Here, we used post-natal rat derived OLs and OPCs to assess the impact of RNS60 on the response of OLs to metabolic stress in vitro (glucose-nutrient deprivation, referred to as 'NG') and on OPC differentiation capacity. Under the NG condition, our findings indicate that RNS60 decreases caspases 3/7 activation. Respirometric analyses revealed that RNS60 increased spare glycolytic capacity (SGC) under normal culture conditions. However, RNS60 enhanced OL spare respiratory capacity (SRC) when a metabolic stress was present. Furthermore, we show that RNS60 promotes OPC differentiation under physiological conditions. Our findings provide evidence for the potential therapeutic efficacy of RNS60 through the promotion of OL survival and OPC differentiation.

  13. Potential Benefit of the Charge-Stabilized Nanostructure Saline RNS60 for Myelin Maintenance and Repair

    PubMed Central

    Rao, Vijayaraghava T. S.; Khan, Damla; Jones, Russell G.; Nakamura, Diane S.; Kennedy, Timothy E.; Cui, Qiao-Ling; Rone, Malena B.; Healy, Luke M.; Watson, Richard; Ghosh, Supurna; Antel, Jack P.

    2016-01-01

    Myelin injury in multiple sclerosis (MS) has been attributed both to “outside-in” primary immune mediated and “inside-out” metabolic stress of oligodendrocyte (OL) related mechanisms. Subsequent remyelination is dependent on recruitment and differentiation of oligodendrocyte progenitor cells (OPCs). RNS60 is a physically-modified saline containing charge-stabilized nanobubbles generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. Administration of RNS60 has been shown to reduce the severity of EAE by dampening the immune response and myelin loss. Additionally, RNS60 has been demonstrated to enhance mitochondrial ATP synthesis in neurons. Here, we used post-natal rat derived OLs and OPCs to assess the impact of RNS60 on the response of OLs to metabolic stress in vitro (glucose-nutrient deprivation, referred to as ‘NG’) and on OPC differentiation capacity. Under the NG condition, our findings indicate that RNS60 decreases caspases 3/7 activation. Respirometric analyses revealed that RNS60 increased spare glycolytic capacity (SGC) under normal culture conditions. However, RNS60 enhanced OL spare respiratory capacity (SRC) when a metabolic stress was present. Furthermore, we show that RNS60 promotes OPC differentiation under physiological conditions. Our findings provide evidence for the potential therapeutic efficacy of RNS60 through the promotion of OL survival and OPC differentiation. PMID:27451946

  14. Quantifying why urea is a protein denaturant, whereas glycine betaine is a protein stabilizer

    PubMed Central

    Guinn, Emily J.; Pegram, Laurel M.; Capp, Michael W.; Pollock, Michelle N.; Record, M. Thomas

    2011-01-01

    To explain the large, opposite effects of urea and glycine betaine (GB) on stability of folded proteins and protein complexes, we quantify and interpret preferential interactions of urea with 45 model compounds displaying protein functional groups and compare with a previous analysis of GB. This information is needed to use urea as a probe of coupled folding in protein processes and to tune molecular dynamics force fields. Preferential interactions between urea and model compounds relative to their interactions with water are determined by osmometry or solubility and dissected using a unique coarse-grained analysis to obtain interaction potentials quantifying the interaction of urea with each significant type of protein surface (aliphatic, aromatic hydrocarbon (C); polar and charged N and O). Microscopic local-bulk partition coefficients Kp for the accumulation or exclusion of urea in the water of hydration of these surfaces relative to bulk water are obtained. Kp values reveal that urea accumulates moderately at amide O and weakly at aliphatic C, whereas GB is excluded from both. These results provide both thermodynamic and molecular explanations for the opposite effects of urea and glycine betaine on protein stability, as well as deductions about strengths of amide NH—amide O and amide NH—amide N hydrogen bonds relative to hydrogen bonds to water. Interestingly, urea, like GB, is moderately accumulated at aromatic C surface. Urea m-values for protein folding and other protein processes are quantitatively interpreted and predicted using these urea interaction potentials or Kp values. PMID:21930943

  15. Discovery of Manassantin A Protein Targets Using Large-Scale Protein Folding and Stability Measurements.

    PubMed

    Geer Wallace, M Ariel; Kwon, Do-Yeon; Weitzel, Douglas H; Lee, Chen-Ting; Stephenson, Tesia N; Chi, Jen-Tsan; Mook, Robert A; Dewhirst, Mark W; Hong, Jiyong; Fitzgerald, Michael C

    2016-08-05

    Manassantin A is a natural product that has been shown to have anticancer activity in cell-based assays, but has a largely unknown mode-of-action. Described here is the use of two different energetics-based approaches to identify protein targets of manassantin A. Using the stability of proteins from rates of oxidation technique with an isobaric mass tagging strategy (iTRAQ-SPROX) and the pulse proteolysis technique with a stable isotope labeling with amino acids in cell culture strategy (SILAC-PP), over 1000 proteins in a MDA-MB-231 cell lysate grown under hypoxic conditions were assayed for manassantin A interactions (both direct and indirect). A total of 28 protein hits were identified with manassantin A-induced thermodynamic stability changes. Two of the protein hits (filamin A and elongation factor 1α) were identified using both experimental approaches. The remaining 26 hit proteins were only assayed in either the iTRAQ-SPROX or the SILAC-PP experiment. The 28 potential protein targets of manassantin A identified here provide new experimental avenues along which to explore the molecular basis of manassantin A's mode of action. The current work also represents the first application iTRAQ-SPROX and SILAC-PP to the large-scale analysis of protein-ligand binding interactions involving a potential anticancer drug with an unknown mode-of-action.

  16. The maintenance requirements of mandibular overdentures stabilized by Astra Tech implants using three different attachment mechanisms--balls, magnets, and bars; 3-year results.

    PubMed

    Davis, D M; Packer, M E

    2000-12-01

    Thirty seven people were provided with mandibular overdentures stabilized by Astra Tech implants. Thirteen people were provided with ball attachments, 12 with magnet attachments and 12 with bar attachments. The patients were followed for 3 years and detailed records were kept of the extent of maintenance required after placement of the dentures. The bar attachment mechanism required 9 episodes of maintenance, compared to 38 for the ball attachment mechanism and 23 for the magnet attachment mechanism. There was no difference between the three groups for the amount of maintenance required by the overdentures. The design of the ball attachment changed during the study from a two piece to a one piece abutment. It is likely that had this "all in one" attachment been available from the beginning of the study, the amount of maintenance required by the ball attachment mechanism would have been considerably less.

  17. Small-molecule stabilization of protein-protein interactions: an underestimated concept in drug discovery?

    PubMed

    Thiel, Philipp; Kaiser, Markus; Ottmann, Christian

    2012-02-27

    The modulation of protein-protein interactions (PPIs) has been recognized as one of the most challenging tasks in drug discovery. While their systematic development has long been considered as intractable, this view has changed over the last years, with the first drug candidates undergoing clinical studies. To date, the vast majority of PPI modulators are interaction inhibitors. However, in many biological contexts a prolonged lifespan of a PPI might be desirable, calling for the complementary approach of PPI stabilization. In fact, nature offers impressive examples of this concept and some PPI-stabilizing natural products have already found application as important drugs. Moreover, directed small-molecule stabilization has recently been demonstrated. Therefore, it is time to take a closer look at the constructive side of modulating PPIs. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Cementing proteins provide extra mechanical stabilization to viral cages

    NASA Astrophysics Data System (ADS)

    Hernando-Pérez, M.; Lambert, S.; Nakatani-Webster, E.; Catalano, C. E.; de Pablo, P. J.

    2014-07-01

    The study of virus shell stability is key not only for gaining insights into viral biological cycles but also for using viral capsids in materials science. The strength of viral particles depends profoundly on their structural changes occurring during maturation, whose final step often requires the specific binding of ‘decoration’ proteins (such as gpD in bacteriophage lambda) to the viral shell. Here we characterize the mechanical stability of gpD-free and gpD-decorated bacteriophage lambda capsids. The incorporation of gpD into the lambda shell imparts a major mechanical reinforcement that resists punctual deformations. We further interrogate lambda particle stability with molecular fatigue experiments that resemble the sub-lethal Brownian collisions of virus shells with macromolecules in crowded environments. Decorated particles are especially robust against collisions of a few kBT (where kB is the Boltzmann’s constant and T is the temperature ~300 K), which approximate those anticipated from molecular insults in the environment.

  19. Effect of Ramipril on Urinary Protein Excretion in Maintenance Renal Transplant Patients Converted to Sirolimus.

    PubMed

    Mandelbrot, D A; Alberú, J; Barama, A; Marder, B A; Silva, H T; Flechner, S M; Flynn, A; Healy, C; Li, H; Tortorici, M A; Schulman, S L

    2015-12-01

    This prospective, randomized, double-blind, placebo-controlled study evaluated the effects of ramipril on urinary protein excretion in renal transplant patients treated with sirolimus following conversion from a calcineurin inhibitor. Patients received ramipril or placebo for up to 6 weeks before conversion and 52 weeks thereafter. Doses were increased if patients developed proteinuria (urinary protein/creatinine ratio ≥0.5); losartan was given as rescue therapy for persistent proteinuria. The primary end point was time to losartan initiation. Of 295 patients randomized, 264 met the criteria for sirolimus conversion (ramipril, 138; placebo, 126). At 52 weeks, the cumulative rate of losartan initiation was significantly lower with ramipril (6.2%) versus placebo (23.2%) (p < 0.001). No significant differences were observed between ramipril and placebo for change in glomerular filtration rate from baseline (p = 0.148) or in the number of patients with biopsy-confirmed acute rejection (13 vs. 5, respectively; p = 0.073). One patient in the placebo group died due to cerebrovascular accident. Treatment-emergent adverse events were consistent with the known safety profile of sirolimus and were not potentiated by ramipril co-administration. Ramipril was effective in reducing the incidence of proteinuria for up to 1 year following conversion to sirolimus in maintenance renal transplant patients.

  20. Selective inhibition of protein arginine methyltransferase 5 blocks initiation and maintenance of B-cell transformation

    PubMed Central

    Alinari, Lapo; Mahasenan, Kiran V.; Yan, Fengting; Karkhanis, Vrajesh; Chung, Ji-Hyun; Smith, Emily M.; Quinion, Carl; Smith, Porsha L.; Kim, Lisa; Patton, John T.; Lapalombella, Rosa; Yu, Bo; Wu, Yun; Roy, Satavisha; De Leo, Alessandra; Pileri, Stefano; Agostinelli, Claudio; Ayers, Leona; Bradner, James E.; Chen-Kiang, Selina; Elemento, Olivier; Motiwala, Tasneem; Majumder, Sarmila; Byrd, John C.; Jacob, Samson; Sif, Said; Li, Chenglong

    2015-01-01

    Epigenetic events that are essential drivers of lymphocyte transformation remain incompletely characterized. We used models of Epstein-Barr virus (EBV)–induced B-cell transformation to document the relevance of protein arginine methyltransferase 5 (PRMT5) to regulation of epigenetic-repressive marks during lymphomagenesis. EBV+ lymphomas and transformed cell lines exhibited abundant expression of PRMT5, a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues on histone tails. PRMT5 expression was limited to EBV-transformed cells, not resting or activated B lymphocytes, validating it as an ideal therapeutic target. We developed a first-in-class, small-molecule PRMT5 inhibitor that blocked EBV-driven B-lymphocyte transformation and survival while leaving normal B cells unaffected. Inhibition of PRMT5 led to lost recruitment of a PRMT5/p65/HDAC3-repressive complex on the miR96 promoter, restored miR96 expression, and PRMT5 downregulation. RNA-sequencing and chromatin immunoprecipitation experiments identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which became silenced during EBV-driven B-cell transformation. Enhanced PTPROt expression following PRMT5 inhibition led to dephosphorylation of kinases that regulate B-cell receptor signaling. We conclude that PRMT5 is critical to EBV-driven B-cell transformation and maintenance of the malignant phenotype, and that PRMT5 inhibition shows promise as a novel therapeutic approach for B-cell lymphomas. PMID:25742700

  1. FAD Regulates CRYPTOCHROME Protein Stability and Circadian Clock in Mice.

    PubMed

    Hirano, Arisa; Braas, Daniel; Fu, Ying-Hui; Ptáček, Louis J

    2017-04-11

    The circadian clock generates biological rhythms of metabolic and physiological processes, including the sleep-wake cycle. We previously identified a missense mutation in the flavin adenine dinucleotide (FAD) binding pocket of CRYPTOCHROME2 (CRY2), a clock protein that causes human advanced sleep phase. This prompted us to examine the role of FAD as a mediator of the clock and metabolism. FAD stabilized CRY proteins, leading to increased protein levels. In contrast, knockdown of Riboflavin kinase (Rfk), an FAD biosynthetic enzyme, enhanced CRY degradation. RFK protein levels and FAD concentrations oscillate in the nucleus, suggesting that they are subject to circadian control. Knockdown of Rfk combined with a riboflavin-deficient diet altered the CRY levels in mouse liver and the expression profiles of clock and clock-controlled genes (especially those related to metabolism including glucose homeostasis). We conclude that light-independent mechanisms of FAD regulate CRY and contribute to proper circadian oscillation of metabolic genes in mammals. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  2. Cerebral klotho protein as a humoral factor for maintenance of baroreflex.

    PubMed

    Chen, L-J; Cheng, M-F; Ku, P-M; Cheng, J-T

    2015-02-01

    The klotho protein produced by the choroid plexus is known as a humoral factor in central nervous system. Many hormones affecting the baroreflex sensitivity have been introduced in the brain. However, role of klotho in the baroreflex sensitivity is still unknown. Recently, mutations in the klotho gene have been linked to cardiovascular diseases in both animals and human subjects. Also, silencing of brain klotho has been reported to enhance cold-induced elevation of blood pressure. Thus, we investigated the role of klotho in maintenance of central cardiovascular reflex sensitivity. Male Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) were used. Either klotho shRNA or scramble shRNA was also ICV-infused into the brains of WKY rats to investigate the role of klotho in brain. Recombinant klotho or rat IgG was infused into the cerebral paraventricle (ICV) of SHRs for further understanding the role of klotho in hypertension. The baroreflex sensitivity was detected using the challenge with a depressor dose of sodium nitroprusside (SNP, 50 μg/kg) or with a pressor dose of phenylephrine (PE, 8 μg/kg). We found that silencing of klotho expression in the brain decreased the baroreflex sensitivity in WKY rats. Also, modulation of the blood pressure for one week altered the cardiovascular homeostasis and resulted in an increased expression of klotho in medulla oblongata. Moreover, the baroreflex sensitivity was restored in SHRs that received recombinant klotho through ICV brain. Thus, klotho is involved in the maintenance of baroreflex sensitivity in the brain.

  3. Stability and structure of protein-polysaccharide coacervates in the presence of protein aggregates.

    PubMed

    Sanchez, Christian; Renard, Denis

    2002-08-21

    We have studied at pH 4.2 and three protein (Pr):polysaccharide (Pol) weight ratios (8:1, 2:1 and 1:1) the structure and stability of beta-lactoglobulin/acacia gum/water dispersions containing protein aggregates (BLG/AG/W) or free from aggregates (AF-BLG/AG/W). Phase diagrams were characteristic of complex coacervation. BLG/AG/W dispersions displayed a larger biphasic area than AF-BLG/AG/W dispersions, that moved towards the protein axis. It was concluded that protein aggregates affected complex coacervation both by entropic (size and molecular masses of aggregates) and enthalpic (surface properties of aggregates) effects. Laser light scattering measurements revealed that the particles diameter (d(43)) induced by demixing was controlled by protein aggregates in AF-BLG/AG/W dispersions. At 1 wt.% biopolymer concentration, particles were 15-20 times larger in AF-BLG/AG/W dispersions than in BLG/AG/W dispersions at (Pr:Pol) ratios of 2:1 or 1:1. Confocal scanning laser microscopy showed that AF-BLG/AG/W dispersions only contained spherical coacervates. BLG/AG/W dispersions contained both coacervates and aggregates coated with AG or/and BLG/AG coacervates. At a (Pr:Pol) ratio of 2:1 and 1:1, coacervates were vesicular or multivesicular. Coacervates were smaller in BLG/AG/W dispersions than in AF-BLG/AG/W dispersions. It was concluded that protein aggregates have the intrinsic ability to stabilize complex coacervates and could be used to design multifunctional delivery systems. This study showed that composite dispersions containing both protein aggregates embedded in protein-polysaccharide coacervates and free coacervates may be performed. In this respect, the design of protein aggregates with controlled size distribution and surface properties could open new possibilities both in the non-chemical control of coacervates stability and in the development of multifunctional delivery systems.

  4. Protein attributes contribute to halo-stability, bioinformatics approach

    PubMed Central

    2011-01-01

    Halophile proteins can tolerate high salt concentrations. Understanding halophilicity features is the first step toward engineering halostable crops. To this end, we examined protein features contributing to the halo-toleration of halophilic organisms. We compared more than 850 features for halophilic and non-halophilic proteins with various screening, clustering, decision tree, and generalized rule induction models to search for patterns that code for halo-toleration. Up to 251 protein attributes selected by various attribute weighting algorithms as important features contribute to halo-stability; from them 14 attributes selected by 90% of models and the count of hydrogen gained the highest value (1.0) in 70% of attribute weighting models, showing the importance of this attribute in feature selection modeling. The other attributes mostly were the frequencies of di-peptides. No changes were found in the numbers of groups when K-Means and TwoStep clustering modeling were performed on datasets with or without feature selection filtering. Although the depths of induced trees were not high, the accuracies of trees were higher than 94% and the frequency of hydrophobic residues pointed as the most important feature to build trees. The performance evaluation of decision tree models had the same values and the best correctness percentage recorded with the Exhaustive CHAID and CHAID models. We did not find any significant difference in the percent of correctness, performance evaluation, and mean correctness of various decision tree models with or without feature selection. For the first time, we analyzed the performance of different screening, clustering, and decision tree algorithms for discriminating halophilic and non-halophilic proteins and the results showed that amino acid composition can be used to discriminate between halo-tolerant and halo-sensitive proteins. PMID:21592393

  5. Concise review: Fragile X proteins in stem cell maintenance and differentiation.

    PubMed

    Li, Yue; Zhao, Xinyu

    2014-07-01

    Fragile X syndrome (FXS), the most common genetic form of autism spectrum disorder, is caused by deficiency of the fragile X mental retardation protein (FMRP). Despite extensive research and scientific progress, understanding how FMRP regulates brain development and function remains a major challenge. FMRP is a neuronal RNA-binding protein that binds about a third of messenger RNAs in the brain and controls their translation, stability, and cellular localization. The absence of FMRP results in increased protein synthesis, leading to enhanced signaling in a number of intracellular pathways, including the mTOR, mGLuR5, ERK, Gsk3β, PI3K, and insulin pathways. Until recently, FXS was largely considered a deficit of mature neurons; however, a number of new studies have shown that FMRP may also play important roles in stem cells, among them neural stem cells, germline stem cells, and pluripotent stem cells. In this review, we will cover these newly discovered functions of FMRP, as well as the other two fragile X-related proteins, in stem cells. We will also discuss the literature on the use of stem cells, particularly neural stem cells and induced pluripotent stem cells, as model systems for studying the functions of FMRP in neuronal development.

  6. Maintenance of pluripotency in human embryonic stem cells stably over-expressing enhanced green fluorescent protein.

    PubMed

    Liu, Yi-Ping; Dovzhenko, Oksana V; Garthwaite, Mark A; Dambaeva, Svetlana V; Durning, Maureen; Pollastrini, Leah M; Golos, Thaddeus G

    2004-12-01

    The availability of human embryonic stem (HES) cells with a readily evaluated genetic marker such as green fluorescent protein (GFP) could facilitate a number of experimental opportunities. We constructed a novel plasmid with two elongation factor-1alpha (EF-1alpha) promoters (YPL2) to obtain a vector with mammalian promoters for simultaneous transgene expression in HES cells. An enhanced green fluorescent protein (EGFP) cDNA was inserted under the control of the first EF-1alpha promoter to construct plasmid YPL2-EGFP. The second EF1-alpha promoter was upstream of the neomycin resistance gene. H1 HES cells were transfected with YPL2-EGFP using Fugene 6. Following 100 microg/ml neomycin selection, individual colonies demonstrating stable EGFP expression were observed. After 4 months of passage under neomycin selection, the cells continued to maintain typical HES cell morphology. Undifferentiated cells showed no change in EGFP expression as determined by FACS analysis. Immunostaining demonstrated maintenance of Oct-3/4 expression in undifferentiated H1EGFP cells that was indistinguishable from wild-type HES cells. Addition of 10 ng/ml bone morphogenic protein-4 (BMP-4) to the cells provoked morphological and functional differentiation to trophoblasts, but no loss of EGFP expression. Following injection of EGFP-HES cells into immunodeficient mice, there was robust formation of teratomas that demonstrated a broad range of morphological pluripotency with widespread EGFP expression. EGFP expression was also maintained in differentiating embryoid bodies formed from EGFP-HES cells. This report demonstrates that ES cells carrying EGFP will be useful in diverse areas of embryonic stem cell research.

  7. Soy protein polymers: Enhancing the water stability property

    NASA Astrophysics Data System (ADS)

    Srinivasan, Gowrishankar

    Soy protein based plastics have been processed in the past by researchers for various short-term applications; however a common issue is the high water sensitivity of these plastics. This work concentrates on resolving this water sensitivity issue of soy protein polymers by employing chemical and mechanical interaction at the molecular level during extrusion. The primary chemical interactions employed were anhydride chemistries such as maleic anhydride (MA), phthalic anhydride (PTA), and butylated hydroxyanisole (BHA). These were respectively used in conjunction with glycerol as a plasticizer to produce relatively water stable soy protein based plastics. Formulations with varying additive levels of the chemistries were extruded and injection molded to form the samples for characterization. The additive levels of anhydrides were varied between 3-10% tw/tw (total mass). Results indicated that phthalic anhydride formulations resulted in highest water stability. Plastic formulations with concentration up to 10% phthalic anhydride were observed to have water absorption as low as 21.5% after 24 hrs of exposure to water with respect to 250% for the control formulation. Fourier transform infrared spectroscopy (FTIR) was utilized to characterize and confirm the fundamental mechanisms of water stability achieved by phthalic and maleic anhydride chemistries. In addition, the anhydride formulations were modified by inclusion of cotton fibers and pretreated cotton powder in order to improve mechanical properties. The incorporation of cotton fibers improved the dry strength by 18%, but did not significantly improve the wet state strength of the plastics. It was also observed that the butylated-hydroxy anisole (BHA) formulation exhibited high extension values in the dry state and had inferior water absorption properties in comparison with anhydride formulations.

  8. Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein.

    PubMed

    Rocha, Carolina S; Luz, Dirce F; Oliveira, Marli L; Baracat-Pereira, Maria C; Medrano, Francisco Javier; Fontes, Elizabeth P B

    2007-03-01

    The sucrose binding protein (SBP) belongs to the cupin family of proteins and is structurally related to vicilin-like storage proteins. In this investigation, a SBP isoform (GmSBP2/S64) was expressed in E. coli and large amounts of the protein accumulated in the insoluble fraction as inclusion bodies. The renatured protein was studied by circular dichroism (CD), intrinsic fluorescence, and binding of the hydrophobic probes ANS and Bis-ANS. The estimated content of secondary structure of the renatured protein was consistent with that obtained by theoretical modeling with a large predominance of beta-strand structure (42%) over the alpha-helix (9.9%). The fluorescence emission maximum of 303 nm for SBP2 indicated that the fluorescent tryptophan was completely buried within a highly hydrophobic environment. We also measured the equilibrium dissociation constant (K(d)) of sucrose binding by fluorescence titration using the refolded protein. The low sucrose binding affinity (K(d)=2.79+/-0.22 mM) of the renatured protein was similar to that of the native protein purified from soybean seeds. Collectively, these results indicate that the folded structure of the renatured protein was similar to the native SBP protein. As a member of the bicupin family of proteins, which includes highly stable seed storage proteins, SBP2 was fairly stable at high temperatures. Likewise, it remained folded to a similar extent in the presence or absence of 7.6M urea or 6.7 M GdmHCl. The high stability of the renatured protein may be a reminiscent property of SBP from its evolutionary relatedness to the seed storage proteins.

  9. Protein's native state stability in a chemically induced denaturation mechanism.

    PubMed

    Olivares-Quiroz, L; Garcia-Colin, L S

    2007-05-21

    In this work, we present a generalization of Zwanzig's protein unfolding analysis [Zwanzig, R., 1997. Two-state models of protein folding kinetics. Proc. Natl Acad. Sci. USA 94, 148-150; Zwanzig, R., 1995. Simple model of protein folding kinetics. Proc. Natl Acad. Sci. USA 92, 9801], in order to calculate the free energy change Delta(N)(D)F between the protein's native state N and its unfolded state D in a chemically induced denaturation. This Extended Zwanzig Model (EZM) is both based on an equilibrium statistical mechanics approach and the inclusion of experimental denaturation curves. It enables us to construct a suitable partition function Z and to derive an analytical formula for Delta(N)(D)F in terms of the number K of residues of the macromolecule, the average number nu of accessible states for each single amino acid and the concentration C(1/2) where the midpoint of the N<==>D transition occurs. The results of the EZM for proteins where chemical denaturation follows a sigmoidal-type profile, as it occurs for the case of the T70N human variant of lysozyme (PDB code: T70N) [Esposito, G., et al., 2003. J. Biol. Chem. 278, 25910-25918], can be splitted into two lines. First, EZM shows that for sigmoidal denaturation profiles, the internal degrees of freedom of the chain play an outstanding role in the stability of the native state. On the other hand, that under certain conditions DeltaF can be written as a quadratic polynomial on concentration C(1/2), i.e., DeltaF approximately aC(1/2)(2)+bC(1/2)+c, where a,b,c are constant coefficients directly linked to protein's size K and the averaged number of non-native conformations nu. Such functional form for DeltaF has been widely known to fit experimental measures in chemically induced protein denaturation [Yagi, M., et al., 2003. J. Biol. Chem. 278, 47009-47015; Asgeirsson, B., Guojonsdottir, K., 2006. Biochim. Biophys. Acta 1764, 190-198; Sharma, S., et al., 2006. Protein Pept. Lett. 13(4), 323-329; Salem, M., et

  10. beta-sitosterol decreases irradiation-induced thymocyte early damage by regulation of the intracellular redox balance and maintenance of mitochondrial membrane stability.

    PubMed

    Li, Chun Rong; Zhou, Zhe; Lin, Ru Xin; Zhu, Dan; Sun, Yu Ning; Tian, Lin Lin; Li, Lu; Gao, Yue; Wang, Sheng Qi

    2007-10-15

    Both radiation injury and oxidation toxicity occur when cells are exposed to ion irradiation (IR), ultimately leading to apoptosis. This study was designed to determine the effect of beta-sitosterol (BSS) on early cellular damage in irradiated thymocytes and a possible mechanism of effect on irradiation-mediated activation of the apoptotic pathways. Thymocytes were irradiated (6 Gy) with or without BSS. Cell apoptosis and apoptosis-related proteins were evaluated. BSS decreased irradiation-induced cell death and nuclear DNA strand breaks while attenuating intracellular reactive oxygen species (ROS) and increasing the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). BSS decreased the release of cytochrome c from mitochondria to the cytosol and the mitochondrio-nuclear translocation of apoptosis-inducing factor (AIF). Furthermore, BSS partially inhibited the radiation-induced increase of cleaved caspase 3 and cleaved PARP, and attenuated the activation of JNK and AP-1. In addition, evidence suggests that ROS generated by irradiation are involved in this course of cell damage. The results indicate that BSS confers a radioprotective effect on thymocytes by regulation of the intracellular redox balance which is carried out via the scavenging of ROS and maintenance of mitochondrial membrane stability.

  11. Minichromosome maintenance protein 7 regulates phagocytosis in kuruma shrimp Marsupenaeus japonicas against white spot syndrome virus.

    PubMed

    Wang, Zhi; Zhu, Fei

    2016-08-01

    Minichromosome maintenance protein (MCM7) belongs to the MCM protein family and participates in the MCM complex by playing a role in the cell replication cycle and chromosome initiation of eukaryotes. Previously, we found that several genes, including MCM7, were over-expressed in Drosophila melanogaster after white spot syndrome virus (WSSV) infection. In this study, we aimed to further research the MCM7 of kuruma shrimp, Marsupenaeus japonicus (mjMCM7) and determine its role in the innate immune system. To this end, we cloned the entire 2307-bp mjMCM7 sequence, including a 1974-bp open reading frame (ORF) encoding a 658-aa-long protein. Real-time PCR showed that the gene was primarily expressed in the hemolymph and hepatopancreas and over-expressed in shrimp challenged with WSSV. Gene function study was carried out by knocking down the expression of MCM7 using small interference RNA (siRNA). The results revealed that β-actin, hemocyanin, prophenoloxidase (proPO) and tumor necrosis factor-α (TNF-α) were up-regulated while the cytoskeleton proteins such as myosin and Rho were significantly down-regulated at 24 h after treatment. The results indicate a possible relationship between mjMCM7 and the innate immune system, and suggest that mjMCM7 may play a role in phagocytosis. After WSSV challenge, WSSV copies and mortality count were both higher in the MCM7-siRNA-treated groups at 60 h after treatment, and the mortality count approached that of the control groups over time. The phagocytosis rate was significantly lower in the MCM7-siRNA-treated group than in the WSSV group. The findings of this study confirm that mjMCM7 positively regulates phagocytosis and plays an important role against WSSV. These results could help researchers to further understand the function of the MCM7 protein and reveal its potential role in the innate immunity of invertebrates.

  12. Regulation of the p73 protein stability and degradation

    SciTech Connect

    Oberst, Andrew; Salomoni, Paolo; Pandolfi, Pier Paolo; Oren, Moshe; Melino, Gerry; Bernassola, Francesca . E-mail: bernasso@uniroma2.it

    2005-06-10

    p73, a homologue to the tumor suppressor gene p53, is involved in tumorigenesis, though its specific role remains unclear. The gene has two distinct promoters which allow the formation of two protein isoforms with opposite effects: full-length transactivating (TA) p73 shows pro-apoptotic effects, while the shorter {delta}Np73, which lacks the N-terminal transactivating domain, has an evident anti-apoptotic function. Unlike p53, the p73 gene is rarely mutated in human cancers. However, alterations in the relative levels of TA and {delta}Np73 have been shown to correlate with prognosis in several human cancers, suggesting that the fine regulation of these two isoforms is of pivotal importance in controlling proliferation and cell death. Much effort is currently focused on the elucidation of the mechanisms that differentially control TA and {delta}Np73 activity and protein stability, a process complicated by the finding that both proteins are regulated by a similar suite of complex post-translational modifications that include ubiquitination, sequential phosphorylation, prolyl-isomerization, recruitment into the PML-nuclear body (PML-NB), and acetylation. Here we shall consider the main regulatory partners of p73, with particular attention to the recently discovered Itch- and Nedd8-mediated degradation pathways, along with the emerging roles of PML, p38 MAP kinase, Pin1, and p300 in p73 transcriptional activation, and possible mechanisms for the differential regulation of the TAp73 and {delta}Np73 isoforms.

  13. Solubilizing and Stabilizing Proteins in Anhydrous Ionic Liquids through Formation of Protein-Polymer Surfactant Nanoconstructs.

    PubMed

    Brogan, Alex P S; Hallett, Jason P

    2016-04-06

    Nonaqueous biocatalysis is rapidly becoming a desirable tool for chemical and fuel synthesis in both the laboratory and industry. Similarly, ionic liquids are increasingly popular anhydrous reaction media for a number of industrial processes. Consequently, the use of enzymes in ionic liquids as efficient, environment-friendly, commercial biocatalysts is highly attractive. However, issues surrounding the poor solubility and low stability of enzymes in truly anhydrous media remain a significant challenge. Here, we demonstrate for the first time that engineering the surface of a protein to yield protein-polymer surfactant nanoconstructs allows for dissolution of dry protein into dry ionic liquids. Using myoglobin as a model protein, we show that this method can deliver protein molecules with near native structure into both hydrophilic and hydrophobic anhydrous ionic liquids. Remarkably, using temperature-dependent synchrotron radiation circular dichroism spectroscopy to measure half-denaturation temperatures, our results show that protein stability increases by 55 °C in the ionic liquid as compared to aqueous solution, pushing the solution thermal denaturation beyond the boiling point of water. Therefore, the work presented herein could provide a platform for the realization of biocatalysis at high temperatures or in anhydrous solvent systems.

  14. British Dietetic Association evidence-based guidelines for the protein requirements of adults undergoing maintenance haemodialysis or peritoneal dialysis.

    PubMed

    Naylor, H L; Jackson, H; Walker, G H; Macafee, S; Magee, K; Hooper, L; Stewart, L; MacLaughlin, H L

    2013-08-01

    Existing nutritional guidelines suggest that protein requirements of adults with stage five chronic kidney disease undergoing haemodialysis (HD) or peritoneal dialysis (PD) are increased as a result of protein losses during dialysis. The present review aimed to update previous guidance and develop evidence-based practice guidelines on the protein requirements of adults undergoing maintenance dialysis. Following a PICO approach (Participants or Population, Intervention or Exposure, Comparison and Outcome), four research questions were formulated to investigate the total protein requirement and protein quality required by adults undergoing HD and PD. A comprehensive, systematic review was undertaken using the databases Medline, EMBASE and the Cochrane Library from 2005 to September 2009 for HD studies and from 1997 to September 2009 for PD studies. The literature search yielded 2931 studies, which were assessed for inclusion. Following appraisal, 19 studies in HD and 18 studies in PD met the inclusion criteria and were systematically reviewed. Limited good quality evidence supports the recommendations that: (i) adults undergoing maintenance HD require a minimum protein intake of 1.1 g kg(-1) ideal body weight (IBW) per day; and (ii) adults undergoing maintenance PD require a minimum protein intake of 1.0-1.2 kg(-1) IBW per day, in conjunction with an adequate energy intake. There were no studies that addressed the quality of protein for either HD or PD. Evidence suggests that nutritional status may be maintained with lower protein intakes than previously recommended. However, the evidence base is limited and further randomised controlled trials are required to establish the optimal protein intake for dialysis patients. © 2013 The Authors Journal of Human Nutrition and Dietetics © 2013 The British Dietetic Association Ltd.

  15. The kinesin related motor protein, Eg5, is essential for maintenance of pre-implantation embryogenesis

    SciTech Connect

    Castillo, Andrew; Justice, Monica J. . E-mail: mjustice@bcm.tmc.edu

    2007-06-08

    Eg5 is a plus end directed kinesin related motor protein (KRP) previously shown to be involved in the assembly and maintenance of the mitotic spindle. KRPs are molecular motors capable of generating forces upon microtubules (MTs) in dividing cells and driving structural rearrangements necessary in the developing spindle. In vitro experiments demonstrate that loss of Eg5 results in cell cycle arrest and defective centrosome separation resulting in the development of monopolar spindles. Here we describe mice with a genetrap insertion in Eg5. Heterozygous mutant mice appear phenotypically normal. In contrast, embryos homozygous for the Eg5 null allele recovered at embryonic days 2.5-3.5 display signs of a proliferation defect as reduced cell numbers and failure of compaction and progression to the blastocyst stage was observed. These data, in conjunction with previous in vitro data, suggest that loss of Eg5 results in abnormal spindle structure, cell cycle arrest and thereby reduced cell proliferation of early cleavage pre-implantation embryos. These observations further support the conclusion that Eg5 is essential for cell division early in mouse development, and that maternal contribution may sustain the embryo through the maternal to zygotic transition at which point supplies of functional Eg5 are exhausted, preventing further cell cleavage.

  16. Checkpoint proteins influence telomeric silencing and length maintenance in budding yeast.

    PubMed Central

    Longhese, M P; Paciotti, V; Neecke, H; Lucchini, G

    2000-01-01

    A complex network of surveillance mechanisms, called checkpoints, interrupts cell cycle progression when damage to the genome is detected or when cells fail to complete DNA replication, thus ensuring genetic integrity. In budding yeast, components of the DNA damage checkpoint regulatory network include the RAD9, RAD17, RAD24, MEC3, DDC1, RAD53, and MEC1 genes that are proposed to be involved in different aspects of DNA metabolism. We provide evidence that some DNA damage checkpoint components play a role in maintaining telomere integrity. In fact, rad53 mutants specifically enhance repression of telomere-proximal transcription via the Sir-mediated pathway, suggesting that Rad53 might be required for proper chromatin structure at telomeres. Moreover, Rad53, Mec1, Ddc1, and Rad17 are necessary for telomere length maintenance, since mutations in all of these genes cause a decrease in telomere size. The telomeric shortening in rad53 and mec1 mutants is further enhanced in the absence of SIR genes, suggesting that Rad53/Mec1 and Sir proteins contribute to chromosome end protection by different pathways. The finding that telomere shortening, but not increased telomeric repression of gene expression in rad53 mutants, can be suppressed by increasing dNTP synthetic capacity in these strains suggests that transcriptional silencing and telomere integrity involve separable functions of Rad53. PMID:10924458

  17. Maintenance of Membrane Integrity and Permeability Depends on a Patched-Related Protein in Caenorhabditis elegans.

    PubMed

    Choi, Myung-Kyu; Son, Sangwon; Hong, Mingi; Choi, Min Sung; Kwon, Jae Young; Lee, Junho

    2016-04-01

    Membrane integrity is critical for cell survival, defects of which cause pathological symptoms such as metabolic diseases. In this study, we used ethanol sensitivity of the nematode Caenorhabditis elegans to identify genetic factors involved in membrane integrity. InC. elegans, acute exposure to a high concentration (7% v/v) of ethanol changes membrane permeability, as measured by propidium iodide staining, and causes paralysis. We used the timing of complete paralysis as an indicator for alteration of membrane integrity in our genetic screen, and identified ptr-6 as a gene that confers ethanol resistance when mutated. PTR-6 is a patched-related protein and contains a sterol sensing domain. Inhibition of two PTR-encoding genes,ptr-15 and ptr-23, and mboa-1, encoding an Acyl Co-A: cholesterol acyltransferase homolog, restored ethanol sensitivity of the ptr-6 mutant, suggesting that these ptr genes and mboa-1 are involved in the maintenance of membrane integrity and permeability. Our results suggest that C. elegans can be used as a model system to identify factors involved in metabolic diseases and to screen for therapeutic drugs. Copyright © 2016 by the Genetics Society of America.

  18. The protein kinase TOUSLED is required for maintenance of transcriptional gene silencing in Arabidopsis

    PubMed Central

    Wang, Yu; Liu, Jun; Xia, Ran; Wang, Junguo; Shen, Jie; Cao, Rui; Hong, Xuhui; Zhu, Jian-Kang; Gong, Zhizhong

    2007-01-01

    TOUSLED-like kinases (TLKs) are highly conserved in plants and animals, but direct evidence linking TLKs and transcriptional gene silencing is lacking. We isolated two new alleles of TOUSLED (TSL). Mutations of TSL in ros1 reactivate the transcriptionally silent 35S-NPTII transgene and the transcriptionally silent endogenous loci TSI (TRANSCRIPTIONAL SILENCING INFORMATION). Chromatin immunoprecipitation (ChIP) analysis shows that histone H3Lys9 dimethylation is decreased in the reactivated transgene and endogenous TSI loci in the tsl ros1 mutant. However, there is no change in DNA methylation in the affected loci. Western blot and ChIP assay suggest that TSL might not be responsible for histone H3Ser10 phosphorylation. The tsl seedlings were more sensitive to DNA damage reagent methyl methanesulphonate and UV-B light. Our results provide direct evidence for a crucial role of the TOUSLED protein kinase in the maintenance of transcriptional gene silencing in some genomic regions in a DNA-methylation-independent manner in Arabidopsis. PMID:17110953

  19. The protein kinase TOUSLED is required for maintenance of transcriptional gene silencing in Arabidopsis.

    PubMed

    Wang, Yu; Liu, Jun; Xia, Ran; Wang, Junguo; Shen, Jie; Cao, Rui; Hong, Xuhui; Zhu, Jian-Kang; Gong, Zhizhong

    2007-01-01

    TOUSLED-like kinases (TLKs) are highly conserved in plants and animals, but direct evidence linking TLKs and transcriptional gene silencing is lacking. We isolated two new alleles of TOUSLED (TSL). Mutations of TSL in ros1 reactivate the transcriptionally silent 35S-NPTII transgene and the transcriptionally silent endogenous loci TSI (TRANSCRIPTIONAL SILENCING INFORMATION). Chromatin immunoprecipitation (ChIP) analysis shows that histone H3Lys9 dimethylation is decreased in the reactivated transgene and endogenous TSI loci in the tsl ros1 mutant. However, there is no change in DNA methylation in the affected loci. Western blot and ChIP assay suggest that TSL might not be responsible for histone H3Ser10 phosphorylation. The tsl seedlings were more sensitive to DNA damage reagent methyl methanesulphonate and UV-B light. Our results provide direct evidence for a crucial role of the TOUSLED protein kinase in the maintenance of transcriptional gene silencing in some genomic regions in a DNA-methylation-independent manner in Arabidopsis.

  20. Role of Homer Proteins in the Maintenance of Sleep-Wake States

    PubMed Central

    Naidoo, Nirinjini; Ferber, Megan; Galante, Raymond J.; McShane, Blake; Hu, Jia Hua; Zimmerman, John; Maislin, Greg; Cater, Jacqui; Wyner, Abraham

    2012-01-01

    Sleep is an evolutionarily conserved process that is linked to diurnal cycles and normal daytime wakefulness. Healthy sleep and wakefulness are integral to a healthy lifestyle; this occurs when an organism is able to maintain long bouts of both sleep and wake. Homer proteins, which function as adaptors for group 1 metabotropic glutamate receptors, have been implicated in genetic studies of sleep in both Drosophila and mouse. Drosophila express a single Homer gene product that is upregulated during sleep. By contrast, vertebrates express Homer as both constitutive and immediate early gene (H1a) forms, and H1a is up-regulated during wakefulness. Genetic deletion of Homer in Drosophila results in fragmented sleep and in failure to sustain long bouts of sleep, even under increased sleep drive. However, deletion of Homer1a in mouse results in failure to sustain long bouts of wakefulness. Further evidence for the role of Homer1a in the maintenance of wake comes from the CREB alpha delta mutant mouse, which displays a reduced wake phenotype similar to the Homer1a knockout and fails to up-regulate Homer1a upon sleep loss. Homer1a is a gene whose expression is induced by CREB. Sustained behaviors of the sleep/wake cycle are created by molecular pathways that are distinct from those for arousal or short bouts, and implicate an evolutionarily-conserved role for Homer in sustaining these behaviors. PMID:22532843

  1. Distribution, Transition and Thermodynamic Stability of Protein Conformations in the Denaturant-Induced Unfolding of Proteins

    PubMed Central

    Bian, Liujiao; Ji, Xu

    2014-01-01

    Background Extensive and intensive studies on the unfolding of proteins require appropriate theoretical model and parameter to clearly illustrate the feature and characteristic of the unfolding system. Over the past several decades, four approaches have been proposed to describe the interaction between proteins and denaturants, but some ambiguity and deviations usually occur in the explanation of the experimental data. Methodology/Principal Findings In this work, a theoretical model was presented to show the dependency of the residual activity ratio of the proteins on the molar denaturant concentration. Through the characteristic unfolding parameters ki and Δmi in this model, the distribution, transition and thermodynamic stability of protein conformations during the unfolding process can be quantitatively described. This model was tested with the two-state unfolding of bovine heart cytochrome c and the three-state unfolding of hen egg white lysozyme induced by both guanidine hydrochloride and urea, the four-state unfolding of bovine carbonic anhydrase b induced by guanidine hydrochloride and the unfolding of some other proteins induced by denaturants. The results illustrated that this model could be used accurately to reveal the distribution and transition of protein conformations in the presence of different concentrations of denaturants and to evaluate the unfolding tendency and thermodynamic stability of different conformations. In most denaturant-induced unfolding of proteins, the unfolding became increasingly hard in next transition step and the proteins became more unstable as they attained next successive stable conformation. Conclusions/Significance This work presents a useful method for people to study the unfolding of proteins and may be used to describe the unfolding and refolding of other biopolymers induced by denaturants, inducers, etc. PMID:24603868

  2. Dual effects of Tween 80 on protein stability.

    PubMed

    Wang, Wei; Wang, Y John; Wang, D Q

    2008-01-22

    In this paper, we used IL-2 mutein as a model protein and evaluated the effect of Tween 80, a non-ionic surfactant. In summary, we found that the dual effects of Tween 80 on the stability of IL-2SA, such as that shaking-induced aggregation of IL-2 mutein was significantly inhibited in the presence of Tween 80. However, this surfactant adversely affected the stability of IL-2 mutein in solution during storage in terms of both oxidation and aggregation. These adverse effects are strongly temperature and formulation-dependent. Data particularly showed that IL-2 mutein in solution forms soluble aggregates to a different degree in different formulations during storage at 40 degrees C for 2 months. Aggregation was barely detectable during storage at 5 degrees C for 22 months. Addition of 0.1% Tween 80 significantly increased the rate of IL-2 mutein aggregation during storage. The IL-2 mutein aggregates are linked by both disulfide and non-disulfide bonds and their relative contribution is temperature-dependent. IL-2 mutein can be oxidized also to a different degree in different formulations during storage and the oxidation rate is strongly temperature-dependent with an activation energy between 21 and 25 kcal/mol. Addition of 0.1% Tween 80 not only increased the rate of oxidation in general but also altered the temperature-dependency of IL-2 mutein oxidation.

  3. Nuclear HMGA1 nonhistone chromatin proteins directly influence mitochondrial transcription, maintenance, and function

    SciTech Connect

    Dement, Gregory A.; Maloney, Scott C.; Reeves, Raymond . E-mail: reevesr@mail.wsu.edu

    2007-01-01

    We have previously demonstrated that HMGA1 proteins translocate from the nucleus to mitochondria and bind to mitochondrial DNA (mtDNA) at the D-loop control region [G.A. Dement, N.R. Treff, N.S. Magnuson, V. Franceschi, R. Reeves, Dynamic mitochondrial localization of nuclear transcription factor HMGA1, Exp. Cell Res. 307 (2005) 388-401.] [11]. To elucidate possible physiological roles for such binding, we employed methods to analyze mtDNA transcription, mitochondrial maintenance, and other organelle functions in transgenic human MCF-7 cells (HA7C) induced to over-express an HA-tagged HMGA1 protein and control (parental) MCF-7 cells. Quantitative real-time (RT) PCR analyses demonstrated that mtDNA levels were reduced approximately 2-fold in HMGA1 over-expressing HA7C cells and flow cytometric analyses further revealed that mitochondrial mass was significantly reduced in these cells. Cellular ATP levels were also reduced in HA7C cells and survival studies showed an increased sensitivity to killing by 2-deoxy-D-glucose, a glycolysis-specific inhibitor. Flow cytometric analyses revealed additional mitochondrial abnormalities in HA7C cells that are consistent with a cancerous phenotype: namely, increased reactive oxygen species (ROS) and increased mitochondrial membrane potential ({delta}{psi}{sub m}). Additional RT-PCR analyses demonstrated that gene transcripts from both the heavy (ND2, COXI, ATP6) and light (ND6) strands of mtDNA were up-regulated approximately 3-fold in HA7C cells. Together, these mitochondrial changes are consistent with many previous reports and reveal several possible mechanisms by which HMGA1 over-expression, a common feature of naturally occurring cancers, may affect tumor progression.

  4. Role of the polarity protein Scribble for podocyte differentiation and maintenance.

    PubMed

    Hartleben, Björn; Widmeier, Eugen; Wanner, Nicola; Schmidts, Miriam; Kim, Sung Tae; Schneider, Lisa; Mayer, Britta; Kerjaschki, Dontscho; Miner, Jeffrey H; Walz, Gerd; Huber, Tobias B

    2012-01-01

    The kidney filter represents a unique assembly of podocyte epithelial cells that tightly enwrap the glomerular capillaries with their complex foot process network. While deficiency of the polarity proteins Crumbs and aPKC result in impaired podocyte foot process architecture, the function of basolateral polarity proteins for podocyte differentiation and maintenance remained unclear. Here we report, that Scribble is expressed in developing podocytes, where it translocates from the lateral aspects of immature podocytes to the basal cell membrane and foot processes of mature podocytes. Immunogold electron microscopy reveals membrane associated localisation of Scribble predominantly at the basolateral site of foot processes. To further study the role of Scribble for podocyte differentiation Scribble(flox/flox) mice were generated by introducing loxP-sites into the Scribble introns 1 and 8 and these mice were crossed to NPHS2.Cre mice and Cre deleter mice. Podocyte-specific Scribble knockout mice develop normally and display no histological, ultrastructural or clinical abnormalities up to 12 months of age. In addition, no increased susceptibility to glomerular stress could be detected in these mice. In contrast, constitutive Scribble knockout animals die during embryonic development indicating the fundamental importance of Scribble for embryogenesis. Like in podocyte-specific Scribble knockout mice, the development of podocyte foot processes and the slit diaphragm was unaffected in kidney cultures from constitutive Scribble knockout animals. In summary these results indicate that basolateral polarity signaling via Scribble is dispensable for podocyte function, highlighting the unique feature of podocyte development with its significant apical membrane expansions being dominated by apical polarity complexes rather than by basolateral polarity signaling.

  5. Topoisomerase II-associated protein PAT1H1 is involved in the root stem cell niche maintenance in Arabidopsis thaliana.

    PubMed

    Yu, Qianqian; Liu, Jiajia; Zheng, Huihui; Jia, Yuebin; Tian, Huiyu; Ding, Zhaojun

    2016-06-01

    PAT1H1, one of the homologues of Topoisomerase II-associated protein, is involved in the maintenance of root stem cell niche through the interaction with NINJA. The root stem cell niche, which possesses four mitotically inactive quiescent cells (QC) and the surrounding mitotically active stem cells, is critical for root development in Arabidopsis thaliana. However, the molecular regulation of the maintenance of root stem cell niche identity is still not fully understood. Here we show that one of the homologues of Topoisomerase II-associated protein, here named as PAT1H1, could regulate root stem cell niche identity. The pat1h1 mutant showed higher frequency of QC cell division and root distal stem cell (DSC) differentiation. With a high expression in roots, PAT1H1 was found to interact with the jasmonic acid (JA) signalling negative regulator Novel Interactor of JAZ (NINJA) and thus regulate root DSC niche identity. Consistent with the active QC cell division, which rarely occurs in wild-type controls, the pat1h1 mutant displayed higher expression of CYCB1 in the root stem cell niche. Together our data reveals that PAT1H1 maintains root stem cell niche stability through the interaction with NINJA and the regulation of cell division.

  6. Conservation of Oxidative Protein Stabilization in an Insect Homologue of Parkinsonism-Associated Protein DJ-1

    SciTech Connect

    Lin, Jiusheng; Prahlad, Janani; Wilson, Mark A.

    2012-08-21

    DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1{alpha} and DJ-1{beta}) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1{beta}. The structure of D. melanogaster DJ-1{beta} is similar to that of human DJ-1, although two important residues in the human protein, Met26 and His126, are not conserved in DJ-1{beta}. His126 in human DJ-1 is substituted with a tyrosine in DJ-1{beta}, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO{sub 2{sup -}}) results in considerable thermal stabilization of both Drosophila DJ-1{beta} and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.

  7. Mathematics, Thermodynamics, and Modeling to Address Ten Common Misconceptions about Protein Structure, Folding, and Stability

    ERIC Educational Resources Information Center

    Robic, Srebrenka

    2010-01-01

    To fully understand the roles proteins play in cellular processes, students need to grasp complex ideas about protein structure, folding, and stability. Our current understanding of these topics is based on mathematical models and experimental data. However, protein structure, folding, and stability are often introduced as descriptive, qualitative…

  8. Mathematics, Thermodynamics, and Modeling to Address Ten Common Misconceptions about Protein Structure, Folding, and Stability

    ERIC Educational Resources Information Center

    Robic, Srebrenka

    2010-01-01

    To fully understand the roles proteins play in cellular processes, students need to grasp complex ideas about protein structure, folding, and stability. Our current understanding of these topics is based on mathematical models and experimental data. However, protein structure, folding, and stability are often introduced as descriptive, qualitative…

  9. Osmolytes stabilize ribonuclease S by stabilizing its fragments S protein and S peptide to compact folding-competent states.

    PubMed

    Ratnaparkhi, G S; Varadarajan, R

    2001-08-03

    Osmolytes stabilize proteins to thermal and chemical denaturation. We have studied the effects of the osmolytes sarcosine, betaine, trimethylamine-N-oxide, and taurine on the structure and stability of the protein.peptide complex RNase S using x-ray crystallography and titration calorimetry, respectively. The largest degree of stabilization is achieved with 6 m sarcosine, which increases the denaturation temperatures of RNase S and S pro by 24.6 and 17.4 degrees C, respectively, at pH 5 and protects both proteins against tryptic cleavage. Four crystal structures of RNase S in the presence of different osmolytes do not offer any evidence for osmolyte binding to the folded state of the protein or any perturbation in the water structure surrounding the protein. The degree of stabilization in 6 m sarcosine increases with temperature, ranging from -0.52 kcal mol(-1) at 20 degrees C to -5.4 kcal mol(-1) at 60 degrees C. The data support the thesis that osmolytes that stabilize proteins, do so by perturbing unfolded states, which change conformation to a compact, folding competent state in the presence of osmolyte. The increased stabilization thus results from a decrease in conformational entropy of the unfolded state.

  10. Stabilization of methionine-rich protein in Saccharomyces cerevisiae: targeting of BZN protein into the peroxisome.

    PubMed

    Nicaud, J M; Raynal, A; Beyou, A; Merkamm, M; Ito, H; Labat, N

    1994-01-01

    We have constructed a gene coding for the 12-kDa intermediate form of the 2s methionine-rich protein from Bertholletia excelsa seeds. This protein, expressed intracellularly in yeast, is characterised by a 20-min half-life. By adding 11 amino acids corresponding to the peroxisome-targeting sequence (PTSc) of luciferase, we have significantly increased its half-life. This stabilization allowed accumulation of the BZN protein into the peroxisome as judged by cell fractionation. Accumulation of the 12-kDa protein results in a significant increase of the total methionine content in yeast cells (30%) indicating that such a microorganism could represent a practicable protected shuttle for an animal-feed additive.

  11. Chromatin Association of Human Origin Recognition Complex, Cdc6, and Minichromosome Maintenance Proteins during the Cell Cycle: Assembly of Prereplication Complexes in Late Mitosis

    PubMed Central

    Méndez, Juan; Stillman, Bruce

    2000-01-01

    Evidence obtained from studies with yeast and Xenopus indicate that the initiation of DNA replication is a multistep process. The origin recognition complex (ORC), Cdc6p, and minichromosome maintenance (MCM) proteins are required for establishing prereplication complexes, upon which initiation is triggered by the activation of cyclin-dependent kinases and the Dbf4p-dependent kinase Cdc7p. The identification of human homologues of these replication proteins allows investigation of S-phase regulation in mammalian cells. Using centrifugal elutriation of several human cell lines, we demonstrate that whereas human Orc2 (hOrc2p) and hMcm proteins are present throughout the cell cycle, hCdc6p levels vary, being very low in early G1 and accumulating until cells enter mitosis. hCdc6p can be polyubiquitinated in vivo, and it is stabilized by proteasome inhibitors. Similar to the case for hOrc2p, a significant fraction of hCdc6p is present on chromatin throughout the cell cycle, whereas hMcm proteins alternate between soluble and chromatin-bound forms. Loading of hMcm proteins onto chromatin occurs in late mitosis concomitant with the destruction of cyclin B, indicating that the mitotic kinase activity inhibits prereplication complex formation in human cells. PMID:11046155

  12. Alpha-haemoglobin stabilizing protein (AHSP) stabilizes apo-α-haemoglobin in a partially folded state

    PubMed Central

    Krishna Kumar, Kaavya; Dickson, Claire F.; Weiss, Mitchell J.; Mackay, Joel P.; Gell, David A.

    2015-01-01

    SYNOPSIS To produce functional haemoglobin, nascent α-globin (αo) and β-globin (βo) chains must each bind a single haem molecule (to form αh and βh) and interact together to form heterodimers. The precise sequence of binding events is unknown, and it has been suggested that additional factors might enhance the efficiency of Hb folding. The α-haemoglobin stabilizing protein (AHSP) has previously been shown to bind αh and regulate redox activity of the haem iron. Here, we use a combination of classical and dynamic light scattering and NMR spectroscopy to demonstrate that AHSP forms a heterodimeric complex with αo that inhibits αo aggregation and promotes αo folding in the absence of haem. These findings indicate that AHSP may function as an αo-specific chaperone, and suggest an important role for αo in guiding Hb assembly by stabilizing βo and inhibiting off-pathway self-association of βh. PMID:20860551

  13. α-Hemoglobin stabilizing protein: a modulating factor in thalassemias?

    PubMed

    Wajcman, Henri; Vasseur, Corinne; Pissard, Serge; Baudin-Creuza, Veronique

    2011-01-01

    α-Hemoglobin stabilizing protein (AHSP) is a small protein of 102 residues induced by GATA-1, Oct-1- and EKLF. It is synthesized at a high level in the red blood cell precursors and acts as a chaperone protecting the α-hemoglobin (α-Hb) chains against precipitation. α-Hemoglobin stabilizing protein forms a heterodimer complex with α-Hb, then displaying modified oxygen binding kinetics. In the absence of AHSP, α-Hb oxidizes and precipitates within the erythrocyte precursors of bone marrow leading to apoptosis and defective erythropoiesis. Several α-Hb variants with a structural abnormality, frequently located in the contact area between α-Hb and AHSP, exhibit instability and a thalassemia-like syndrome when they are associated with another α-thalassemia (α-thal) determinant. We suggest that this disorder could result from a disturbed interaction between the abnormal α-Hb chains and AHSP. Hb Groene Hart (Pro119>Ser) was one of the first examples in which we observed this abnormality. We later verified this mechanism in a list of several variants, now considered as being nondeletional α-thalassemias. Conversely, it was hypothesized from studies on knock-out mice, that a defect affecting AHSP could cause a thalassemia-like syndrome. This was supported in man by studies showing that a decreased expression of AHSP linked to specific genetic clades could act as a modulating factor in some thalassemia phenotypes. It was also supported by our observation of a family from Southeast Asia, in which a child homozygous for an AHSP mutant (Val56>Gly) displayed, in his first year of life, a moderate thalassemia syndrome. This mutant AHSP was expressed in vitro and demonstrated by biochemical and biophysical studies to display a clear defective interaction with α-Hb, which could support the hypothesis that the reb blood cell (RBC) disorders of the child resulted from this abnormality. It therefore appears that AHSP is a factor with a key role in the formation of Hb

  14. Food proteins as novel nanosuspension stabilizers for poorly water-soluble drugs.

    PubMed

    He, Wei; Lu, Yi; Qi, Jianping; Chen, Lingyun; Hu, Fuqiang; Wu, Wei

    2013-01-30

    Nanonization of the poorly water-soluble drugs is a promising strategy to improve dissolution and oral bioavailability. To stabilize the drug nanosuspensions, stabilizers are usually used; however, the use of common stabilizers is limited by weak stabilization effect and toxicological concerns for long-term treatment. The present work was to investigate the potential of food proteins as novel safe stabilizers for nanosuspensions using indomethacin as a model drug. The nanosuspensions stabilized by food proteins (soybean protein isolate, whey protein isolate and β-lactoglobulin) were prepared by the nanoprecipitation-ultrasonication method. The particle size could be easily reduced to 100-400 nm with bimodal particle-size distribution through monitoring the preparative variables. The exposure of buried hydrophobic moieties due to heat-denaturation and subsequent adsorption onto the surface of drug particles was assumed to contribute to their efficient stabilization effect. In comparison with conventional stabilizers, food proteins are superior in stabilization efficiency. The dissolution was enhanced significantly owing to particle size reduction. The protein-stabilized nanosuspensions could be easily freeze-fried and reconstituted into nanosuspensions, keeping the original mean particle size and particle-size distribution. It is concluded that the three denatured proteins perform as efficient stabilizers for indomethacin nanosuspensions. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Stability constraints and protein evolution: the role of chain length, composition and disulfide bonds.

    PubMed

    Bastolla, U; Demetrius, Lloyd

    2005-09-01

    Stability of the native state is an essential requirement in protein evolution and design. Here we investigated the interplay between chain length and stability constraints using a simple model of protein folding and a statistical study of the Protein Data Bank. We distinguish two types of stability of the native state: with respect to the unfolded state (unfolding stability) and with respect to misfolded configurations (misfolding stability). Several contributions to stability are evaluated and their correlations are disentangled through principal components analysis, with the following main results. (1) We show that longer proteins can fulfil more easily the requirements of unfolding and misfolding stability, because they have a higher number of native interactions per residue. Consistently, in longer proteins native interactions are weaker and they are less optimized with respect to non-native interactions. (2) Stability against misfolding is negatively correlated with the strength of native interactions, which is related to hydrophobicity. Hence there is a trade-off between unfolding and misfolding stability. This trade-off is influenced by protein length: less hydrophobic sequences are observed in very long proteins. (3) The number of disulfide bonds is positively correlated with the deficit of free energy stabilizing the native state. Chain length and the number of disulfide bonds per residue are negatively correlated in proteins with short chains and uncorrelated in proteins with long chains. (4) The number of salt bridges per residue and per native contact increases with chain length. We interpret these observations as an indication that the constraints imposed by unfolding stability are less demanding in long proteins and they are further reduced by the competing requirement for stability against misfolding. In particular, disulfide bonds appear to be positively selected in short proteins, whereas they evolve in an effectively neutral way in long proteins.

  16. Effect of cosolvent on protein stability: A theoretical investigation

    SciTech Connect

    Chalikian, Tigran V.

    2014-12-14

    We developed a statistical thermodynamic algorithm for analyzing solvent-induced folding/unfolding transitions of proteins. The energetics of protein transitions is governed by the interplay between the cavity formation contribution and the term reflecting direct solute-cosolvent interactions. The latter is viewed as an exchange reaction in which the binding of a cosolvent to a solute is accompanied by release of waters of hydration to the bulk. Our model clearly differentiates between the stoichiometric and non-stoichiometric interactions of solvent or co-solvent molecules with a solute. We analyzed the urea- and glycine betaine (GB)-induced conformational transitions of model proteins of varying size which are geometrically approximated by a sphere in their native state and a spherocylinder in their unfolded state. The free energy of cavity formation and its changes accompanying protein transitions were computed based on the concepts of scaled particle theory. The free energy of direct solute-cosolvent interactions were analyzed using empirical parameters previously determined for urea and GB interactions with low molecular weight model compounds. Our computations correctly capture the mode of action of urea and GB and yield realistic numbers for (∂ΔG°/∂a{sub 3}){sub T,P} which are related to the m-values of protein denaturation. Urea is characterized by negative values of (∂ΔG°/∂a{sub 3}){sub T,P} within the entire range of urea concentrations analyzed. At concentrations below ∼1 M, GB exhibits positive values of (∂ΔG°/∂a{sub 3}){sub T,P} which turn positive at higher GB concentrations. The balance between the thermodynamic contributions of cavity formation and direct solute-cosolvent interactions that, ultimately, defines the mode of cosolvent action is extremely subtle. A 20% increase or decrease in the equilibrium constant for solute-cosolvent binding may change the sign of (∂ΔG°/∂a{sub 3}){sub T,P} thereby altering the mode of

  17. Effect of cosolvent on protein stability: A theoretical investigation

    NASA Astrophysics Data System (ADS)

    Chalikian, Tigran V.

    2014-12-01

    We developed a statistical thermodynamic algorithm for analyzing solvent-induced folding/unfolding transitions of proteins. The energetics of protein transitions is governed by the interplay between the cavity formation contribution and the term reflecting direct solute-cosolvent interactions. The latter is viewed as an exchange reaction in which the binding of a cosolvent to a solute is accompanied by release of waters of hydration to the bulk. Our model clearly differentiates between the stoichiometric and non-stoichiometric interactions of solvent or co-solvent molecules with a solute. We analyzed the urea- and glycine betaine (GB)-induced conformational transitions of model proteins of varying size which are geometrically approximated by a sphere in their native state and a spherocylinder in their unfolded state. The free energy of cavity formation and its changes accompanying protein transitions were computed based on the concepts of scaled particle theory. The free energy of direct solute-cosolvent interactions were analyzed using empirical parameters previously determined for urea and GB interactions with low molecular weight model compounds. Our computations correctly capture the mode of action of urea and GB and yield realistic numbers for (∂ΔG°/∂a3)T,P which are related to the m-values of protein denaturation. Urea is characterized by negative values of (∂ΔG°/∂a3)T,P within the entire range of urea concentrations analyzed. At concentrations below ˜1 M, GB exhibits positive values of (∂ΔG°/∂a3)T,P which turn positive at higher GB concentrations. The balance between the thermodynamic contributions of cavity formation and direct solute-cosolvent interactions that, ultimately, defines the mode of cosolvent action is extremely subtle. A 20% increase or decrease in the equilibrium constant for solute-cosolvent binding may change the sign of (∂ΔG°/∂a3)T,P thereby altering the mode of cosolvent action (stabilizing to destabilizing or vice

  18. Dissecting the role of protein-protein and protein-nucleic acid interactions in MS2 bacteriophage stability.

    PubMed

    Lima, Sheila M B; Vaz, Ana Carolina Q; Souza, Theo L F; Peabody, David S; Silva, Jerson L; Oliveira, Andréa C

    2006-04-01

    To investigate the role of protein-protein and protein-nucleic acid interactions in virus assembly, we compared the stabilities of native bacteriophage MS2, virus-like particles (VLPs) containing nonviral RNAs, and an assembly-defective coat protein mutant (dlFG) and its single-chain variant (sc-dlFG). Physical (high pressure) and chemical (urea and guanidine hydrochloride) agents were used to promote virus disassembly and protein denaturation, and the changes in virus and protein structure were monitored by measuring tryptophan intrinsic fluorescence, bis-ANS probe fluorescence, and light scattering. We found that VLPs dissociate into capsid proteins that remain folded and more stable than the proteins dissociated from authentic particles. The proposed model is that the capsid disassembles but the protein remains bound to the heterologous RNA encased by VLPs. The dlFG dimerizes correctly, but fails to assemble into capsids, because it lacks the 15-amino acid FG loop involved in inter-dimer interactions at the viral fivefold and quasi-sixfold axes. This protein was very unstable and, when compared with the dissociation/denaturation of the VLPs and the wild-type virus, it was much more susceptible to chemical and physical perturbation. Genetic fusion of the two subunits of the dimer in the single-chain dimer sc-dlFG stabilized the protein, as did the presence of 34-bp poly(GC) DNA. These studies reveal mechanisms by which interactions in the capsid lattice can be sufficiently stable and specific to ensure assembly, and they shed light on the processes that lead to the formation of infectious viral particles.

  19. StaRProtein, A Web Server for Prediction of the Stability of Repeat Proteins

    PubMed Central

    Xu, Yongtao; Zhou, Xu; Huang, Meilan

    2015-01-01

    Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins. PMID:25807112

  20. Reduced native state stability in crowded cellular environment due to protein-protein interactions

    PubMed Central

    Harada, Ryuhei; Tochio, Naoya; Kigawa, Takanori; Sugita, Yuji; Feig, Michael

    2013-01-01

    The effect of cellular crowding environments on protein structure and stability is a key issue in molecular and cellular biology. The classical view of crowding emphasizes the volume exclusion effect that generally favors compact, native states. Here, results from molecular dynamics simulations and NMR experiments show that protein crowders may destabilize native states via protein-protein interactions. In the model system considered here, mixtures of villin head piece and protein G at high concentrations, villin structures become increasingly destabilized upon increasing crowder concentrations. The denatured states observed in the simulation involve partial unfolding as well as more subtle conformational shifts. The unfolded states remain overall compact and only partially overlap with unfolded ensembles at high temperature and in the presence of urea. NMR measurements on the same systems confirm structural changes upon crowding based on changes of chemical shifts relative to dilute conditions. An analysis of protein-protein interactions and energetic aspects suggests the importance of enthalpic and solvation contributions to the crowding free energies that challenge an entropic-centered view of crowding effects. PMID:23402619

  1. Minichromosome maintenance 7 protein is a reliable biological marker for human cervical progressive disease

    PubMed Central

    Lobato, Soraya; Tafuri, Alexandre; Fernandes, Paula Ávila; Caliari, Marcelo Vidigal; Silva, Marcos Xavier; Xavier, Marcelo Antônio Pascoal

    2012-01-01

    Objective This study focused on comparing the expression levels of p16, Ki-67, and minichromosome maintenance 7 (MCM7) protein in normal and affected cervical epithelium to ascertain the biological significance of these markers in detecting progressive cervical disease. Methods A quantitative and based on-scanning-microscopy analysis of the three markers expression was performed in normal and cervical intraepithelial neoplasia (CIN) I, II, and III tissues. p16 area as well as p16, Ki-67, and MCM7 positive cells or nuclei were evaluated according to their distribution and extent through the cervical epithelium. Results A clear p16 over-expression was observed in all the dysplastic epithelium tissue samples. The quantitative analysis of p16 area as well as the number of p16 positive cells was able to better discriminate the CIN lesions grades than the usual semi-quantitative analysis. The average Ki-67 labeling indexes for the normal epithelium, CIN I, CIN II, and CIN III groups were 19.8%, 27.3%, 32.8%, and 37.1%, respectively, whereas the mean MCM7 labeling indexes for the correspondent grades were 27.0%, 30.4%, 50.5%, and 67.2%. The Ki-67 and MCM7 labeling indexes were closely correlated with the CIN histological grade, with higher labeling indexe values obtained from the more severe lesions (p<0.05), being the MCM7 labeling indexes the highest values in all the CIN categories (p<0.05). Conclusion We observed a good correlation among the p16, Ki-67, and MCM7 data. In addition, MCM7 demonstrated to be a more efficient and sensitive marker to assess disease progression in the uterine cervix. PMID:22355461

  2. Regulation of myocardin factor protein stability by the LIM-only protein FHL2

    PubMed Central

    Hinson, Jeremiah S.; Medlin, Matt D.; Taylor, Joan M.; Mack, Christopher P.

    2008-01-01

    Extensive evidence indicates that serum response factor (SRF) regulates muscle-specific gene expression and that myocardin family SRF cofactors are critical for smooth muscle cell differentiation. In a yeast two hybrid screen for novel SRF binding partners expressed in aortic SMC, we identified four and a half LIM domain protein 2 (FHL2) and confirmed this interaction by GST pull-down and coimmunoprecipitation assays. FHL2 also interacted with all three myocardin factors and enhanced myocardin and myocardin-related transcription factor (MRTF)-A-dependent transactivation of smooth muscle α-actin, SM22, and cardiac atrial natriuretic factor promoters in 10T1/2 cells. The expression of FHL2 increased myocardin and MRTF-A protein levels, and, importantly, this effect was due to an increase in protein stability not due to an increase in myocardin factor mRNA expression. Treatment of cells with proteasome inhibitors MG-132 and lactacystin strongly upregulated endogenous MRTF-A protein levels and resulted in a substantial increase in ubiquitin immunoreactivity in MRTF-A immunoprecipitants. Interestingly, the expression of FHL2 attenuated the effects of RhoA and MRTF-B on promoter activity, perhaps through decreased MRTF-B nuclear localization or decreased SRF-CArG binding. Taken together, these data indicate that myocardin factors are regulated by proteasome-mediated degradation and that FHL2 regulates SRF-dependent transcription by multiple mechanisms, including stabilization of myocardin and MRTF-A. PMID:18586895

  3. Small-Molecule Stabilization of 14-3-3 Protein-Protein Interactions Stimulates Axon Regeneration.

    PubMed

    Kaplan, Andrew; Morquette, Barbara; Kroner, Antje; Leong, SooYuen; Madwar, Carolin; Sanz, Ricardo; Banerjee, Sara L; Antel, Jack; Bisson, Nicolas; David, Samuel; Fournier, Alyson E

    2017-03-08

    Damaged central nervous system (CNS) neurons have a poor ability to spontaneously regenerate, causing persistent functional deficits after injury. Therapies that stimulate axon growth are needed to repair CNS damage. 14-3-3 adaptors are hub proteins that are attractive targets to manipulate cell signaling. We identify a positive role for 14-3-3s in axon growth and uncover a developmental regulation of the phosphorylation and function of 14-3-3s. We show that fusicoccin-A (FC-A), a small-molecule stabilizer of 14-3-3 protein-protein interactions, stimulates axon growth in vitro and regeneration in vivo. We show that FC-A stabilizes a complex between 14-3-3 and the stress response regulator GCN1, inducing GCN1 turnover and neurite outgrowth. These findings show that 14-3-3 adaptor protein complexes are druggable targets and identify a new class of small molecules that may be further optimized for the repair of CNS damage.

  4. Purification and characterization of ensconsin, a novel microtubule stabilizing protein.

    PubMed

    Bulinski, J C; Bossler, A

    1994-10-01

    In previous studies (Bulinski and Borisy (1979). Proc. Nat. Acad. Sci. 76, 293-297; Weatherbee et al. (1980). Biochemistry 19, 4116-4123) a microtubule-associated protein (MAP) of M(r) approximately 125,000 was identified as a prominent MAP in HeLa cells. We set out to perform a biochemical characterization of this protein, and to determine its in vitro functions and in vivo distribution. We determined that, like the assembly-promoting MAPs, tau, MAP2 and MAP4, the 125 kDa MAP was both proteolytically sensitive and thermostable. An additional property of this MAP; namely, its unusually tight association with a calcium-insensitive population of MTs in the presence of taxol, was exploited in devising an efficient purification strategy. Because of the MAP's tenacious association with a stable population of MTs, and because it appeared to contribute to the stability of this population of MTs in vitro, we have named this protein ensconsin. We examined the binding of purified ensconsin to MTs; ensconsin exhibited binding that saturated its MT binding sites at an approximate molar ratio of 1:6 (ensconsin:tubulin). Unlike other MAPs characterized to date, ensconsin's binding to MTs was insensitive to moderate salt concentrations (< or = 0.6 M). We further characterized ensconsin in immunoblotting experiments using mouse polyclonal anti-ensconsin antibodies and antibodies reactive with previously described MAPs, such as high molecular mass tau isoforms, dynamin, STOP, CLIP-170 and kinesin. These experiments demonstrated that ensconsin is distinct from other proteins of similar M(r) that may be present in association with MTs. Immunofluorescence with anti-ensconsin antibodies demonstrated that ensconsin was detectable in association with most or all of the MTs of several lines of human epithelial, fibroblastic and muscle cells; its in vivo properties and distribution, especially in response to drug or other treatments of cells, were found to be different from those of MAP4

  5. Stability and Immunogenicity of Hypoallergenic Peanut Protein-Polyphenol Complexes During In Vitro Pepsin Digestion

    USDA-ARS?s Scientific Manuscript database

    Allergenic peanut proteins are relatively resistant to digestion, and if digested, metabolized peptides tend to remain large and immunoreactive, triggering allergic reactions in sensitive individuals. In this study, the stability of hypoallergenic peanut protein-polyphenol complexes was evaluated d...

  6. Diagnosis of bladder cancer by immunocytochemical detection of minichromosome maintenance protein-2 in cells retrieved from urine.

    PubMed

    Saeb-Parsy, K; Wilson, A; Scarpini, C; Corcoran, M; Chilcott, S; McKean, M; Thottakam, B; Rai, B; Nabi, G; Rana, D; Perera, M; Stewart, K; Laskey, R A; Neal, D E; Coleman, N

    2012-10-09

    We tested the accuracy of immunocytochemistry (ICC) for minichromosome maintenance protein-2 (MCM-2) in diagnosing bladder cancer, using cells retrieved from urine. Adequate samples were obtained from 497 patients, the majority presenting with gross haematuria (GH) or undergoing cystoscopic surveillance (CS) following previous bladder cancer. We performed an initial study of 313 patients, followed by a validation study of 184 patients. In all cases, presence/absence of bladder cancer was established by cystoscopy/biopsy. In the initial study, receiver operator characteristic analysis showed an area under the curve of 0.820 (P<0.0005) for the GH group and 0.821 (P<0.01) for the CS group. Optimal sensitivity/specificity were provided by threshold values of 50+ MCM-2-positive cells in GH samples and 200+ cells in CS samples, based on a minimum total cell number of 5000. Applying these thresholds to the validation data set gave 81.3% sensitivity, 76.0% specificity and 92.7% negative predictive value (NPV) in GH and 63.2% sensitivity, 89.9% specificity and 89.9% NPV in CS. Minichromosome maintenance protein-2 ICC provided clinically relevant improvements over urine cytology, with greater sensitivity in GH and greater specificity in CS (P=0.05). Minichromosome maintenance protein-2 ICC is a reproducible and accurate test that is suitable for both GH and CS patient groups.

  7. Genetic selection designed to stabilize proteins uncovers a chaperone called Spy.

    PubMed

    Quan, Shu; Koldewey, Philipp; Tapley, Tim; Kirsch, Nadine; Ruane, Karen M; Pfizenmaier, Jennifer; Shi, Rong; Hofmann, Stephan; Foit, Linda; Ren, Guoping; Jakob, Ursula; Xu, Zhaohui; Cygler, Miroslaw; Bardwell, James C A

    2011-03-01

    To optimize the in vivo folding of proteins, we linked protein stability to antibiotic resistance, thereby forcing bacteria to effectively fold and stabilize proteins. When we challenged Escherichia coli to stabilize a very unstable periplasmic protein, it massively overproduced a periplasmic protein called Spy, which increases the steady-state levels of a set of unstable protein mutants up to 700-fold. In vitro studies demonstrate that the Spy protein is an effective ATP-independent chaperone that suppresses protein aggregation and aids protein refolding. Our strategy opens up new routes for chaperone discovery and the custom tailoring of the in vivo folding environment. Spy forms thin, apparently flexible cradle-shaped dimers. The structure of Spy is unlike that of any previously solved chaperone, making it the prototypical member of a new class of small chaperones that facilitate protein refolding in the absence of energy cofactors.

  8. Genetic selection designed to stabilize proteins uncovers a chaperone called Spy

    PubMed Central

    Quan, Shu; Koldewey, Philipp; Tapley, Tim; Kirsch, Nadine; Ruane, Karen M.; Pfizenmaier, Jennifer; Shi, Rong; Hofmann, Stephan; Foit, Linda; Ren, Guoping; Jakob, Ursula; Xu, Zhaohui; Cygler, Miroslaw; Bardwell, James C. A.

    2011-01-01

    To optimize the in vivo folding of proteins, we linked protein stability to antibiotic resistance, thereby forcing bacteria to effectively fold and stabilize proteins. When we challenged Escherichia coli to stabilize a very unstable periplasmic protein, it massively overproduced a periplasmic protein called Spy, which increases the steady-state levels of a set of unstable protein mutants up to 700-fold. In vitro studies demonstrate that the Spy protein is an effective ATP-independent chaperone that suppresses protein aggregation and aids protein refolding. Our strategy opens up new routes for chaperone discovery and the custom tailoring of the in vivo folding environment. Spy forms thin, apparently flexible cradle-shaped dimers. Spy is unlike the structure of any previously solved chaperone, making it the prototypical member of a new class of small chaperones that facilitate protein refolding in the absence of energy cofactors. PMID:21317898

  9. Telomere binding of checkpoint sensor and DNA repair proteins contributes to maintenance of functional fission yeast telomeres.

    PubMed Central

    Nakamura, Toru M; Moser, Bettina A; Russell, Paul

    2002-01-01

    Telomeres, the ends of linear chromosomes, are DNA double-strand ends that do not trigger a cell cycle arrest and yet require checkpoint and DNA repair proteins for maintenance. Genetic and biochemical studies in the fission yeast Schizosaccharomyces pombe were undertaken to understand how checkpoint and DNA repair proteins contribute to telomere maintenance. On the basis of telomere lengths of mutant combinations of various checkpoint-related proteins (Rad1, Rad3, Rad9, Rad17, Rad26, Hus1, Crb2, Chk1, Cds1), Tel1, a telomere-binding protein (Taz1), and DNA repair proteins (Ku70, Rad32), we conclude that Rad3/Rad26 and Tel1/Rad32 represent two pathways required to maintain telomeres and prevent chromosome circularization. Rad1/Rad9/Hus1/Rad17 and Ku70 are two additional epistasis groups, which act in the Rad3/Rad26 pathway. However, Rad3/Rad26 must have additional target(s), as cells lacking Tel1/Rad32, Rad1/Rad9/Hus1/Rad17, and Ku70 groups did not circularize chromosomes. Cells lacking Rad3/Rad26 and Tel1/Rad32 senesced faster than a telomerase trt1Delta mutant, suggesting that these pathways may contribute to telomere protection. Deletion of taz1 did not suppress chromosome circularization in cells lacking Rad3/Rad26 and Tel1/Rad32, also suggesting that two pathways protect telomeres. Chromatin immunoprecipitation analyses found that Rad3, Rad1, Rad9, Hus1, Rad17, Rad32, and Ku70 associate with telomeres. Thus, checkpoint sensor and DNA repair proteins contribute to telomere maintenance and protection through their association with telomeres. PMID:12196391

  10. Computational tools help improve protein stability but with a solubility tradeoff.

    PubMed

    Broom, Aron; Jacobi, Zachary; Trainor, Kyle; Meiering, Elizabeth M

    2017-09-01

    Accurately predicting changes in protein stability upon amino acid substitution is a much sought after goal. Destabilizing mutations are often implicated in disease, whereas stabilizing mutations are of great value for industrial and therapeutic biotechnology. Increasing protein stability is an especially challenging task, with random substitution yielding stabilizing mutations in only ∼2% of cases. To overcome this bottleneck, computational tools that aim to predict the effect of mutations have been developed; however, achieving accuracy and consistency remains challenging. Here, we combined 11 freely available tools into a meta-predictor (meieringlab.uwaterloo.ca/stabilitypredict/). Validation against ∼600 experimental mutations indicated that our meta-predictor has improved performance over any of the individual tools. The meta-predictor was then used to recommend 10 mutations in a previously designed protein of moderate thermodynamic stability, ThreeFoil. Experimental characterization showed that four mutations increased protein stability and could be amplified through ThreeFoil's structural symmetry to yield several multiple mutants with >2-kcal/mol stabilization. By avoiding residues within functional ties, we could maintain ThreeFoil's glycan-binding capacity. Despite successfully achieving substantial stabilization, however, almost all mutations decreased protein solubility, the most common cause of protein design failure. Examination of the 600-mutation data set revealed that stabilizing mutations on the protein surface tend to increase hydrophobicity and that the individual tools favor this approach to gain stability. Thus, whereas currently available tools can increase protein stability and combining them into a meta-predictor yields enhanced reliability, improvements to the potentials/force fields underlying these tools are needed to avoid gaining protein stability at the cost of solubility. © 2017 by The American Society for Biochemistry and

  11. Identification of Multiple Proteins Coupling Transcriptional Gene Silencing to Genome Stability in Arabidopsis thaliana.

    PubMed

    Hale, Christopher J; Potok, Magdalena E; Lopez, Jennifer; Do, Truman; Liu, Ao; Gallego-Bartolome, Javier; Michaels, Scott D; Jacobsen, Steven E

    2016-06-01

    Eukaryotic genomes are regulated by epigenetic marks that act to modulate transcriptional control as well as to regulate DNA replication and repair. In Arabidopsis thaliana, mutation of the ATXR5 and ATXR6 histone methyltransferases causes reduction in histone H3 lysine 27 monomethylation, transcriptional upregulation of transposons, and a genome instability defect in which there is an accumulation of excess DNA corresponding to pericentromeric heterochromatin. We designed a forward genetic screen to identify suppressors of the atxr5/6 phenotype that uncovered loss-of-function mutations in two components of the TREX-2 complex (AtTHP1, AtSAC3B), a SUMO-interacting E3 ubiquitin ligase (AtSTUbL2) and a methyl-binding domain protein (AtMBD9). Additionally, using a reverse genetic approach, we show that a mutation in a plant homolog of the tumor suppressor gene BRCA1 enhances the atxr5/6 phenotype. Through characterization of these mutations, our results suggest models for the production atxr5 atxr6-induced extra DNA involving conflicts between the replicative and transcriptional processes in the cell, and suggest that the atxr5 atxr6 transcriptional defects may be the cause of the genome instability defects in the mutants. These findings highlight the critical intersection of transcriptional silencing and DNA replication in the maintenance of genome stability of heterochromatin.

  12. Ultra-High Pressure Homogenization improves oxidative stability and interfacial properties of soy protein isolate-stabilized emulsions.

    PubMed

    Fernandez-Avila, C; Trujillo, A J

    2016-10-15

    Ultra-High Pressure Homogenization (100-300MPa) has great potential for technological, microbiological and nutritional aspects of fluid processing. Its effect on the oxidative stability and interfacial properties of oil-in-water emulsions prepared with 4% (w/v) of soy protein isolate and soybean oil (10 and 20%, v/v) were studied and compared to emulsions treated by conventional homogenization (15MPa). Emulsions were characterized by particle size, emulsifying activity index, surface protein concentration at the interface and by transmission electron microscopy. Primary and secondary lipid oxidation products were evaluated in emulsions upon storage. Emulsions with 20% oil treated at 100 and 200MPa exhibited the most oxidative stability due to higher amount of oil and protein surface load at the interface. This manuscript addresses the improvement in oxidative stability in emulsions treated by UHPH when compared to conventional emulsions.

  13. Using state diagrams for predicting colloidal stability of whey protein beverages.

    PubMed

    Wagoner, Ty B; Ward, Loren; Foegeding, E Allen

    2015-05-06

    A method for evaluating aspects of colloidal stability of whey protein beverages after thermal treatment was established. Three state diagrams for beverages (pH 3-7) were developed representing protein solubility, turbidity, and macroscopic state after two ultrahigh-temperature (UHT) treatments. Key transitions of stability in the state diagrams were explored using electrophoresis and chromatography to determine aggregation propensities of β-lactoglobulin, α-lactalbumin, bovine serum albumin, and glycomacropeptide. The state diagrams present an overlapping view of high colloidal stability at pH 3 accompanied by high solubility of individual whey proteins. At pH 5, beverages were characterized by poor solubility, high turbidity, and aggregation/gelation of whey proteins with the exception of glycomacropeptide. Stability increased at pH 6, due to increased solubility of α-lactalbumin. The results indicate that combinations of state diagrams can be used to identify key regions of stability for whey protein containing beverages.

  14. β-Catenin Accumulation Is Associated With Increased Expression of Nanog Protein and Predicts Maintenance of MSC Self-Renewal.

    PubMed

    Yu, Sang-Jin; Kim, Hyun-Je; Lee, Eui Seok; Park, Chung-Gyu; Cho, Su Jin; Jeon, Soung-Hoo

    2017-02-16

    Human mesenchymal stem cells (hMSCs) are self-renewing cells with the ability to differentiate into organized, functional network of cells. Recent studies have revealed that activation of the Wnt/β-catenin pathway by a glycogen synthase kinase (GSK)-3-specific pharmacological inhibitor, Bio, results in the maintenance of self-renewal in both mouse and human ES cells. The molecular mechanism behind the maintenance of hMSCs by these factors, however, is not fully understood. We found that rEGF enhances the level of β-catenin, a component of the Wnt/β-catenin signaling pathway. Furthermore, it was found that β-catenin upregulates Nanog. EGF activates the β-catenin pathway via the Ras protein and also increased the Nanog protein and gene expression levels 2 h after rEGF treatment. These results suggest that adding EGF can enhance β-catenin and Nanog expression in MSCs and facilitate EGF-mediated maintenance of MSC self-renewal. EGF was shown to augment MSC proliferation while preserving early progenitors within MSC population and thus did not induce differentiation. Thus, EGF not only can be used to expand MSC in vitro but also be utilized to autologous transplantation of MSCs in vivo.

  15. Involvement of protein kinase ζ in the maintenance of hippocampal long-term potentiation in rats with chronic visceral hypersensitivity.

    PubMed

    Chen, Aiqin; Bao, Chengjia; Tang, Ying; Luo, Xiaoqing; Guo, Lixia; Liu, Bin; Lin, Chun

    2015-05-01

    The hippocampal long-term potentiation (LTP) was implicated in the formation of visceral hypersensitivity in rats with irritable bowel syndrome in our previous study. Recent studies have shown that protein kinase M ζ (PKMζ) may be responsible for the maintenance of LTP in memory formation. However, it remains unclear whether PKMζ is involved in the visceral hypersensitivity. In this study, a rat model of visceral hypersensitivity was generated by neonatal maternal separation (NMS). The visceral hypersensitivity was assessed by recording responses of the external oblique abdominal muscle to colorectal distension. Our results demonstrated that hippocampal LTP and visceral hypersensitivity were enhanced significantly in rats of NMS. ζ-Pseudosubstrate inhibitory peptide (ZIP) could dose dependently inhibit the maintenance of Cornu Ammonis area 1 LTP in rats of NMS. Furthermore, Western blot data showed that the expression of hippocampal phosphorylated PKMζ (p-PKMζ) significantly increased in rats of NMS. In addition, bilateral intrahippocampal injections of ZIP attenuated the visceral hypersensitivity dose dependently in rats of NMS. The maximal inhibition was observed at 30 min, and significant inhibition lasted for 1.5-2 h after ZIP application. Besides, data from the open-field test and Morris water maze showed that ZIP did not influence the movement and spatial procedural memory in rats of NMS. In conclusion, p-PKMζ might be a critical protein in the maintenance of hippocampal LTP, which could result in visceral hypersensitivity.

  16. Involvement of protein kinase ζ in the maintenance of hippocampal long-term potentiation in rats with chronic visceral hypersensitivity

    PubMed Central

    Chen, Aiqin; Bao, Chengjia; Tang, Ying; Luo, Xiaoqing; Guo, Lixia; Liu, Bin

    2015-01-01

    The hippocampal long-term potentiation (LTP) was implicated in the formation of visceral hypersensitivity in rats with irritable bowel syndrome in our previous study. Recent studies have shown that protein kinase M ζ (PKMζ) may be responsible for the maintenance of LTP in memory formation. However, it remains unclear whether PKMζ is involved in the visceral hypersensitivity. In this study, a rat model of visceral hypersensitivity was generated by neonatal maternal separation (NMS). The visceral hypersensitivity was assessed by recording responses of the external oblique abdominal muscle to colorectal distension. Our results demonstrated that hippocampal LTP and visceral hypersensitivity were enhanced significantly in rats of NMS. ζ-Pseudosubstrate inhibitory peptide (ZIP) could dose dependently inhibit the maintenance of Cornu Ammonis area 1 LTP in rats of NMS. Furthermore, Western blot data showed that the expression of hippocampal phosphorylated PKMζ (p-PKMζ) significantly increased in rats of NMS. In addition, bilateral intrahippocampal injections of ZIP attenuated the visceral hypersensitivity dose dependently in rats of NMS. The maximal inhibition was observed at 30 min, and significant inhibition lasted for 1.5–2 h after ZIP application. Besides, data from the open-field test and Morris water maze showed that ZIP did not influence the movement and spatial procedural memory in rats of NMS. In conclusion, p-PKMζ might be a critical protein in the maintenance of hippocampal LTP, which could result in visceral hypersensitivity. PMID:25761958

  17. Importance of mutant position in Ramachandran plot for predicting protein stability of surface mutations.

    PubMed

    Gromiha, M Michael; Oobatake, Motohisa; Kono, Hidetoshi; Uedaira, Hatsuho; Sarai, Akinori

    2002-08-05

    Understanding the mechanisms by which mutations affect protein stability is one of the most important problems in molecular biology. In this work, we analyzed the relationship between changes in protein stability caused by surface mutations and changes in 49 physicochemical, energetic, and conformational properties of amino acid residues. We found that the hydration entropy was the major contributor to the stability of surface mutations in helical segments; other properties responsible for size and volume of molecule also correlated significantly with stability. Classification of coil mutations based on their locations in the (phi-psi) map improved the correlation significantly, demonstrating the existence of a relationship between stability and strain energy, which indicates that the role of strain energy is very important for the stability of surface mutations. We observed that the inclusion of sequence and structural information raised the correlation, indicating the influence of surrounding residues on the stability of surface mutations. Further, we examined the previously reported "inverse relationship" between stability and hydrophobicity, and observed that the inverse hydrophobic effect was generally applicable only to coil mutations. The present study leads to a simple method for predicting protein stability changes caused by amino acid substitutions, which will be useful for protein engineering in designing novel proteins with increased stability and altered function.

  18. Comparison of the colloidal stability, bioaccessibility and antioxidant activity of corn protein hydrolysate and sodium caseinate stabilized curcumin nanoparticles.

    PubMed

    Wang, Yong-Hui; Yuan, Yang; Yang, Xiao-Quan; Wang, Jin-Mei; Guo, Jian; Lin, Yuan

    2016-07-01

    The aims of this work were to construct corn protein hydrolysate (CPH)-based curcumin nanoparticles (Cur NPs) and to compare the colloidal stability, bioaccessibility and antioxidant activity of the Cur NPs stabilized CPH and sodium caseinate (NaCas) respectively. The results indicated that Cur solubility could be considerably improved after the Cur NPs fabrication. The spectroscopy results demonstrated that the solubilization of Cur should be attributed to its complexation with CPH or NaCas. The Cur NPs exhibited good colloidal stability after 1 week's storage but showed smaller (40 nm) size in CPH than in NaCas (100 nm). After lyophilization, the Cur NPs powders showed good rehydration properties and chemical stability, and compared with NaCas, the size of Cur NPs stabilized by CPH was still smaller. Additionally, the Cur NPs exhibited higher chemical stability against the temperature compared with free Cur, and the CPH could protect Cur from degradation more efficiently. Comparing with NaCas, the Cur NPs stabilized by CPH exhibited better bioaccessibility and antioxidant activity. This study demonstrated that CPH may be better than NaCas in Cur NPs fabrication and it opens up the possibility of using hydrophobic protein hydrolysate to construct the NPs delivery system.

  19. Increasing protein stability: Importance of ΔCp and the denatured state

    PubMed Central

    Fu, Hailong; Grimsley, Gerald; Scholtz, J Martin; Pace, C Nick

    2010-01-01

    Increasing the conformational stability of proteins is an important goal for both basic research and industrial applications. In vitro selection has been used successfully to increase protein stability, but more often site-directed mutagenesis is used to optimize the various forces that contribute to protein stability. In previous studies, we showed that improving electrostatic interactions on the protein surface and improving the β-turn sequences were good general strategies for increasing protein stability, and used them to increase the stability of RNase Sa. By incorporating seven of these mutations in RNase Sa, we increased the stability by 5.3 kcal/mol. Adding one more mutation, D79F, gave a total increase in stability of 7.7 kcal/mol, and a melting temperature 28°C higher than the wild-type enzyme. Surprisingly, the D79F mutation lowers the change in heat capacity for folding, ΔCp, by 0.6 kcal/mol/K. This suggests that this mutation stabilizes structure in the denatured state ensemble. We made other mutants that give some insight into the structure present in the denatured state. Finally, the thermodynamics of folding of these stabilized variants of RNase Sa are compared with those observed for proteins from thermophiles. PMID:20340133

  20. Increasing protein stability: importance of DeltaC(p) and the denatured state.

    PubMed

    Fu, Hailong; Grimsley, Gerald; Scholtz, J Martin; Pace, C Nick

    2010-05-01

    Increasing the conformational stability of proteins is an important goal for both basic research and industrial applications. In vitro selection has been used successfully to increase protein stability, but more often site-directed mutagenesis is used to optimize the various forces that contribute to protein stability. In previous studies, we showed that improving electrostatic interactions on the protein surface and improving the beta-turn sequences were good general strategies for increasing protein stability, and used them to increase the stability of RNase Sa. By incorporating seven of these mutations in RNase Sa, we increased the stability by 5.3 kcal/mol. Adding one more mutation, D79F, gave a total increase in stability of 7.7 kcal/mol, and a melting temperature 28 degrees C higher than the wild-type enzyme. Surprisingly, the D79F mutation lowers the change in heat capacity for folding, DeltaC(p), by 0.6 kcal/mol/K. This suggests that this mutation stabilizes structure in the denatured state ensemble. We made other mutants that give some insight into the structure present in the denatured state. Finally, the thermodynamics of folding of these stabilized variants of RNase Sa are compared with those observed for proteins from thermophiles.

  1. Protein stability in stored decellularized heart valve scaffolds and diffusion kinetics of protective molecules.

    PubMed

    Wang, Shangping; Oldenhof, Harriëtte; Dai, Xiaolei; Haverich, Axel; Hilfiker, Andres; Harder, Michael; Wolkers, Willem F

    2014-02-01

    Decellularized tissues can be used as matrix implants. The aims of this study were to investigate protein stability and solvent accessibility in decellularized pulmonary heart valve tissues. Protein denaturation profiles of tissues were studied by differential scanning calorimetry. Protein solvent accessibility of tissue exposed to D2O, and diffusion kinetics of various protective molecules were studied by Fourier transform infrared spectroscopy. Little changes were observed in the protein denaturation temperature during storage, at either 5 or 40°C. Glycerol was found to stabilize proteins; it increased the protein denaturation temperature. The stabilizing effect of glycerol disappeared after washing the sample with saline solution. Hydrogen-to-deuterium exchange rates of protein amide groups were fastest in leaflet tissue, followed by artery and muscle tissue. Diffusion of glycerol was found to be fastest in muscle tissue, followed by artery and leaflet tissue. Diffusion coefficients were derived and used to estimate the time needed to reach saturation. Fixation of tissue with glutaraldehyde had little effects on exchange and diffusion rates. Diffusion rates decreased with increasing molecular size. Proteins in decellularized heart valve tissue are stable during storage. Glycerol increases protein stability in a reversible manner. Solvent accessibility studies of protein amide groups provide an additional tool to study proteins in tissues. Diffusion coefficients can be derived to simulate diffusion kinetics of protective molecules in tissues. This study provides novel tools to evaluate protein stability and solvent accessibility in tissues, which can be used to develop biopreservation strategies.

  2. Protein engineering to stabilize soluble amyloid β-protein aggregates for structural and functional studies.

    PubMed

    Härd, Torleif

    2011-10-01

    The molecular biology underlying protein aggregation and neuronal death in Alzheimer's disease is not yet completely understood, but small soluble nonamyloid aggregates of the amyloid β-protein (Aβ) have been shown to play a fundamental neurotoxic role. The composition and biological action of such aggregates, known as oligomers and protofibrils, are therefore areas of intense study. However, research is complicated by the multitude of different interconverting aggregates that Aβ can form in vitro and in vivo, and by the inhomogeneity and instability of in vitro preparations. Here we review recent studies in which protein engineering, and in particular disulfide engineering, has been applied to stabilize different Aβ aggregates. For example, several techniques now exist to obtain stable and neurotoxic protofibrillar forms of Aβ, and engineered Aβ dimers, or larger aggregates formed by these, have been shown to specifically induce neuronal damage in a way that mimics Alzheimer's disease pathology. Disulfide engineering has also revealed structural properties of neurotoxic aggregates, for instance that Aβ in protofibrils and globular oligomers adopts a β-hairpin conformation that is similar to, but topologically distinct from, the conformation of Aβ in mature amyloid fibrils. Protein engineering is therefore a workable strategy to address many of the outstanding questions relating to the structure, interconversion and biological effects of oligomers and protofibrils of Aβ.

  3. Conjugation Strategy Strongly Impacts the Conformational Stability of a PEG-Protein Conjugate.

    PubMed

    Lawrence, Paul B; Billings, Wendy M; Miller, McKenzie B; Pandey, Brijesh K; Stephens, Andrew R; Langlois, Minnie I; Price, Joshua L

    2016-07-15

    Site-specific PEGylation is an important strategy for enhancing the pharmacokinetic properties of protein drugs, and has been enabled by the recent development of many chemoselective reactions for protein side-chain modification. However, the impact of these different conjugation strategies on the properties of PEG-protein conjugates is poorly understood. Here we show that the ability of PEG to enhance protein conformational stability depends strongly on the identity of the PEG-protein linker, with the most stabilizing linkers involving conjugation of PEG to planar polar groups near the peptide backbone. We also find that branched PEGs provide superior stabilization relative to their linear counterparts, suggesting additional applications for branched PEGs in protein stabilization.

  4. Structural and functional stabilization of protein entities: state-of-the-art.

    PubMed

    Balcão, Victor M; Vila, Marta M D C

    2015-10-01

    Within the context of biomedicine and pharmaceutical sciences, the issue of (therapeutic) protein stabilization assumes particular relevance. Stabilization of protein and protein-like molecules translates into preservation of both structure and functionality during storage and/or targeting, and such stabilization is mostly attained through establishment of a thermodynamic equilibrium with the (micro)environment. The basic thermodynamic principles that govern protein structural transitions and the interactions of the protein molecule with its (micro)environment are, therefore, tackled in a systematic fashion. Highlights are given to the major classes of (bio)therapeutic molecules, viz. enzymes, recombinant proteins, (macro)peptides, (monoclonal) antibodies and bacteriophages. Modification of the microenvironment of the biomolecule via multipoint covalent attachment onto a solid surface followed by hydrophilic polymer co-immobilization, or physical containment within nanocarriers, are some of the (latest) strategies discussed aiming at full structural and functional stabilization of said biomolecules.

  5. Denatured state aggregation parameters derived from concentration dependence of protein stability.

    PubMed

    Schön, Arne; Clarkson, Benjamin R; Siles, Rogelio; Ross, Patrick; Brown, Richard K; Freire, Ernesto

    2015-11-01

    Protein aggregation is a major issue affecting the long-term stability of protein preparations. Proteins exist in equilibrium between the native and denatured or partially denatured conformations. Often denatured or partially denatured conformations are prone to aggregate because they expose to solvent the hydrophobic core of the protein. The aggregation of denatured protein gradually shifts the protein equilibrium toward increasing amounts of denatured and ultimately aggregated protein. Recognizing and quantitating the presence of denatured protein and its aggregation at the earliest possible time will bring enormous benefits to the identification and selection of optimal solvent conditions or the engineering of proteins with the best stability/aggregation profile. In this article, a new approach that allows simultaneous determination of structural stability and the amount of denatured and aggregated protein is presented. This approach is based on the analysis of the concentration dependence of the Gibbs energy (ΔG) of protein stability. It is shown that three important quantities can be evaluated simultaneously: (i) the population of denatured protein, (ii) the population of aggregated protein, and (iii) the fraction of denatured protein that is aggregated.

  6. Evolutionary stabilization of the gene-3-protein of phage fd reveals the principles that govern the thermodynamic stability of two-domain proteins.

    PubMed

    Martin, Andreas; Schmid, Franz X

    2003-05-09

    The gene-3-protein (G3P) of filamentous phage is essential for their propagation. It consists of three domains. The CT domain anchors G3P in the phage coat, the N2 domain binds to the F pilus of Escherichia coli and thus initiates infection, and the N1 domain continues by interacting with the TolA receptor. Phage are thus only infective when the three domains of G3P are tightly linked, and this requirement is exploited by Proside, an in vitro selection method for proteins with increased stability. In Proside, a repertoire of variants of the protein to be stabilized is inserted between the N2 and the CT domains of G3P. Stabilized variants can be selected because they resist cleavage by a protease and thus maintain the essential linkage between the domains. The method is limited by the proteolytic stability of G3P itself. We improved the stability of G3P by subjecting the phage without a guest protein to rounds of random in vivo mutagenesis and proteolytic Proside selections. Variants of G3P with one to four mutations were selected, and the temperature at which the corresponding phage became accessible for a protease increased in a stepwise manner from 40 degrees C to almost 60 degrees C. The N1-N2 fragments of wild-type gene-3-protein and of the four selected variants were purified and their stabilities towards thermal and denaturant-induced unfolding were determined. In the biphasic transitions of these proteins domain dissociation and unfolding of N2 occur in a concerted reaction in the first step, followed by the independent unfolding of domain N1 in the second step. N2 is thus less stable than N1, and it unfolds when the interactions with N1 are broken. The strongest stabilizations were caused by mutations in domain N2, in particular in its hinge subdomain, which provides many stabilizing interactions between the N1 and N2 domains. These results reveal how the individual domains and their assembly contribute to the overall stability of two-domain proteins and

  7. Conserved interaction of Ctf18-RFC with DNA polymerase ε is critical for maintenance of genome stability in Saccharomyces cerevisiae.

    PubMed

    Okimoto, Hiroko; Tanaka, Seiji; Araki, Hiroyuki; Ohashi, Eiji; Tsurimoto, Toshiki

    2016-05-01

    Human Ctf18-RFC, a PCNA loader complex, interacts with DNA polymerase ε (Polε) through a structure formed by the Ctf18, Dcc1 and Ctf8 subunits. The C-terminal stretch of Ctf18, which is highly conserved from yeast to human, is necessary to form the Polε-capturing structure. We found that in the budding yeast Saccharomyces cerevisiae, Ctf18, Dcc1 and Ctf8 formed the same structure through the conserved C-terminus and interacted specifically with Polε. Thus, the specific interaction of Ctf18-RFC with Polε is a conserved feature between these proteins. A C-terminal deletion mutant of Ctf18 (ctf18(ΔC) ) exhibited the same high sensitivity to hydroxyurea as the complete deletion strain (ctf18Δ) or ATPase-deficient mutant (ctf18(K189A) ), but was somewhat less sensitive to methyl methanesulfonate than either of them. These phenotypes were also observed in dcc1Δ and ctf8Δ, predicted to be deficient in the interaction with Polε. Furthermore, both plasmid loss and gross chromosomal rearrangement (GCR) rates were increased in ctf18(ΔC) cells to the same extent as in ctf18Δ cells. These results indicate that the Ctf18-RFC/Polε interaction plays a crucial role in maintaining genome stability in budding yeast, probably through recruitment of this PCNA loader to the replication fork.

  8. Effects of Protein Stabilizing Agents on Thermal Backbone Motions: A Disulfide Trapping Study†

    PubMed Central

    Butler, Scott L.; Falke, Joseph J.

    2010-01-01

    Chemical stabilizers are widely used to enhance protein stability, both in nature and in the laboratory. Here, the molecular mechanism of chemical stabilizers is studied using a disulfide trapping assay to measure the effects of stabilizers on thermal backbone dynamics in the Escherichia coli galactose/glucose binding protein. Two types of backbone fluctuations are examined: (a) relative movements of adjacent surface α-helices within the same domain and (b) interdomain twisting motions. Both types of fluctuations are significantly reduced by all six stabilizers tested (glycerol, sucrose, trehalose, l-glucose, d-glucose, and d-galactose), and in each case larger amplitude motions are inhibited more than smaller ones. Motional inhibition does not require a high-affinity stabilizer binding site, indicating that the effects of stabilizers are nonspecific. Overall, the results support the theory that effective stabilizing agents act by favoring the most compact structure of a protein, thereby reducing local backbone fluctuations away from the fully folded state. Such inhibition of protein backbone dynamics may be a general mechanism of protein stabilization in extreme thermal or chemical environments. PMID:8718847

  9. Protein thermal stability and phospholipid-protein interaction investigated by capillary isoelectric focusing with whole column imaging detection.

    PubMed

    Bo, Tao; Pawliszyn, Janusz

    2006-05-01

    CIEF with whole column imaging detection (WCID) is an attractive technique for studying protein reaction and protein-ligand interaction due to its fast separation, simple operation, and high efficiency. In this study, two interesting applications by the CIEF-WCID were developed, involving the study of protein thermal stability and phospholipid-protein interaction. Four proteins (beta-lactoglobulin B, trypsin inhibitor, phosphorylase b, and trypsinogen) with different pI, and two types of phospholipids, including phosphatidylcholine (PC) and phosphatidylserine (PS), were used for this purpose. First, the altered CIEF profiles of four proteins were exhibited due to conformational changes resulting from protein denaturation induced by a high incubation temperature at 60 degrees C. It was demonstrated that the addition of a zwitterionic phospholipid (PC) played a crucial role in the thermal stability of targeted proteins, especially for trypsin inhibitor whose thermal stability was promoted with the addition of the PC vesicles at 60 degrees C. Second, the zwitterionic (PC) and acidic (PS) phospholipids displayed completely different interactions with the proteins. The addition of PS vesicles modified the zwitterionic phospholipids to carry negative charges, which correspondingly changed the interaction between the phospholipid and the protein. Our study demonstrates that the CIEF-WCID is a powerful approach to study protein reaction and protein-ligand interaction with high efficiency, high selectivity, and fast separation.

  10. A Multi-layered Protein Network Stabilizes the Escherichia coli FtsZ-ring and Modulates Constriction Dynamics

    PubMed Central

    Buss, Jackson; Coltharp, Carla; Shtengel, Gleb; Yang, Xinxing; Hess, Harald; Xiao, Jie

    2015-01-01

    The prokaryotic tubulin homolog, FtsZ, forms a ring-like structure (FtsZ-ring) at midcell. The FtsZ-ring establishes the division plane and enables the assembly of the macromolecular division machinery (divisome). Although many molecular components of the divisome have been identified and their interactions extensively characterized, the spatial organization of these proteins within the divisome is unclear. Consequently, the physical mechanisms that drive divisome assembly, maintenance, and constriction remain elusive. Here we applied single-molecule based superresolution imaging, combined with genetic and biophysical investigations, to reveal the spatial organization of cellular structures formed by four important divisome proteins in E. coli: FtsZ, ZapA, ZapB and MatP. We show that these interacting proteins are arranged into a multi-layered protein network extending from the cell membrane to the chromosome, each with unique structural and dynamic properties. Further, we find that this protein network stabilizes the FtsZ-ring, and unexpectedly, slows down cell constriction, suggesting a new, unrecognized role for this network in bacterial cell division. Our results provide new insight into the structure and function of the divisome, and highlight the importance of coordinated cell constriction and chromosome segregation. PMID:25848771

  11. Associations between dairy protein intake and body weight and risk markers of diabetes and CVD during weight maintenance.

    PubMed

    Bendtsen, Line Q; Lorenzen, Janne K; Larsen, Thomas M; van Baak, Marleen; Papadaki, Angeliki; Martinez, J Alfredo; Handjieva-Darlenska, Teodora; Jebb, Susan A; Kunešová, Marie; Pfeiffer, Andreas F H; Saris, Wim H M; Astrup, Arne; Raben, Anne

    2014-03-14

    Dairy products have previously been reported to be associated with beneficial effects on body weight and metabolic risk markers. Moreover, primary data from the Diet, Obesity and Genes (DiOGenes) study indicate a weight-maintaining effect of a high-protein-low-glycaemic index diet. The objective of the present study was to examine putative associations between consumption of dairy proteins and changes in body weight and metabolic risk markers after weight loss in obese and overweight adults. Results were based on secondary analyses of data obtained from overweight and obese adults who completed the DiOGenes study. The study consisted of an 8-week weight-loss phase and a 6-month weight-maintenance (WM) phase, where the subjects were given five different diets varying in protein content and glycaemic index. In the present study, data obtained from all the subjects were pooled. Dairy protein intake was estimated from 3 d dietary records at two time points (week 4 and week 26) during the WM phase. Body weight and metabolic risk markers were determined at baseline (week -9 to -11) and before and at the end of the WM phase (week 0 and week 26). Overall, no significant associations were found between consumption of dairy proteins and changes in body weight and metabolic risk markers. However, dairy protein intake tended to be negatively associated with body weight gain (P=0·08; β=-0·17), but this was not persistent when controlled for total protein intake, which indicates that dairy protein adds no additional effect to the effect of total protein. Therefore, the present study does not report that dairy proteins are more favourable than other proteins for body weight regulation.

  12. A rapid, ensemble and free energy based method for engineering protein stabilities.

    PubMed

    Naganathan, Athi N

    2013-05-02

    Engineering the conformational stabilities of proteins through mutations has immense potential in biotechnological applications. It is, however, an inherently challenging problem given the weak noncovalent nature of the stabilizing interactions. In this regard, we present here a robust and fast strategy to engineer protein stabilities through mutations involving charged residues using a structure-based statistical mechanical model that accounts for the ensemble nature of folding. We validate the method by predicting the absolute changes in stability for 138 experimental mutations from 16 different proteins and enzymes with a correlation of 0.65 and importantly with a success rate of 81%. Multiple point mutants are predicted with a higher success rate (90%) that is validated further by comparing meosphile-thermophile protein pairs. In parallel, we devise a methodology to rapidly engineer mutations in silico which we benchmark against experimental mutations of ubiquitin (correlation of 0.95) and check for its feasibility on a larger therapeutic protein DNase I. We expect the method to be of importance as a first and rapid step to screen for protein mutants with specific stability in the biotechnology industry, in the construction of stability maps at the residue level (i.e., hot spots), and as a robust tool to probe for mutations that enhance the stability of protein-based drugs.

  13. Predicting stability of alpha-helical, orthogonal-bundle proteins on surfaces

    NASA Astrophysics Data System (ADS)

    Wei, Shuai; Knotts, Thomas A.

    2010-09-01

    The interaction of proteins with surfaces is a key phenomenon in many applications, but current understanding of the biophysics involved is lacking. At present, rational design of such emerging technologies is difficult as no methods or theories exist that correctly predict how surfaces influence protein behavior. Using molecular simulation and a coarse-grain model, this study illustrates for the first time that stability of proteins on surfaces can be correlated with tertiary structural elements for alpha-helical, orthogonal-bundle proteins. Results show that several factors contribute to stability on surfaces including the nature of the loop region where the tether is placed and the ability of the protein to freely rotate on the surface. A thermodynamic analysis demonstrates that surfaces stabilize proteins entropically and that any destabilization is an enthalpic effect. Moreover, the entropic effects are concentrated on the unfolded state of the protein while the ethalpic effects are focused on the folded state.

  14. Predicting stability of alpha-helical, orthogonal-bundle proteins on surfaces.

    PubMed

    Wei, Shuai; Knotts, Thomas A

    2010-09-21

    The interaction of proteins with surfaces is a key phenomenon in many applications, but current understanding of the biophysics involved is lacking. At present, rational design of such emerging technologies is difficult as no methods or theories exist that correctly predict how surfaces influence protein behavior. Using molecular simulation and a coarse-grain model, this study illustrates for the first time that stability of proteins on surfaces can be correlated with tertiary structural elements for alpha-helical, orthogonal-bundle proteins. Results show that several factors contribute to stability on surfaces including the nature of the loop region where the tether is placed and the ability of the protein to freely rotate on the surface. A thermodynamic analysis demonstrates that surfaces stabilize proteins entropically and that any destabilization is an enthalpic effect. Moreover, the entropic effects are concentrated on the unfolded state of the protein while the ethalpic effects are focused on the folded state.

  15. The role of maintenance proteins in the preservation of epithelial cell identity during mammary gland remodeling and breast cancer initiation.

    PubMed

    Coradini, Danila; Oriana, Saro

    2014-02-01

    During normal postnatal mammary gland development and adult remodeling related to the menstrual cycle, pregnancy, and lactation, ovarian hormones and peptide growth factors contribute to the delineation of a definite epithelial cell identity. This identity is maintained during cell replication in a heritable but DNA-independent manner. The preservation of cell identity is fundamental, especially when cells must undergo changes in response to intrinsic and extrinsic signals. The maintenance proteins, which are required for cell identity preservation, act epigenetically by regulating gene expression through DNA methylation, histone modification, and chromatin remodeling. Among the maintenance proteins, the Trithorax (TrxG) and Polycomb (PcG) group proteins are the best characterized. In this review, we summarize the structures and activities of the TrxG and PcG complexes and describe their pivotal roles in nuclear estrogen receptor activity. In addition, we provide evidence that perturbations in these epigenetic regulators are involved in disrupting epithelial cell identity, mammary gland remodeling, and breast cancer initiation.

  16. A functional protein retention and release multilayer with high stability

    NASA Astrophysics Data System (ADS)

    Nie, Kun; An, Qi; Zhang, Yihe

    2016-04-01

    Effective and robust interfacial protein retention lies at the heart of the fabrication of protein-based functional interfaces, which is potentially applicable in catalysis, medical therapy, antifouling, and smart devices, but remains challenging due to the sensitive nature of proteins. This study reports a general protein retention strategy to spatial-temporally confine various types of proteins at interfacial regions. The proteins were preserved in mesoporous silica nanoparticles embedded in covalently woven multilayers. It is worth noting that the protein retention strategy effectively preserves the catalytic capabilities of the proteins, and the multilayer structure is robust enough to withstand the bubbling catalytic reactions and could be repeatedly used due to conservation of proteins. The spatiotemporal retention of proteins could be adjusted by varying the number of capping layers. Furthermore, we demonstrate that the protein-loaded interfacial layers could not only be used to construct catalytic-active interfaces, but also be integrated as the power-generating unit to propel a macroscopic floating device.Effective and robust interfacial protein retention lies at the heart of the fabrication of protein-based functional interfaces, which is potentially applicable in catalysis, medical therapy, antifouling, and smart devices, but remains challenging due to the sensitive nature of proteins. This study reports a general protein retention strategy to spatial-temporally confine various types of proteins at interfacial regions. The proteins were preserved in mesoporous silica nanoparticles embedded in covalently woven multilayers. It is worth noting that the protein retention strategy effectively preserves the catalytic capabilities of the proteins, and the multilayer structure is robust enough to withstand the bubbling catalytic reactions and could be repeatedly used due to conservation of proteins. The spatiotemporal retention of proteins could be adjusted by

  17. Measuring the interaction of urea and protein-stabilizing osmolytes with the nonpolar surface of hydroxypropylcellulose.

    PubMed

    Stanley, Christopher; Rau, Donald C

    2008-06-24

    The interaction of urea and several naturally occurring protein-stabilizing osmolytes, glycerol, sorbitol, glycine betaine, trimethylamine oxide (TMAO), and proline, with condensed arrays of a hydrophobically modified polysaccharide, hydroxypropylcellulose (HPC), has been inferred from the effect of these solutes on the forces acting between HPC polymers. Urea interacts only very weakly. The protein-stabilizing osmolytes are strongly excluded. The observed energies indicate that the exclusion of the protein-stabilizing osmolytes from protein hydrophobic side chains would add significantly to protein stability. The temperature dependence of exclusion indicates a significant contribution of enthalpy to the interaction energy in contrast to expectations from "molecular crowding" theories based on steric repulsion. The dependence of exclusion on the distance between HPC polymers rather indicates that perturbations of water structuring or hydration forces underlie exclusion.

  18. Fungal Hydrophobin Proteins Produce Self-Assembling Protein Films with Diverse Structure and Chemical Stability

    PubMed Central

    Lo, Victor C.; Ren, Qin; Pham, Chi L. L.; Morris, Vanessa K.; Kwan, Ann H.; Sunde, Margaret

    2014-01-01

    Hydrophobins are small proteins secreted by fungi and which spontaneously assemble into amphipathic layers at hydrophilic-hydrophobic interfaces. We have examined the self-assembly of the Class I hydrophobins EAS∆15 and DewA, the Class II hydrophobin NC2 and an engineered chimeric hydrophobin. These Class I hydrophobins form layers composed of laterally associated fibrils with an underlying amyloid structure. These two Class I hydrophobins, despite showing significant conformational differences in solution, self-assemble to form fibrillar layers with very similar structures and require a hydrophilic-hydrophobic interface to trigger self-assembly. Addition of additives that influence surface tension can be used to manipulate the fine structure of the protein films. The Class II hydrophobin NC2 forms a mesh-like protein network and the engineered chimeric hydrophobin displays two multimeric forms, depending on assembly conditions. When formed on a graphite surface, the fibrillar EAS∆15 layers are resistant to alcohol, acid and basic washes. In contrast, the NC2 Class II monolayers are dissociated by alcohol treatment but are relatively stable towards acid and base washes. The engineered chimeric Class I/II hydrophobin shows increased stability towards alcohol and acid and base washes. Self-assembled hydrophobin films may have extensive applications in biotechnology where biocompatible; amphipathic coatings facilitate the functionalization of nanomaterials.

  19. Modulation of protein stability and aggregation properties by surface charge engineering.

    PubMed

    Raghunathan, Govindan; Sokalingam, Sriram; Soundrarajan, Nagasundarapandian; Madan, Bharat; Munussami, Ganapathiraman; Lee, Sun-Gu

    2013-09-01

    An attempt to alter protein surface charges through traditional protein engineering approaches often affects the native protein structure significantly and induces misfolding. This limitation is a major hindrance in modulating protein properties through surface charge variations. In this study, as a strategy to overcome such a limitation, we attempted to co-introduce stabilizing mutations that can neutralize the destabilizing effect of protein surface charge variation. Two sets of rational mutations were designed; one to increase the number of surface charged amino acids and the other to decrease the number of surface charged amino acids by mutating surface polar uncharged amino acids and charged amino acids, respectively. These two sets of mutations were introduced into Green Fluorescent Protein (GFP) together with or without stabilizing mutations. The co-introduction of stabilizing mutations along with mutations for surface charge modification allowed us to obtain functionally active protein variants (s-GFP(+15-17) and s-GFP(+5-6)). When the protein properties such as fluorescent activity, folding rate and kinetic stability were assessed, we found the possibility that the protein stability can be modulated independently of activity and folding by engineering protein surface charges. The aggregation properties of GFP could also be altered through the surface charge engineering.

  20. Adhesive–cohesive model for protein compressibility: An alternative perspective on stability

    PubMed Central

    Dadarlat, Voichita M.; Post, Carol Beth

    2003-01-01

    As a dynamic property of folded proteins, protein compressibility provides important information about the forces that govern structural stability. We relate intrinsic compressibility to stability by using molecular dynamics to identify a molecular basis for the variation in compressibility among globular proteins. We find that excess surface charge accounts for this variation not only for the proteins simulated by molecular dynamics but also for a larger set of globular proteins. This dependence on charge distribution forms the basis for an adhesive–cohesive model of protein compressibility in which attractive forces from solvent compete with tertiary interactions that favor folding. Further, a newly recognized correlation between compressibility and the heat capacity of unfolding infers a link between compressibility and the enthalpy of unfolding. This linkage, together with the adhesive–cohesive model for compressibility, leads to the conclusion that folded proteins can gain enthalpic stability from a uniform distribution of charged atoms, as opposed to partitioning charge to the protein surface. Whether buried charged groups can be energetically stabilizing is a fundamental, yet controversial, question regarding protein structure. The analysis reported here implies that one mechanism to gain enthalpic stability involves positioning charge inside the protein in an optimal structural arrangement. PMID:14638940

  1. Mapping the stabilome: a novel computational method for classifying metabolic protein stability

    PubMed Central

    2012-01-01

    Background The half-life of a protein is regulated by a range of system properties, including the abundance of components of the degradative machinery and protein modifiers. It is also influenced by protein-specific properties, such as a protein’s structural make-up and interaction partners. New experimental techniques coupled with powerful data integration methods now enable us to not only investigate what features govern protein stability in general, but also to build models that identify what properties determine each protein’s metabolic stability. Results In this work we present five groups of features useful for predicting protein stability: (1) post-translational modifications, (2) domain types, (3) structural disorder, (4) the identity of a protein’s N-terminal residue and (5) amino acid sequence. We incorporate these features into a predictive model with promising accuracy. At a 20% false positive rate, the model exhibits an 80% true positive rate, outperforming the only previously proposed stability predictor. We also investigate the impact of N-terminal protein tagging as used to generate the data set, in particular the impact it may have on the measurements for secreted and transmembrane proteins; we train and test our model on a subset of the data with those proteins removed, and show that the model sustains high accuracy. Finally, we estimate system-wide metabolic stability by surveying the whole human proteome. Conclusions We describe a variety of protein features that are significantly over- or under-represented in stable and unstable proteins, including phosphorylation, acetylation and destabilizing N-terminal residues. Bayesian networks are ideal for combining these features into a predictive model with superior accuracy and transparency compared to the only other proposed stability predictor. Furthermore, our stability predictions of the human proteome will find application in the analysis of functionally related proteins, shedding new light

  2. 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

  3. Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution.

    PubMed

    He, Yi-Ming; Ma, Bin-Guang

    2016-05-25

    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.

  4. The Efficacy of U.S. and U.S.S.R. Arms Transfers for the Maintenance of Regime Stability in the Third World

    DTIC Science & Technology

    1990-09-01

    WORLD 12 PERSONAL AUTHORS) Miranda, Enrique F. 13a TYPE OF REPORT 13b TIME COVERED 114 DATE OF REPORT (Year, Month, Day) 115 PAGE CCK.NT Master’s Thesis...for the Maintenance of Regime Stability in the Third World by Enrique Franco miranda Lieutenant, United States Navy B.S., United States Naval Academy...Easy Choice (Cambridge: Harvard University Press, 1976), p. 17. 7. Black, Cyril E. The Dynamics of Modernization (New York: Harper and Row, 1966), pp. 3

  5. Stability Curve Prediction of Homologous Proteins Using Temperature-Dependent Statistical Potentials

    PubMed Central

    Pucci, Fabrizio; Rooman, Marianne

    2014-01-01

    The unraveling and control of protein stability at different temperatures is a fundamental problem in biophysics that is substantially far from being quantitatively and accurately solved, as it requires a precise knowledge of the temperature dependence of amino acid interactions. In this paper we attempt to gain insight into the thermal stability of proteins by designing a tool to predict the full stability curve as a function of the temperature for a set of 45 proteins belonging to 11 homologous families, given their sequence and structure, as well as the melting temperature () and the change in heat capacity () of proteins belonging to the same family. Stability curves constitute a fundamental instrument to analyze in detail the thermal stability and its relation to the thermodynamic stability, and to estimate the enthalpic and entropic contributions to the folding free energy. In summary, our approach for predicting the protein stability curves relies on temperature-dependent statistical potentials derived from three datasets of protein structures with targeted thermal stability properties. Using these potentials, the folding free energies () at three different temperatures were computed for each protein. The Gibbs-Helmholtz equation was then used to predict the protein's stability curve as the curve that best fits these three points. The results are quite encouraging: the standard deviations between the experimental and predicted 's, 's and folding free energies at room temperature () are equal to 13 , 1.3 ) and 4.1 , respectively, in cross-validation. The main sources of error and some further improvements and perspectives are briefly discussed. PMID:25032839

  6. Stability curve prediction of homologous proteins using temperature-dependent statistical potentials.

    PubMed

    Pucci, Fabrizio; Rooman, Marianne

    2014-07-01

    The unraveling and control of protein stability at different temperatures is a fundamental problem in biophysics that is substantially far from being quantitatively and accurately solved, as it requires a precise knowledge of the temperature dependence of amino acid interactions. In this paper we attempt to gain insight into the thermal stability of proteins by designing a tool to predict the full stability curve as a function of the temperature for a set of 45 proteins belonging to 11 homologous families, given their sequence and structure, as well as the melting temperature (Tm) and the change in heat capacity (ΔCP) of proteins belonging to the same family. Stability curves constitute a fundamental instrument to analyze in detail the thermal stability and its relation to the thermodynamic stability, and to estimate the enthalpic and entropic contributions to the folding free energy. In summary, our approach for predicting the protein stability curves relies on temperature-dependent statistical potentials derived from three datasets of protein structures with targeted thermal stability properties. Using these potentials, the folding free energies (ΔG) at three different temperatures were computed for each protein. The Gibbs-Helmholtz equation was then used to predict the protein's stability curve as the curve that best fits these three points. The results are quite encouraging: the standard deviations between the experimental and predicted Tm's, ΔCP's and folding free energies at room temperature (ΔG25) are equal to 13° C, 1.3 kcal/(mol° C) and 4.1 kcal/mol, respectively, in cross-validation. The main sources of error and some further improvements and perspectives are briefly discussed.

  7. Folding and Stabilization of Native-Sequence-Reversed Proteins

    PubMed Central

    Zhang, Yuanzhao; Weber, Jeffrey K; Zhou, Ruhong

    2016-01-01

    Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols. PMID:27113844

  8. Folding and Stabilization of Native-Sequence-Reversed Proteins

    NASA Astrophysics Data System (ADS)

    Zhang, Yuanzhao; Weber, Jeffrey K.; Zhou, Ruhong

    2016-04-01

    Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols.

  9. Metabolic Turnover of Synaptic Proteins: Kinetics, Interdependencies and Implications for Synaptic Maintenance

    PubMed Central

    Cohen, Laurie D.; Zuchman, Rina; Sorokina, Oksana; Müller, Anke; Dieterich, Daniela C.; Armstrong, J. Douglas; Ziv, Tamar; Ziv, Noam E.

    2013-01-01

    Chemical synapses contain multitudes of proteins, which in common with all proteins, have finite lifetimes and therefore need to be continuously replaced. Given the huge numbers of synaptic connections typical neurons form, the demand to maintain the protein contents of these connections might be expected to place considerable metabolic demands on each neuron. Moreover, synaptic proteostasis might differ according to distance from global protein synthesis sites, the availability of distributed protein synthesis facilities, trafficking rates and synaptic protein dynamics. To date, the turnover kinetics of synaptic proteins have not been studied or analyzed systematically, and thus metabolic demands or the aforementioned relationships remain largely unknown. In the current study we used dynamic Stable Isotope Labeling with Amino acids in Cell culture (SILAC), mass spectrometry (MS), Fluorescent Non–Canonical Amino acid Tagging (FUNCAT), quantitative immunohistochemistry and bioinformatics to systematically measure the metabolic half-lives of hundreds of synaptic proteins, examine how these depend on their pre/postsynaptic affiliation or their association with particular molecular complexes, and assess the metabolic load of synaptic proteostasis. We found that nearly all synaptic proteins identified here exhibited half-lifetimes in the range of 2–5 days. Unexpectedly, metabolic turnover rates were not significantly different for presynaptic and postsynaptic proteins, or for proteins for which mRNAs are consistently found in dendrites. Some functionally or structurally related proteins exhibited very similar turnover rates, indicating that their biogenesis and degradation might be coupled, a possibility further supported by bioinformatics-based analyses. The relatively low turnover rates measured here (∼0.7% of synaptic protein content per hour) are in good agreement with imaging-based studies of synaptic protein trafficking, yet indicate that the metabolic load

  10. Effect of protein load on stability of immobilized enzymes.

    PubMed

    Fernandez-Lopez, Laura; Pedrero, Sara G; Lopez-Carrobles, Nerea; Gorines, Beatriz C; Virgen-Ortíz, Jose J; Fernandez-Lafuente, Roberto

    2017-03-01

    Different lipases have been immobilized on octyl agarose beads at 1mg/g and at maximum loading, via physical interfacial activation versus the octyl layer on the support. The stability of the preparations was analyzed. Most biocatalysts had the expected result: the apparent stability increased using the highly loaded preparations, due to the diffusional limitations that reduced the initial observed activity. However, lipase B from Candida antarctica (CALB) was significantly more stable using the lowly loaded preparation than the maximum loaded one. This negative effect of the enzyme crowding on enzyme stability was found in inactivations at pH 5, 7 or 9, but not in inactivations in the presence of organic solvents. The immobilization using ethanol to reduce the immobilization rate had no effect on the stability of the lowly loaded preparation, while the highly loaded enzyme biocatalysts increased their stabilities, becoming very similar to that of the lowly loaded preparation. Results suggested that CALB molecules immobilized on octyl agarose may be closely packed together due to the high immobilization rate and this produced some negative interactions between immobilized enzyme molecules during enzyme thermal inactivation. Slowing-down the immobilization rate may be a solution for this unexpected problem. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Salts Enhance Both Protein Stability and Amyloid Formation of an Immunoglobulin Light Chain

    PubMed Central

    Sikkink, Laura A.; Ramirez-Alvarado, Marina

    2008-01-01

    Amyloid fibrils are associated with sulfated glycosaminoglycans in the extracellular matrix. The presence of sulfated glycosaminoglycans is known to promote amyloid formation in vitro and in vivo, with the sulfate groups playing a role in this process. In order to understand the role that sulfate plays in amyloid formation, we have studied the effect of salts from the Hofmeister series on the protein structure, stability and amyloid formation of an amyloidogenic light chain protein, AL-12. We have been able to show for the first time a direct correlation between protein stability and amyloid formation enhancement by salts from the Hofmeister series, where SO42−conferred the most protein stability and enhancement of amyloid formation. Our study emphasizes the importance of the effect of ions in the protein bound water properties and downplays the role of specific interactions between the protein and ions. PMID:18395318

  12. The Stability and Formation of Native Proteins from Unfolded Monomers Is Increased through Interactions with Unrelated Proteins

    PubMed Central

    Rodríguez-Almazán, Claudia; Torner, Francisco J.; Costas, Miguel; Pérez-Montfort, Ruy; de Gómez-Puyou, Marieta Tuena; Puyou, Armando Gómez

    2007-01-01

    The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins. PMID:17551578

  13. Role of G-protein-coupled estrogen receptor (GPER/GPR30) in maintenance of meiotic arrest in fish oocytes.

    PubMed

    Thomas, Peter

    2017-03-01

    An essential role for GPER (formerly known as GPR30) in regulating mammalian reproduction has not been identified to date, although it has shown to be involved in the regulation a broad range of other estrogen-dependent functions. In contrast, an important reproductive role for GPER in the maintenance of oocyte meiotic arrest has been identified in teleost fishes, which is briefly reviewed here. Recent studies have clearly shown that ovarian follicle production of estradiol-17β (E2) maintains meiotic arrest in several teleost species through activation of GPER coupled to a stimulatory G protein (Gs) on oocyte plasma membranes resulting in stimulation of cAMP production and maintenance of elevated cAMP levels. Studies with denuded zebrafish oocytes and with microinjection of GPER antisense oligonucleotides into oocytes have demonstrated the requirement for both ovarian follicle production of estrogens and expression of GPER on the oocyte surface for maintenance of meiotic arrest. This inhibitory action of E2 on the resumption of meiosis is mimicked by the GPER-selective agonist G-1, by the GPER agonists and nuclear ER antagonists, ICI 182,780 and tamoxifen, and also by the xenoestrogen bisphenol-A (BPA) and related alkylphenols. GPER also maintains meiotic arrest of zebrafish oocytes through estrogen- and BPA-dependent GPER activation of epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase (MAPK) signaling. Interestingly, progesterone receptor component 1 (PGRMC1) is also involved in estrogen maintenance of meiotic arrest through regulation of EGFR expression on the oocyte plasma membrane. The preovulatory surge in LH secretion induces the ovarian synthesis of progestin hormones that activate a membrane progestin receptor alpha (mPRα)/inhibitory G protein (Gi) pathway. It also increases ovarian synthesis of the catecholestrogen, 2-hydroxy-estradiol-17β (2-OHE2) which inhibits the GPER/Gs/adenylyl cyclase pathway. Both of these LH actions

  14. Knotted and topologically complex proteins as models for studying folding and stability

    PubMed Central

    Yeates, Todd O.; Norcross, Todd S.; King, Neil P.

    2008-01-01

    SUMMARY Among proteins of known three dimensional structure, only a few possess complex topological features such as knotted or interlinked (catenated) protein backbones. Such unusual proteins offer potentially unique insights into folding pathways and stabilization mechanisms. They also present special challenges for both theorists and computational scientists interested in understanding and predicting protein folding behavior. Here we review complex topological features in proteins with a focus on recent progress on the identification and characterization of knotted and interlinked protein systems. Also, an approach is described for designing an expanded set of knotted proteins. PMID:17967433

  15. The cohesin complex prevents the end-joining of distant DNA double-strand ends in S phase: Consequences on genome stability maintenance.

    PubMed

    Gelot, Camille; Guirouilh-Barbat, Josée; Lopez, Bernard S

    2016-07-03

    DNA double-strand break (DSB) repair is essential for genome stability maintenance, but the joining of distant DNA double strand ends (DSEs) inevitably leads to genome rearrangements. Therefore, DSB repair should be tightly controlled to secure genome stability while allowing genetic variability. Tethering of the proximal ends of a 2-ended DSB limits their mobility, protecting thus against their joining with a distant DSE. However, replication stress generates DSBs with only one DSE, on which tethering is impossible. Consistently, we demonstrated that the joining of 2 DSBs only 3.2 kb apart is repressed in the S, but not the G1, phase, revealing an additional mechanism limiting DNA ends mobility in S phase. The cohesin complex, by maintaining the 2 sister chromatids linked, limits DSEs mobility and thus represses the joining of distant DSEs, while allowing that of adjacent DSEs. At the genome scale, the cohesin complex protects against deletions, inversions, translocations and chromosome fusion.

  16. Long-term manure amendments reduced soil aggregate stability via redistribution of the glomalin-related soil protein in macroaggregates

    PubMed Central

    Xie, Hongtu; Li, Jianwei; Zhang, Bin; Wang, Lianfeng; Wang, Jingkuan; He, Hongbo; Zhang, Xudong

    2015-01-01

    Glomalin-related soil protein (GRSP) contributes to the formation and maintenance of soil aggregates, it is however remains unclear whether long-term intensive manure amendments alter soil aggregates stability and whether GRSP regulates these changes. Based on a three-decade long fertilization experiment in northeast China, this study examined the impact of long-term manure input on soil organic carbon (SOC), total and easily extractable GRSP (GRSPt and GRSPe) and their respective allocations in four soil aggregates (>2000 μm; 2000–250 μm; 250–53 μm; and <53 μm). The treatments include no fertilization (CK), low and high manure amendment (M1, M2), chemical nitrogen, phosphorus and potassium fertilizers (NPK), and combined manure and chemical fertilizers (NPKM1, NPKM2). Though SOC, GRSPe and GRSPt in soil and SOC in each aggregate generally increased with increasing manure input, GRSPt and GRSPe in each aggregate showed varying changes with manure input. Both GRSP in macroaggregates (2000–250 μm) were significantly higher under low manure input, a pattern consistent with changes in soil aggregate stability. Constituting 38~49% of soil mass, macroaggregates likely contributed to the nonlinear changes of aggregate stability under manure amendments. The regulatory process of GRSP allocations in soil aggregates has important implications for manure management under intensive agriculture. PMID:26423355

  17. Long-term manure amendments reduced soil aggregate stability via redistribution of the glomalin-related soil protein in macroaggregates.

    PubMed

    Xie, Hongtu; Li, Jianwei; Zhang, Bin; Wang, Lianfeng; Wang, Jingkuan; He, Hongbo; Zhang, Xudong

    2015-10-01

    Glomalin-related soil protein (GRSP) contributes to the formation and maintenance of soil aggregates, it is however remains unclear whether long-term intensive manure amendments alter soil aggregates stability and whether GRSP regulates these changes. Based on a three-decade long fertilization experiment in northeast China, this study examined the impact of long-term manure input on soil organic carbon (SOC), total and easily extractable GRSP (GRSPt and GRSPe) and their respective allocations in four soil aggregates (>2000 μm; 2000-250 μm; 250-53 μm; and <53 μm). The treatments include no fertilization (CK), low and high manure amendment (M1, M2), chemical nitrogen, phosphorus and potassium fertilizers (NPK), and combined manure and chemical fertilizers (NPKM1, NPKM2). Though SOC, GRSPe and GRSPt in soil and SOC in each aggregate generally increased with increasing manure input, GRSPt and GRSPe in each aggregate showed varying changes with manure input. Both GRSP in macroaggregates (2000-250 μm) were significantly higher under low manure input, a pattern consistent with changes in soil aggregate stability. Constituting 38~49% of soil mass, macroaggregates likely contributed to the nonlinear changes of aggregate stability under manure amendments. The regulatory process of GRSP allocations in soil aggregates has important implications for manure management under intensive agriculture.

  18. Long-term manure amendments reduced soil aggregate stability via redistribution of the glomalin-related soil protein in macroaggregates

    NASA Astrophysics Data System (ADS)

    Xie, Hongtu; Li, Jianwei; Zhang, Bin; Wang, Lianfeng; Wang, Jingkuan; He, Hongbo; Zhang, Xudong

    2015-10-01

    Glomalin-related soil protein (GRSP) contributes to the formation and maintenance of soil aggregates, it is however remains unclear whether long-term intensive manure amendments alter soil aggregates stability and whether GRSP regulates these changes. Based on a three-decade long fertilization experiment in northeast China, this study examined the impact of long-term manure input on soil organic carbon (SOC), total and easily extractable GRSP (GRSPt and GRSPe) and their respective allocations in four soil aggregates (>2000 μm 2000-250 μm 250-53 μm and <53 μm). The treatments include no fertilization (CK), low and high manure amendment (M1, M2), chemical nitrogen, phosphorus and potassium fertilizers (NPK), and combined manure and chemical fertilizers (NPKM1, NPKM2). Though SOC, GRSPe and GRSPt in soil and SOC in each aggregate generally increased with increasing manure input, GRSPt and GRSPe in each aggregate showed varying changes with manure input. Both GRSP in macroaggregates (2000-250 μm) were significantly higher under low manure input, a pattern consistent with changes in soil aggregate stability. Constituting 38~49% of soil mass, macroaggregates likely contributed to the nonlinear changes of aggregate stability under manure amendments. The regulatory process of GRSP allocations in soil aggregates has important implications for manure management under intensive agriculture.

  19. Identification of VPS13C as a Galectin-12-Binding Protein That Regulates Galectin-12 Protein Stability and Adipogenesis

    PubMed Central

    Yang, Ri-Yao; Xue, Huiting; Yu, Lan; Velayos-Baeza, Antonio; Monaco, Anthony P.; Liu, Fu-Tong

    2016-01-01

    Galectin-12, a member of the galectin family of β-galactoside-binding animal lectins, is preferentially expressed in adipocytes and required for adipocyte differentiation in vitro. This protein was recently found to regulate lipolysis, whole body adiposity, and glucose homeostasis in vivo. Here we identify VPS13C, a member of the VPS13 family of vacuolar protein sorting-associated proteins highly conserved throughout eukaryotic evolution, as a major galectin-12-binding protein. VPS13C is upregulated during adipocyte differentiation, and is required for galectin-12 protein stability. Knockdown of Vps13c markedly reduces the steady-state levels of galectin-12 by promoting its degradation through primarily the lysosomal pathway, and impairs adipocyte differentiation. Our studies also suggest that VPS13C may have a broader role in protein quality control. The regulation of galectin-12 stability by VPS13C could potentially be exploited for therapeutic intervention of obesity and related metabolic diseases. PMID:27073999

  20. Identification of VPS13C as a Galectin-12-Binding Protein That Regulates Galectin-12 Protein Stability and Adipogenesis.

    PubMed

    Yang, Ri-Yao; Xue, Huiting; Yu, Lan; Velayos-Baeza, Antonio; Monaco, Anthony P; Liu, Fu-Tong

    2016-01-01

    Galectin-12, a member of the galectin family of β-galactoside-binding animal lectins, is preferentially expressed in adipocytes and required for adipocyte differentiation in vitro. This protein was recently found to regulate lipolysis, whole body adiposity, and glucose homeostasis in vivo. Here we identify VPS13C, a member of the VPS13 family of vacuolar protein sorting-associated proteins highly conserved throughout eukaryotic evolution, as a major galectin-12-binding protein. VPS13C is upregulated during adipocyte differentiation, and is required for galectin-12 protein stability. Knockdown of Vps13c markedly reduces the steady-state levels of galectin-12 by promoting its degradation through primarily the lysosomal pathway, and impairs adipocyte differentiation. Our studies also suggest that VPS13C may have a broader role in protein quality control. The regulation of galectin-12 stability by VPS13C could potentially be exploited for therapeutic intervention of obesity and related metabolic diseases.

  1. Cooperative hydration effect causes thermal unfolding of proteins and water activity plays a key role in protein stability in solutions.

    PubMed

    Miyawaki, Osato; Dozen, Michiko; Hirota, Kaede

    2016-08-01

    The protein unfolding process observed in a narrow temperature range was clearly explained by evaluating the small difference in the enthalpy of hydrogen-bonding between amino acid residues and the hydration of amino acid residue separately. In aqueous solutions, the effect of cosolute on the protein stability is primarily dependent on water activity, aw, the role of which has been long neglected in the literature. The effect of aw on protein stability works as a power law so that a small change in aw is amplified substantially through the cooperative hydration effect. In the present approach, the role of hydrophobic interaction stands behind. This affects protein stability indirectly through the change in solution structure caused by the existence of cosolute.

  2. Role of protein synthesis and DNA methylation in the consolidation and maintenance of long-term memory in Aplysia.

    PubMed

    Pearce, Kaycey; Cai, Diancai; Roberts, Adam C; Glanzman, David L

    2017-01-09

    Previously, we reported that long-term memory (LTM) in Aplysia can be reinstated by truncated (partial) training following its disruption by reconsolidation blockade and inhibition of PKM (Chen et al., 2014). Here, we report that LTM can be induced by partial training after disruption of original consolidation by protein synthesis inhibition (PSI) begun shortly after training. But when PSI occurs during training, partial training cannot subsequently establish LTM. Furthermore, we find that inhibition of DNA methyltransferase (DNMT), whether during training or shortly afterwards, blocks consolidation of LTM and prevents its subsequent induction by truncated training; moreover, later inhibition of DNMT eliminates consolidated LTM. Thus, the consolidation of LTM depends on two functionally distinct phases of protein synthesis: an early phase that appears to prime LTM; and a later phase whose successful completion is necessary for the normal expression of LTM. Both the consolidation and maintenance of LTM depend on DNA methylation.

  3. Solution stability and variability in a simple model of globular proteins.

    PubMed

    Sear, Richard P

    2004-01-08

    It is well known among molecular biologists that proteins with a common ancestor and that perform the same function in similar organisms, can have rather different amino-acid sequences. Mutations have altered the amino-acid sequences without affecting the function. A simple model of a protein in which the interactions are encoded by sequences of bits is introduced, and used to study how mutations can change these bits, and hence the interactions, while maintaining the stability of the protein solution. This stability is a simple minimal requirement on our model proteins which mimics part of the requirement on a real protein to be functional. The properties of our model protein, such as its second virial coefficient, are found to vary significantly from one model protein to another. It is suggested that this may also be the case for real proteins in vivo. (c) 2004 American Institute of Physics

  4. Alignment of Homologous Chromosomes and Effective Repair of Programmed DNA Double-Strand Breaks during Mouse Meiosis Require the Minichromosome Maintenance Domain Containing 2 (MCMDC2) Protein.

    PubMed

    Finsterbusch, Friederike; Ravindranathan, Ramya; Dereli, Ihsan; Stanzione, Marcello; Tränkner, Daniel; Tóth, Attila

    2016-10-01

    Orderly chromosome segregation during the first meiotic division requires meiotic recombination to form crossovers between homologous chromosomes (homologues). Members of the minichromosome maintenance (MCM) helicase family have been implicated in meiotic recombination. In addition, they have roles in initiation of DNA replication, DNA mismatch repair and mitotic DNA double-strand break repair. Here, we addressed the function of MCMDC2, an atypical yet conserved MCM protein, whose function in vertebrates has not been reported. While we did not find an important role for MCMDC2 in mitotically dividing cells, our work revealed that MCMDC2 is essential for fertility in both sexes due to a crucial function in meiotic recombination. Meiotic recombination begins with the introduction of DNA double-strand breaks into the genome. DNA ends at break sites are resected. The resultant 3-prime single-stranded DNA overhangs recruit RAD51 and DMC1 recombinases that promote the invasion of homologous duplex DNAs by the resected DNA ends. Multiple strand invasions on each chromosome promote the alignment of homologous chromosomes, which is a prerequisite for inter-homologue crossover formation during meiosis. We found that although DNA ends at break sites were evidently resected, and they recruited RAD51 and DMC1 recombinases, these recombinases were ineffective in promoting alignment of homologous chromosomes in the absence of MCMDC2. Consequently, RAD51 and DMC1 foci, which are thought to mark early recombination intermediates, were abnormally persistent in Mcmdc2-/- meiocytes. Importantly, the strand invasion stabilizing MSH4 protein, which marks more advanced recombination intermediates, did not efficiently form foci in Mcmdc2-/- meiocytes. Thus, our work suggests that MCMDC2 plays an important role in either the formation, or the stabilization, of DNA strand invasion events that promote homologue alignment and provide the basis for inter-homologue crossover formation during

  5. Alignment of Homologous Chromosomes and Effective Repair of Programmed DNA Double-Strand Breaks during Mouse Meiosis Require the Minichromosome Maintenance Domain Containing 2 (MCMDC2) Protein

    PubMed Central

    Ravindranathan, Ramya; Dereli, Ihsan; Stanzione, Marcello; Tóth, Attila

    2016-01-01

    Orderly chromosome segregation during the first meiotic division requires meiotic recombination to form crossovers between homologous chromosomes (homologues). Members of the minichromosome maintenance (MCM) helicase family have been implicated in meiotic recombination. In addition, they have roles in initiation of DNA replication, DNA mismatch repair and mitotic DNA double-strand break repair. Here, we addressed the function of MCMDC2, an atypical yet conserved MCM protein, whose function in vertebrates has not been reported. While we did not find an important role for MCMDC2 in mitotically dividing cells, our work revealed that MCMDC2 is essential for fertility in both sexes due to a crucial function in meiotic recombination. Meiotic recombination begins with the introduction of DNA double-strand breaks into the genome. DNA ends at break sites are resected. The resultant 3-prime single-stranded DNA overhangs recruit RAD51 and DMC1 recombinases that promote the invasion of homologous duplex DNAs by the resected DNA ends. Multiple strand invasions on each chromosome promote the alignment of homologous chromosomes, which is a prerequisite for inter-homologue crossover formation during meiosis. We found that although DNA ends at break sites were evidently resected, and they recruited RAD51 and DMC1 recombinases, these recombinases were ineffective in promoting alignment of homologous chromosomes in the absence of MCMDC2. Consequently, RAD51 and DMC1 foci, which are thought to mark early recombination intermediates, were abnormally persistent in Mcmdc2-/- meiocytes. Importantly, the strand invasion stabilizing MSH4 protein, which marks more advanced recombination intermediates, did not efficiently form foci in Mcmdc2-/- meiocytes. Thus, our work suggests that MCMDC2 plays an important role in either the formation, or the stabilization, of DNA strand invasion events that promote homologue alignment and provide the basis for inter-homologue crossover formation during

  6. Phosphorylation at Serines 216 and 221 Is Important for Drosophila HeT-A Gag Protein Stability

    PubMed Central

    Brar, Sukhdev S.; Petrovich, Robert M.; Williams, Jason G.; Mason, James M.

    2013-01-01

    Telomeres from Drosophila appear to be very different from those of other organisms – in size and the mechanism of their maintenance. In the absence of the enzyme telomerase, Drosophila telomeres are maintained by retrotransposition of three elements, HeT-A, TART, and TAHRE, but details of their transposition mechanisms are not known. Here we characterized some biochemical characteristics of the HeT-A Gag protein encoded by the HeT-A element to understand this mechanism. The HeT-A Gag protein when overexpressed in S2 cells was localized to the nucleus but was resistant to high salt, detergents and nuclease extraction treatments. Analysis of the HeT-A Gag protein by tandem mass spectrophotometry revealed that serines 216 and 221 are phosphorylated. Substituting these serines with alanine or aspartic acid by site-directed mutagenesis did not result in any changes in HeT-A Gag translocation across the nucleus, suggesting that phosphorylation of these sites is not associated with HeT-A Gag translocation, but time course experiments showed that these phosphorylation sites are important for Gag-protein stability. PMID:24058682

  7. An optimized strategy to measure protein stability highlights differences between cold and hot unfolded states

    NASA Astrophysics Data System (ADS)

    Alfano, Caterina; Sanfelice, Domenico; Martin, Stephen R.; Pastore, Annalisa; Temussi, Piero Andrea

    2017-05-01

    Macromolecular crowding ought to stabilize folded forms of proteins, through an excluded volume effect. This explanation has been questioned and observed effects attributed to weak interactions with other cell components. Here we show conclusively that protein stability is affected by volume exclusion and that the effect is more pronounced when the crowder's size is closer to that of the protein under study. Accurate evaluation of the volume exclusion effect is made possible by the choice of yeast frataxin, a protein that undergoes cold denaturation above zero degrees, because the unfolded form at low temperature is more expanded than the corresponding one at high temperature. To achieve optimum sensitivity to changes in stability we introduce an empirical parameter derived from the stability curve. The large effect of PEG 20 on cold denaturation can be explained by a change in water activity, according to Privalov's interpretation of cold denaturation.

  8. An Anion-π Interaction Strongly Stabilizes the β-Sheet Protein WW.

    PubMed

    Smith, Mason S; Lawrence, Eliza E K; Billings, Wendy M; Larsen, Kimberlee S; Bécar, Natalie A; Price, Joshua L

    2017-09-14

    Anions have long been known to engage in stabilizing interactions with electron-deficient arenes. However, the precise nature and energetic contribution of anion-π interactions to protein stability remains a subject of debate. Here, we show that placing a negatively charged Asp in close proximity to electron-rich Phe in a reverse turn within the WW domain results in a favorable interaction that increases WW conformational stability by -1.3 kcal/mol.

  9. Membrane Protein Stability Analyses by Means of Protein Energy Profiles in Case of Nephrogenic Diabetes Insipidus

    PubMed Central

    Heinke, Florian; Labudde, Dirk

    2012-01-01

    Diabetes insipidus (DI) is a rare endocrine, inheritable disorder with low incidences in an estimated one per 25,000–30,000 live births. This disease is characterized by polyuria and compensatory polydypsia. The diverse underlying causes of DI can be central defects, in which no functional arginine vasopressin (AVP) is released from the pituitary or can be a result of defects in the kidney (nephrogenic DI, NDI). NDI is a disorder in which patients are unable to concentrate their urine despite the presence of AVP. This antidiuretic hormone regulates the process of water reabsorption from the prourine that is formed in the kidney. It binds to its type-2 receptor (V2R) in the kidney induces a cAMP-driven cascade, which leads to the insertion of aquaporin-2 water channels into the apical membrane. Mutations in the genes of V2R and aquaporin-2 often lead to NDI. We investigated a structure model of V2R in its bound and unbound state regarding protein stability using a novel protein energy profile approach. Furthermore, these techniques were applied to the wild-type and selected mutations of aquaporin-2. We show that our results correspond well to experimental water ux analysis, which confirms the applicability of our theoretical approach to equivalent problems. PMID:22474537

  10. Computational comparison of a calcium-dependent jellyfish protein (apoaequorin) and calmodulin-cholesterol in short-term memory maintenance.

    PubMed

    Morrill, Gene A; Kostellow, Adele B; Gupta, Raj K

    2017-03-06

    Memory reconsolidation and maintenance depend on calcium channels and on calcium/calmodulin-dependent kinases regulating protein turnover in the hippocampus. Ingestion of a jellyfish protein, apoaequorin, reportedly protects and/or improves verbal learning in adults and is currently widely advertised for use by the elderly. Apoaequorin is a member of the EF-hand calcium binding family of proteins that includes calmodulin. Calmodulin-1 (148 residues) differs from Apoaequorin (195 residues) in that it contains four rather than three Ca(2+)-binding sites and three rather than four cholesterol-binding (CRAC, CARC) domains. All three cholesterol-binding CARC domains in calmodulin have a high interaction affinity for cholesterol compared to only two high affinity CARC domains in apoaequorin. Both calmodulin and apoaequorin can form dimers with a potential of eight bound Ca(2+) ions and six high affinity-bound cholesterol molecules in calmodulin with six bound Ca(2+) ions and a mixed population of eight cholesterols bound to both CARC and CRAC domains in apoaqueorin. MEMSAT-SVM analysis indicates that both calmodulin and apoaqueorin have a pore-lining region. The Peptide-Cutter algorithm predicts that calmodulin-1 contains 11 trypsin-specific cleavage sites (compared to 21 in apoaqueorin), four of which are potentially blocked by cholesterol and three are within the Ca-binding domains and/or the pore-lining region. Three are clustered between the third and fourth Ca(2+)-binding sites. Only calmodulin pore-lining regions contain Ca(2+) binding sites and as dimers may insert into the plasma membrane of neural cells and act as Ca(2+) channels. In a dietary supplement, bound cholesterol may protect both apoaequorin and calmodulin from proteolysis in the gut as well as facilitate uptake across the blood-brain barrier. Our results suggest that a physiological calmodulin-cholesterol complex, not cholesterol-free jellyfish protein, may better serve as a dietary supplement to

  11. Analytical model for studying how environmental factors influence protein conformational stability in solution

    NASA Astrophysics Data System (ADS)

    Cheung, Jason K.; Raverkar, Prajakta S.; Truskett, Thomas M.

    2006-12-01

    We introduce an analytical modeling strategy for probing the conformational stability of globular proteins in aqueous solution. In this approach, the intrinsic (i.e., infinite dilution) thermodynamic stability and coarse structural properties of the proteins, as well as the effective protein-protein interactions, derive from a heteropolymer collapse theory that incorporates predicted temperature- and pressure-dependent hydrophobic interactions. Protein concentration effects are estimated by integrating this information into a molecular thermodynamic model, which is an ad hoc generalization of the exact equilibrium theory of a one-dimensional binary mixture of square-well particles that interconvert through an isomerization (i.e., folding) reaction. The end result is an analytical multiscale modeling approach which, although still schematic, can predict that folded proteins exhibit a closed-loop region of stability in the pressure-temperature plane and that protein concentration has a nonmonotonic effect on protein stability, results consistent with qualitative trends observed in both experiments of protein solutions and simulations of coarse-grained protein models.

  12. Stabilization of oil-in-water emulsions by cod protein extracts.

    PubMed

    Petursson, Sigthor; Decker, Eric A; McClements, D Julian

    2004-06-16

    The ability of two protein fractions extracted from cod to form and stabilize oil-in-water emulsions was examined: a high salt extracted fraction (HSE protein) and a pH 3 acid extracted fraction (AE protein). Both fractions consisted of a complex mixture of different proteins, with the predominant one being myosin (200 kDa). The two protein fractions were used to prepare 5 wt % corn oil-in-water emulsions at ambient temperature (pH 3.0, 10 mM citrate-imidazole buffer). Emulsions with relatively small mean droplet diameters (d(3,2) < 1 microm) and good creaming stability (> 9 days) could be produced at protein concentrations > or =0.2 wt % for both fractions. The isoelectric point of droplets stabilized by both protein fractions was pH approximately 5. The emulsions were stable to droplet flocculation and creaming at relatively low pH (< or =4) and NaCl concentrations (< or =150 mM) when stored at room temperature. In the absence of salt, the emulsions were also stable to thermal treatment (30-90 degrees C for 30 min), but in the presence of 100 mM NaCl droplet flocculation and creaming were observed in some of the emulsions, particularly those stabilized by the AE fraction. The results suggest that protein fractions extracted from cod can be used as emulsifiers to form and stabilize food emulsions.

  13. A chloroplast-targeted DnaJ protein contributes to maintenance of photosystem II under chilling stress

    PubMed Central

    Meng, Qingwei

    2014-01-01

    DnaJ proteins act as essential molecular chaperones in protein homeostasis and protein complex stabilization under stress conditions. The roles of a tomato (Lycopersicon esculentum) chloroplast-targeted DnaJ protein (LeCDJ1), whose expression was upregulated by treatment at 4 and 42 °C, and with high light, NaCl, polyethylene glycol, and H2O2, were investigated here using sense and antisense transgenic tomatoes. The sense plants exhibited not only higher chlorophyll content, fresh weight and net photosynthetic rate, but also lower accumulation of reactive oxygen species and membrane damage under chilling stress. Moreover, the maximal photochemistry efficiency of photosystem II (PSII) (F v/F m) and D1 protein content were higher in the sense plants and lower in the antisense plants, and the photoinhibitory quenching was lower in the sense plants and higher in the antisense plants, suggesting that the inhibition of PSII was less severe in the sense plants and more severe in the antisense plants compared with the wild type. Furthermore, the PSII protein complexes were also more stable in the sense plants. Interestingly, the sense plants treated with streptomycin (SM), an inhibitor of organellar translation, still showed higher F v/F m, D1 protein content and PSII stability than the SM-untreated antisense plants. This finding suggested that the protective effect of LeCDJ1 on PSII was, at least partially, independent of D1 protein synthesis. Furthermore, chloroplast heat-shock protein 70 was identified as the partner of LeCDJ1. These results indicate that LeCDJ1 has essential functions in maintaining PSII under chilling stress. PMID:24227338

  14. Hydrophobic environment is a key factor for the stability of thermophilic proteins.

    PubMed

    Gromiha, M Michael; Pathak, Manish C; Saraboji, Kadhirvel; Ortlund, Eric A; Gaucher, Eric A

    2013-04-01

    The stability of thermophilic proteins has been viewed from different perspectives and there is yet no unified principle to understand this stability. It would be valuable to reveal the most important interactions for designing thermostable proteins for such applications as industrial protein engineering. In this work, we have systematically analyzed the importance of various interactions by computing different parameters such as surrounding hydrophobicity, inter-residue interactions, ion-pairs and hydrogen bonds. The importance of each interaction has been determined by its predicted relative contribution in thermophiles versus the same contribution in mesophilic homologues based on a dataset of 373 protein families. We predict that hydrophobic environment is the major factor for the stability of thermophilic proteins and found that 80% of thermophilic proteins analyzed showed higher hydrophobicity than their mesophilic counterparts. Ion pairs, hydrogen bonds, and interaction energy are also important and favored in 68%, 50%, and 62% of thermophilic proteins, respectively. Interestingly, thermophilic proteins with decreased hydrophobic environments display a greater number of hydrogen bonds and/or ion pairs. The systematic elimination of mesophilic proteins based on surrounding hydrophobicity, interaction energy, and ion pairs/hydrogen bonds, led to correctly identifying 95% of the thermophilic proteins in our analyses. Our analysis was also applied to another, more refined set of 102 thermophilic-mesophilic pairs, which again identified hydrophobicity as a dominant property in 71% of the thermophilic proteins. Further, the notion of surrounding hydrophobicity, which characterizes the hydrophobic behavior of residues in a protein environment, has been applied to the three-dimensional structures of elongation factor-Tu proteins and we found that the thermophilic proteins are enriched with a hydrophobic environment. The results obtained in this work highlight the

  15. Stabilizing Protein Effects on the Pressure Sensitivity of Fluorescent Gold Nanoclusters

    DTIC Science & Technology

    2016-01-13

    ARL-TR-7572 ● JAN 2016 US Army Research Laboratory Stabilizing Protein Effects on the Pressure Sensitivity of Fluorescent Gold ...JAN 2016 US Army Research Laboratory Stabilizing Protein Effects on the Pressure Sensitivity of Fluorescent Gold Nanoclusters by Abby...Fluorescent Gold Nanoclusters 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Abby L West, Mark H Griep

  16. Long term weight maintenance after advice to consume low carbohydrate, higher protein diets--a systematic review and meta analysis.

    PubMed

    Clifton, P M; Condo, D; Keogh, J B

    2014-03-01

    Meta analysis of short term trials indicates that a higher protein, lower carbohydrate weight loss diet enhances fat mass loss and limits lean mass loss compared with a normal protein weight loss diet. Whether this benefit persists long term is not clear. We selected weight loss studies in adults with at least a 12 month follow up in which a higher percentage protein/lower carbohydrate diet was either planned or would be expected for either weight loss or weight maintenance. Studies were selected regardless of the success of the advice but difference in absolute and percentage protein intake at 12 months was used as a moderator in the analysis. Data was analysed using Comprehensive Meta analysis V2 using a random effects analysis. As many as 32 studies with 3492 individuals were analysed with data on fat and lean mass, glucose and insulin from 18 to 22 studies and lipids from 28 studies. A recommendation to consume a lower carbohydrate, higher protein diet in mostly short term intensive interventions with long term follow up was associated with better weight and fat loss but the effect size was small-standardised means of 0.14 and 0.22, p = 0.008 and p < 0.001 respectively (equivalent to 0.4 kg for both). A difference of 5% or greater in percentage protein between diets at 12 mo was associated with a 3 fold greater effect size compared with <5% (p = 0.038) in fat mass (0.9 vs. 0.3 kg). Fasting triglyceride and insulin were also lower with high protein diets with effect sizes of 0.17 and 0.22, p = 0.003 and p = 0.042 respectively. Other lipids and glucose were not different. The short term benefit of higher protein diets appears to persist to a small degree long term. Benefits are greater with better compliance to the diet. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

    PubMed

    Tong, X; Kono, T; Evans-Molina, C

    2015-06-18

    The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b

  18. TRAF Family Proteins Regulate Autophagy Dynamics by Modulating AUTOPHAGY PROTEIN6 Stability in Arabidopsis[OPEN

    PubMed Central

    Qi, Hua; Xia, Fan-Nv; Xie, Li-Juan; Yu, Lu-Jun; Chen, Qin-Fang; Jiang, Liwen

    2017-01-01

    Eukaryotic cells use autophagy to recycle cellular components. During autophagy, autophagosomes deliver cytoplasmic contents to the vacuole or lysosome for breakdown. Mammalian cells regulate the dynamics of autophagy via ubiquitin-mediated proteolysis of autophagy proteins. Here, we show that the Arabidopsis thaliana Tumor necrosis factor Receptor-Associated Factor (TRAF) family proteins TRAF1a and TRAF1b (previously named MUSE14 and MUSE13, respectively) help regulate autophagy via ubiquitination. Upon starvation, cytoplasmic TRAF1a and TRAF1b translocated to autophagosomes. Knockout traf1a/b lines showed reduced tolerance to nutrient deficiency, increased salicylic acid and reactive oxygen species levels, and constitutive cell death in rosettes, resembling the phenotypes of autophagy-defective mutants. Starvation-activated autophagosome accumulation decreased in traf1a/b root cells, indicating that TRAF1a and TRAF1b function redundantly in regulating autophagosome formation. TRAF1a and TRAF1b interacted in planta with ATG6 and the RING finger E3 ligases SINAT1, SINAT2, and SINAT6 (with a truncated RING-finger domain). SINAT1 and SINAT2 require the presence of TRAF1a and TRAF1b to ubiquitinate and destabilize AUTOPHAGY PROTEIN6 (ATG6) in vivo. Conversely, starvation-induced SINAT6 reduced SINAT1- and SINAT2-mediated ubiquitination and degradation of ATG6. Consistently, SINAT1/SINAT2 and SINAT6 knockout mutants exhibited increased tolerance and sensitivity, respectively, to nutrient starvation. Therefore, TRAF1a and TRAF1b function as molecular adaptors that help regulate autophagy by modulating ATG6 stability in Arabidopsis. PMID:28351989

  19. Identification of rare slipknots in proteins and their implications for stability and folding.

    PubMed

    King, Neil P; Yeates, Eric O; Yeates, Todd O

    2007-10-12

    Among the thousands of known three-dimensional protein folds, only a few have been found whose backbones are in knotted configurations. The rarity of knotted proteins has important implications for how natural proteins reach their natively folded states. Proteins with such unusual features offer unique opportunities for studying the relationships between structure, folding, and stability. Here we report the identification of a unique slipknot feature in the fold of a well-known thermostable protein, alkaline phosphatase. A slipknot is created when a knot is formed by part of a protein chain, after which the backbone doubles back so that the entire structure becomes unknotted in a mathematical sense. Slipknots are therefore not detected by computational tests that look for knots in complete protein structures. A computational survey looking specifically for slipknots in the Protein Data Bank reveals a few other instances in addition to alkaline phosphatase. Unexpected similarities are noted among some of the proteins identified. In addition, two transmembrane proteins are found to contain slipknots. Finally, mutagenesis experiments on alkaline phosphatase are used to probe the contribution the slipknot feature makes to thermal stability. The trends and conserved features observed in these proteins provide new insights into mechanisms of protein folding and stability.

  20. Molecular origins of surfactant-mediated stabilization of protein drugs.

    PubMed

    Lee, Hyo Jin; McAuley, Arnold; Schilke, Karl F; McGuire, Joseph

    2011-10-01

    Loss of activity through aggregation and surface-induced denaturation is a significant problem in the production, formulation and administration of therapeutic proteins. Surfactants are commonly used in upstream and downstream processing and drug formulation. However, the effectiveness of a surfactant strongly depends on its mechanism(s) of action and properties of the protein and interfaces. Surfactants can modulate adsorption loss and aggregation by coating interfaces and/or participating in protein-surfactant associations. Minimizing protein loss from colloidal and interfacial interaction requires a fundamental understanding of the molecular factors underlying surfactant effectiveness and mechanism. These concepts provide direction for improvements in the manufacture and finishing of therapeutic proteins. We summarize the roles of surfactants, proteins, and surfactant-protein complexes in modulating interfacial behavior and aggregation. These events depend on surfactant properties that may be quantified using a thermodynamic model, to provide physical/chemical direction for surfactant selection or design, and to effectively reduce aggregation and adsorption loss. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. Gap junction proteins: master regulators of the planarian stem cell response to tissue maintenance and injury.

    PubMed

    Peiris, T Harshani; Oviedo, Néstor J

    2013-01-01

    Gap junction (GJ) proteins are crucial mediators of cell-cell communication during embryogenesis, tissue regeneration and disease. GJ proteins form plasma membrane channels that facilitate passage of small molecules across cells and modulate signaling pathways and cellular behavior in different tissues. These properties have been conserved throughout evolution, and in most invertebrates GJ proteins are known as innexins. Despite their critical relevance for physiology and disease, the mechanisms by which GJ proteins modulate cell behavior are poorly understood. This review summarizes findings from recent work that uses planarian flatworms as a paradigm to analyze GJ proteins in the complexity of the whole organism. The planarian model allows access to a large pool of adult somatic stem cells (known as neoblasts) that support physiological cell turnover and tissue regeneration. Innexin proteins are present in planarians and play a fundamental role in controlling neoblast behavior. We discuss the possibility that GJ proteins participate as cellular sensors that inform neoblasts about local and systemic physiological demands. We believe that functional analyses of GJ proteins will bring a complementary perspective to studies that focus on the temporal expression of genes. Finally, integrating functional studies along with molecular genetics and epigenetic approaches would expand our understanding of cellular regulation in vivo and greatly enhance the possibilities for rationally modulating stem cell behavior in their natural environment. This article is part of a Special Issue entitled: The communicating junctions, roles and dysfunctions. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Effect of pasteurization on the protein composition and oxidative stability of beer during storage.

    PubMed

    Lund, Marianne N; Hoff, Signe; Berner, Torben S; Lametsch, René; Andersen, Mogens L

    2012-12-19

    The impacts of pasteurization of a lager beer on protein composition and the oxidative stability were studied during storage at 22 °C for 426 days in the dark. Pasteurization clearly improved the oxidative stability of beer determined by ESR spectroscopy, whereas it had a minor negative effect on the volatile profile by increasing volatile compounds that is generally associated with heat treatment and a loss of fruity ester aroma. A faster rate of radical formation in unpasteurized beer was consistent with a faster consumption of sulfite. Beer proteins in the unpasteurized beer were more degraded, most likely due to proteolytic enzyme activity of yeast remnants and more precipitation of proteins was also observed. The differences in soluble protein content and composition are suggested to result in differences in the contents of prooxidative metals as a consequence of the proteins ability to bind metals. This also contributes to the differences in oxidative stabilities of the beers.

  3. Stabilization of structure in near-infrared fluorescent proteins by binding of biliverdin chromophore

    NASA Astrophysics Data System (ADS)

    Stepanenko, Olesya V.; Stepanenko, Olga V.; Bublikov, G. S.; Kuznetsova, I. M.; Verkhusha, V. V.; Turoverov, K. K.

    2017-07-01

    Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes and their mutants with different location of Cys residues, which able to bind a biliverdin chromophore, or without these Cys residues were studied using intrinsic tryptophan fluorescence, NIR fluorescence and circular dichroism. It was shown that a covalent binding of the biliverdin chromophore to a Cys residue via thioether group substantially stabilizes the spatial structure of NIR FPs. The stability of the protein structure and the chromophore association strength strongly depends on the location of Cys residues and decreases in the following order: a protein with Cys residues in both domains, a protein with Cys in PAS domains, and a protein with Cys in GAF domains. NIR FPs without Cys residues capable to covalently attach biliverdin have the lowest stability, comparable to NIR FP apoforms.

  4. Stability of whey-protein-stabilized oil-in-water emulsions during chilled storage and temperature cycling.

    PubMed

    Kiokias, Sotirios; Reiffers-Magnani, Christel K; Bot, Arjen

    2004-06-16

    The stability of heat-treated and/or acidified, partly-crystalline-fat-based, whey-protein-stabilized oil-in-water (o/w) emulsions against partial coalescence was investigated during chilled storage (at 5 degrees C) and repeated temperature cycling (three times between 5 and 25 degrees C). Experiments focused on the evolution of firmness and droplet size (using pulsed field gradient NMR and scanning electron microscopy). Besides the effects of denaturation and/or acidification, the influence of the droplet size of the dispersed phase on emulsion stability was investigated also. It was found that heat treatment or acidification before emulsification led to unstable emulsions during temperature cycling, whereas heat treatment after acidification resulted in stable emulsions.

  5. Water-in-Oil Microemulsions for Protein Delivery: Loading Optimization and Stability.

    PubMed

    Perinelli, Diego R; Cespi, Marco; Pucciarelli, Stefania; Vincenzetti, Silvia; Casettari, Luca; Lam, Jenny K W; Logrippo, Serena; Canala, Elisa; Soliman, Mahmoud E; Bonacucina, Giulia; Palmieri, Giovanni F

    2017-01-01

    Microemulsions are attractive delivery systems for therapeutic proteins and peptides due to their ability to enhance bioavailability. Although different proteins and peptides have been successfully delivered through such ternary systems, no information can be found about protein loading and the formulation stability when such microemulsions are prepared with pharmaceuticallyapproved oils and surfactants. The aim of this work was to optimise a ternary system consisting of water/ ethyl oleate/Span® 80-Tween® 80 and to determine its protein loading capacity and stability, using bovine serum albumin (BSA) as a model of biomolecule. The optimization was carried out using a Central Composite Design and all the prepared formulations were characterised through dynamic light scattering, rheology, optical and polarized microscopy. Subsequently, the maximum loading capacity was determined and the stability of the final microemulsion with the highest content of protein was followed over six months. To investigate the structural features of the protein, BSA was recovered from the microemulsion and analysed through fluorescence spectroscopy. After incorporation of the protein in the microemulsion, a decrease of its aqueous solubility was observed. However, the formulation remained stable over six months and the native-like state of the recovered protein was demonstrated by fluorescence spectroscopy Conclusion: This study demonstrated the feasibility of preparing microemulsions with the highest content of protein and their long-term stability. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  6. Principles and equations for measuring and interpreting protein stability: From monomer to tetramer.

    PubMed

    Bedouelle, Hugues

    2016-02-01

    The ability to measure the thermodynamic stability of proteins with precision is important for both academic and applied research. Such measurements rely on mathematical models of the protein denaturation profile, i.e. the relation between a global protein signal, corresponding to the folding states in equilibrium, and the variable value of a denaturing agent, either heat or a chemical molecule, e.g. urea or guanidinium hydrochloride. In turn, such models rely on a handful of physical laws: the laws of mass action and conservation, the law that relates the protein signal and concentration, and the one that relates stability and denaturant value. So far, equations have been derived mainly for the denaturation profiles of homomeric proteins. Here, we review the underlying basic physical laws and show in detail how to derive model equations for the unfolding equilibria of homomeric or heteromeric proteins up to trimers and potentially tetramers, with or without folding intermediates, and give full demonstrations. We show that such equations cannot be derived for pentamers or higher oligomers except in special degenerate cases. We expand the method to signals that do not correspond to extensive protein properties. We review and expand methods for uncovering hidden intermediates of unfolding. Finally, we review methods for comparing and interpreting the thermodynamic parameters that derive from stability measurements for cognate wild-type and mutant proteins. This work should provide a robust theoretical basis for measuring the stability of complex proteins.

  7. Molecular basis for polyol-induced protein stability revealed by molecular dynamics simulations.

    PubMed

    Liu, Fu-Feng; Ji, Luo; Zhang, Lin; Dong, Xiao-Yan; Sun, Yan

    2010-06-14

    Molecular dynamics simulations of chymotrypsin inhibitor 2 in different polyols (glycerol, xylitol, sorbitol, trehalose, and sucrose) at 363 K were performed to probe the molecular basis of the stabilizing effect, and the data in water, ethanol, and glycol were compared. It is found that protein protection by polyols is positively correlated with both the molecular volume and the fractional polar surface area, and the former contributes more significantly to the protein's stability. Polyol molecules have only a few direct hydrogen bonds with the protein, and the number of hydrogen bonds between a polyol and the protein is similar for different polyols. Thus, it is concluded that the direct interactions contribute little to the stabilizing effect. It is clarified that the preferential exclusion of the polyols is the origin of their protective effects, and it increases with increasing polyol size. Namely, there is preferential hydration on the protein surface (2 A), and polyol molecules cluster around the protein at a distance of about 4 A. The preferential exclusion of polyols leads to indirect interactions that prevent the protein from thermal unfolding. The water structure becomes more ordered with increasing the polyol size. So, the entropy of water in the first hydration shell decreases, and a larger extent of decrease is observed with increasing polyol size, leading to larger transfer free energy. The findings suggest that polyols protect the protein from thermal unfolding via indirect interactions. The work has thus elucidated the molecular mechanism of structural stability of the protein in polyol solutions.

  8. Molecular basis for polyol-induced protein stability revealed by molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Liu, Fu-Feng; Ji, Luo; Zhang, Lin; Dong, Xiao-Yan; Sun, Yan

    2010-06-01

    Molecular dynamics simulations of chymotrypsin inhibitor 2 in different polyols (glycerol, xylitol, sorbitol, trehalose, and sucrose) at 363 K were performed to probe the molecular basis of the stabilizing effect, and the data in water, ethanol, and glycol were compared. It is found that protein protection by polyols is positively correlated with both the molecular volume and the fractional polar surface area, and the former contributes more significantly to the protein's stability. Polyol molecules have only a few direct hydrogen bonds with the protein, and the number of hydrogen bonds between a polyol and the protein is similar for different polyols. Thus, it is concluded that the direct interactions contribute little to the stabilizing effect. It is clarified that the preferential exclusion of the polyols is the origin of their protective effects, and it increases with increasing polyol size. Namely, there is preferential hydration on the protein surface (2 Å), and polyol molecules cluster around the protein at a distance of about 4 Å. The preferential exclusion of polyols leads to indirect interactions that prevent the protein from thermal unfolding. The water structure becomes more ordered with increasing the polyol size. So, the entropy of water in the first hydration shell decreases, and a larger extent of decrease is observed with increasing polyol size, leading to larger transfer free energy. The findings suggest that polyols protect the protein from thermal unfolding via indirect interactions. The work has thus elucidated the molecular mechanism of structural stability of the protein in polyol solutions.

  9. Accelerated Disease Onset with Stabilized Familial Amyotrophic Lateral Sclerosis (ALS)-linked Mutant TDP-43 Proteins*

    PubMed Central

    Watanabe, Shoji; Kaneko, Kumi; Yamanaka, Koji

    2013-01-01

    Abnormal protein accumulation is a pathological hallmark of neurodegenerative diseases, including accumulation of TAR DNA-binding protein 43 (TDP-43) in amyotrophic lateral sclerosis (ALS). Dominant mutations in the TDP-43 gene are causative for familial ALS; however, the relationship between mutant protein biochemical phenotypes and disease course and their significance to disease pathomechanism are not known. Here, we found that longer half-lives of mutant proteins correlated with accelerated disease onset. Based on our findings, we established a cell model in which chronic stabilization of wild-type TDP-43 protein provoked cytotoxicity and recapitulated pathogenic protein cleavage and insolubility to the detergent Sarkosyl, TDP-43 properties that have been observed in sporadic ALS lesions. Furthermore, these cells showed proteasomal impairment and dysregulation of their own mRNA levels. These results suggest that chronically increased stability of mutant or wild-type TDP-43 proteins results in a gain of toxicity through abnormal proteostasis. PMID:23235148

  10. Modification of proteins with cyclodextrins prevents aggregation and surface adsorption and increases thermal stability.

    PubMed

    Prashar, Deepali; Cui, DaWei; Bandyopadhyay, Debjyoti; Luk, Yan-Yeung

    2011-11-01

    This work describes a general approach for preventing protein aggregation and surface adsorption by modifying proteins with β-cyclodextrins (βCD) via an efficient water-driven ligation. As compared to native unmodified proteins, the cyclodextrin-modified proteins (lysozyme and RNase A) exhibit significant reduction in aggregation, surface adsorption and increase in thermal stability. These results reveal a new chemistry for preventing protein aggregation and surface adsorption that is likely of different mechanisms than that by modifying proteins with poly(ethylene glycol).

  11. Novel regulation of Ski protein stability and endosomal sorting by actin cytoskeleton dynamics in hepatocytes.

    PubMed

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A; Macías-Silva, Marina

    2015-02-13

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration.

  12. Novel Regulation of Ski Protein Stability and Endosomal Sorting by Actin Cytoskeleton Dynamics in Hepatocytes*

    PubMed Central

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R.; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A.; Macías-Silva, Marina

    2015-01-01

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration. PMID:25561741

  13. Rigidity versus flexibility: the dilemma of understanding protein thermal stability.

    PubMed

    Karshikoff, Andrey; Nilsson, Lennart; Ladenstein, Rudolf

    2015-10-01

    The role of fluctuations in protein thermostability has recently received considerable attention. In the current literature a dualistic picture can be found: thermostability seems to be associated with enhanced rigidity of the protein scaffold in parallel with the reduction of flexible parts of the structure. In contradiction to such arguments it has been shown by experimental studies and computer simulation that thermal tolerance of a protein is not necessarily correlated with the suppression of internal fluctuations and mobility. Both concepts, rigidity and flexibility, are derived from mechanical engineering and represent temporally insensitive features describing static properties, neglecting that relative motion at certain time scales is possible in structurally stable regions of a protein. This suggests that a strict separation of rigid and flexible parts of a protein molecule does not describe the reality correctly. In this work the concepts of mobility/flexibility versus rigidity will be critically reconsidered by taking into account molecular dynamics calculations of heat capacity and conformational entropy, salt bridge networks, electrostatic interactions in folded and unfolded states, and the emerging picture of protein thermostability in view of recently developed network theories. Last, but not least, the influence of high temperature on the active site and activity of enzymes will be considered. © 2015 FEBS.

  14. Effect of Oxygen-containing Functional Groups on Protein Stability in Ionic Liquid Solutions

    NASA Technical Reports Server (NTRS)

    Turner, Megan B.; Holbrey, John D.; Spear, Scott K.; Pusey, Marc L.; Rogers, Robin D.

    2004-01-01

    The ability of functionalized ionic liquids (ILs) to provide an environment of increased stability for biomolecules has been studied. Serum albumin is an inexpensive, widely available protein that contributes to the overall colloid osmotic blood pressure within the vascular system. Albumin is used in the present study as a marker of biomolecular stability in the presence of various ILs in a range of concentrations. The incorporation of hydroxyl functionality into the methylimidazolium-based cation leads to increased protein stability detected by fluorescence spectroscopy and circular dichroic (CD) spectrometry.

  15. Effect of Oxygen-containing Functional Groups on Protein Stability in Ionic Liquid Solutions

    NASA Technical Reports Server (NTRS)

    Turner, Megan B.; Holbrey, John D.; Spear, Scott K.; Pusey, Marc L.; Rogers, Robin D.

    2004-01-01

    The ability of functionalized ionic liquids (ILs) to provide an environment of increased stability for biomolecules has been studied. Serum albumin is an inexpensive, widely available protein that contributes to the overall colloid osmotic blood pressure within the vascular system. Albumin is used in the present study as a marker of biomolecular stability in the presence of various ILs in a range of concentrations. The incorporation of hydroxyl functionality into the methylimidazolium-based cation leads to increased protein stability detected by fluorescence spectroscopy and circular dichroic (CD) spectrometry.

  16. A structural-maintenance-of-chromosomes hinge domain-containing protein is required for RNA-directed DNA methylation.

    PubMed

    Kanno, Tatsuo; Bucher, Etienne; Daxinger, Lucia; Huettel, Bruno; Böhmdorfer, Gudrun; Gregor, Wolfgang; Kreil, David P; Matzke, Marjori; Matzke, Antonius J M

    2008-05-01

    RNA-directed DNA methylation (RdDM) is a process in which dicer-generated small RNAs guide de novo cytosine methylation at the homologous DNA region. To identify components of the RdDM machinery important for Arabidopsis thaliana development, we targeted an enhancer active in meristems for methylation, which resulted in silencing of a downstream GFP reporter gene. This silencing system also features secondary siRNAs, which trigger methylation that spreads beyond the targeted enhancer region. A screen for mutants defective in meristem silencing and enhancer methylation retrieved six dms complementation groups, which included the known factors DRD1 (ref. 3; a SNF2-like chromatin-remodeling protein) and Pol IVb subunits. Additionally, we identified a previously unknown gene DMS3 (At3g49250), encoding a protein similar to the hinge-domain region of structural maintenance of chromosomes (SMC) proteins. This finding implicates a putative chromosome architectural protein that can potentially link nucleic acids in facilitating an RNAi-mediated epigenetic modification involving secondary siRNAs and spreading of DNA methylation.

  17. A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluorescent protein.

    PubMed

    Sokalingam, Sriram; Raghunathan, Govindan; Soundrarajan, Nagasundarapandian; Lee, Sun-Gu

    2012-01-01

    Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering.

  18. A Study on the Effect of Surface Lysine to Arginine Mutagenesis on Protein Stability and Structure Using Green Fluorescent Protein

    PubMed Central

    Sokalingam, Sriram; Raghunathan, Govindan; Soundrarajan, Nagasundarapandian; Lee, Sun-Gu

    2012-01-01

    Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering. PMID:22792305

  19. Electronic polarization is important in stabilizing the native structures of proteins.

    PubMed

    Ji, Chang G; Zhang, John Z H

    2009-12-10

    Quantum mechanical computations of proteins based on the molecular fragment approach have been carried out, and polarized protein-specific charges have been derived to provide accurate electrostatic interactions for a benchmark set of proteins. Our study shows that, under the polarized protein-specific force field, the native structure indeed corresponds to the lowest-energy conformation for these proteins. In contrast, when a standard mean-field force field such as AMBER is used, the energies of many decoy structures of proteins could be lower than those of the native structures. Furthermore, MD simulations were carried out and verified that the native structures of these proteins not only are statically more stable but are also dynamically more stable under the polarized protein-specific force field. The present results, together with several recent studies, provide strong evidence that protein polarization is critical to stabilizing the native structures of proteins.

  20. Combined effect of tissue stabilization and protein extraction methods on phosphoprotein analysis.

    PubMed

    Kofanova, Olga A; Fack, Fred; Niclou, Simone P; Betsou, Fay

    2013-06-01

    Preanalytical conditions applied during sample collection and processing can affect the detection or quantification of unstable phosphoprotein biomarkers. We evaluated the consequences of tissue stabilization and protein extraction methods on phosphoprotein analysis. The effects of stabilization techniques (heat stabilization, snap-freezing) and time on the levels of phosphoproteins, including phospho-Akt, p-ERK 1/2, p-IkBα, p-JNK, and p38 MAPK, were evaluated using a BioPlex phosphoprotein assay. Additionally, two different protein extraction protocols, using different extraction buffers (8 M urea buffer, or Bio-Rad buffer without urea) were tested. For snap-frozen samples, protein extraction yields were comparable with the two buffer systems. For heat-stabilized samples, total protein yields were significantly lower following extraction in non-urea buffer. However, the concentrations of specific phosphoproteins were significantly higher in heat-stabilized samples than in the corresponding snap-frozen samples, indicating that this tissue processing method better preserved phosphoproteins. Significant differences were found between the measured phosphoprotein levels in heat-stabilized and snap-frozen tissue, suggesting that alterations occur very rapidly after tissue excision. Our results suggest that heat stabilization can be used as a tissue processing method for subsequent phosphoprotein analyses, but also suggest that the BioPlex phosphoprotein assay could be used as a possible quality control method to assess tissue sample integrity.

  1. Protein modification by acrolein: Formation and stability of cysteine adducts

    PubMed Central

    Cai, Jian; Bhatnagar, Aruni; Pierce, William M.

    2010-01-01

    The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine-acrolein adducts are difficult to identify in vitro and in vivo. In this study, model peptides with cysteine, lysine, and histidine residues were used to examine the reactivity of acrolein. Results from these experiments show that acrolein reacts rapidly with cysteine residues through Michael addition to form M+56 Da adducts. These M+56 adducts are, however, not stable, even though spontaneous dissociation of the adduct is slow. Further studies demonstrated that when acrolein and model peptides are incubated at physiological pH and temperature, the M+56 adducts decreased gradually accompanied by the increase of M+38 adducts, which are formed from intra-molecular Schiff base formation. Adduct formation with the side chains of other amino acid residues (lysine and histidine) was much slower than cysteine and required higher acrolein concentration. When cysteine residues were blocked by reaction with iodoacetamide and higher concentrations of acrolein were used, adducts of the N-terminal amino group or histidyl residues were formed but lysine adducts were not detected. Collectively, these data demonstrate that acrolein reacts avidly with protein cysteine residues and that the apparent loss of protein-acrolein Michael adducts over time may be related to the appearance of a novel (M+38) adduct. These findings may be important in identification of in vivo adducts of acrolein with protein cysteine residues. PMID:19231900

  2. Influence of protein fold stability on immunogenicity and its implications for vaccine design.

    PubMed

    Scheiblhofer, Sandra; Laimer, Josef; Machado, Yoan; Weiss, Richard; Thalhamer, Josef

    2017-05-01

    In modern vaccinology and immunotherapy, recombinant proteins more and more replace whole organisms to induce protective or curative immune responses. Structural stability of proteins is of crucial importance for efficient presentation of antigenic peptides on MHC, which plays a decisive role for triggering strong immune reactions. Areas covered: In this review, we discuss structural stability as a key factor for modulating the potency of recombinant vaccines and its importance for antigen proteolysis, presentation, and stimulation of B and T cells. Moreover, the impact of fold stability on downstream events determining the differentiation of T cells into effector cells is reviewed. We summarize studies investigating the impact of protein fold stability on the outcome of the immune response and provide an overview on computational methods to estimate the effects of point mutations on protein stability. Expert commentary: Based on this information, the rational design of up-to-date vaccines is discussed. A model for predicting immunogenicity of proteins based on their conformational stability at different pH values is proposed.

  3. Stay Wet, Stay Stable? How Internal Water Helps the Stability of Thermophilic Proteins.

    PubMed

    Chakraborty, Debashree; Taly, Antoine; Sterpone, Fabio

    2015-10-08

    We present a systematic computational investigation of the internal hydration of a set of homologous proteins of different stability content and molecular complexities. The goal of the study is to verify whether structural water can be part of the molecular mechanisms ensuring enhanced stability in thermophilic enzymes. Our free-energy calculations show that internal hydration in the thermophilic variants is generally more favorable, and that the cumulated effect of wetting multiple sites results in a meaningful contribution to stability. Moreover, thanks to a more effective capability to retain internal water, some thermophilic proteins benefit by a systematic gain from internal wetting up to their optimal working temperature. Our work supports the idea that internal wetting can be viewed as an alternative molecular variable to be tuned for increasing protein stability.

  4. [Intermolecular hydrogen bond between protein and flavonoid and its contribution to the stability of the flavonoids].

    PubMed

    Fang, Ru; Leng, Xiao-jing; Wu, Xia; Li, Qi; Hao, Rui-fang; Ren, Fa-zheng; Jing, Hao

    2012-01-01

    The interactions between three proteins (BSA, lysozyme and myoglobin) and three flavonoids (quercetin, kaempferol and rutin) were analyzed, using three-dimensional fluorescence spectrometry in combination with UV-Vis spectrometry and Fourier transform infrared (FTIR) spectroscopy. The stabilities of unbound flavonoids and protein-bound flavonoids were compared. The correlation between the interaction and stability was analyzed. The results showed that the hydrophobic interaction was the main binding code in all proteins and flavonoids systems. However, the hydrogen bond has been involved merely in the BSA system. The stability of all three flavonoids (quercetin, kaempferol and rutin) was improved by BSA. There was a great correlation between the hydrogen bonding and the stability of the flavonoids in the presence of BSA. It suggested that the protection of BSA on the flavonoids was due to the intermolecular hydrogen bonding between BSA and flavonoid, and the stronger hydrogen bonding resulted in more protection.

  5. Functional properties of protein isolates extracted from stabilized rice bran by microwave, dry heat, and parboiling.

    PubMed

    Khan, Saima Hafeez; Butt, Masood Sadiq; Sharif, Mian Kamran; Sameen, Ayesha; Mumtaz, Semee; Sultan, Muhammad Tauseef

    2011-03-23

    Protein isolates extracted from differently stabilized rice bran were analyzed to work out the food use potential. Bulk density remained higher for isolates obtained from heat stabilized bran, the treatments were found to have positive impact on the oil absorption properties, while the water absorption was slightly impaired owing to some possible configurational changes. Surface hydrophobicity and emulsion properties were improved with bran stabilization. Isolates exhibited better foaming properties owing to the flexible nature of protein molecules, with less intensive disulfide bonding, that were slightly affected by the stabilization treatment. Nitrogen solubility index followed a curved pattern with the least value near isoelectric point that showed an increasing trend toward basic pH, and parboiled protein isolates exhibited better gelling properties among the isolates.

  6. Stay Wet, Stay Stable? How Internal Water Helps Stability of Thermophilic Proteins

    PubMed Central

    Chakraborty, Debashree; Taly, Antoine; Sterpone, Fabio

    2017-01-01

    We present a systematic computational investigation of the internal hydration of a set of homologous proteins of different stability content and molecular complexities. The goal of the study is to verify whether structural water can be part of the molecular mechanisms ensuring enhanced stability in thermophilic enzymes. Our free energy calculations show that internal hydration in the thermophilic variants is generally more favourable and that the cumulated effect of wetting multiple sites results in a meaningful contribution to stability. Moreover, thanks to a more effective capability to retain internal water some thermophilic proteins benefit of a systematic gain from internal wetting up to their optimal working temperature. Our work supports the idea that internal wetting can be viewed as an alternative molecular variable to be tuned for increasing protein stability. PMID:26335353

  7. Substrate-induced protein stabilization reveals a predominant contribution from mature protein to peptides presented on MHC class I

    PubMed Central

    Colbert, Jeff D.; Farfán-Arribas, Diego J.; Rock, Kenneth L.

    2013-01-01

    The origin of the MHC class I-presented peptides are thought to be primarily from newly synthesized but defective proteins, termed DRiPs. Most of the data supporting this concept come from studies where inhibitors of protein synthesis were found to rapidly block antigen presentation even when cells contained a pool of mature protein. However, these data only indirectly address the origin of presented peptides and in most studies the contribution of mature functional protein to the class I peptide pool has not been directly quantified. In this report we address the efficiency and contribution of mature protein by using a tetracycline-inducible system to express antigen that is conditionally stabilized upon ligand binding. This system circumvents the use of general inhibitors of protein synthesis to control antigen expression. Moreover, by controlling antigen stabilization, we could investigate whether the degradation of mature antigen contributed to antigen presentation at early and/or late time points. We show that mature protein is the major contributor of peptides presented on class I for two distinct antigenic constructs. Furthermore our data show that the protein synthesis inhibitors used previously to test the contribution of defective proteins actually block antigen presentation in ways that are independent from blocking antigen synthesis. These data suggest that for the constructs we have analyzed, mature functional protein rather than DRiPs are the predominant source of MHC class I presented-peptides PMID:24174619

  8. Key challenges for the creation and maintenance of specialist protein resources

    PubMed Central

    Holliday, Gemma L; Bairoch, Amos; Bagos, Pantelis G; Chatonnet, Arnaud; Craik, David J; Finn, Robert D; Henrissat, Bernard; Landsman, David; Manning, Gerard; Nagano, Nozomi; O’Donovan, Claire; Pruitt, Kim D; Rawlings, Neil D; Saier, Milton; Sowdhamini, Ramanathan; Spedding, Michael; Srinivasan, Narayanaswamy; Vriend, Gert; Babbitt, Patricia C; Bateman, Alex

    2015-01-01

    As the volume of data relating to proteins increases, researchers rely more and more on the analysis of published data, thus increasing the importance of good access to these data that vary from the supplemental material of individual articles, all the way to major reference databases with professional staff and long-term funding. Specialist protein resources fill an important middle ground, providing interactive web interfaces to their databases for a focused topic or family of proteins, using specialized approaches that are not feasible in the major reference databases. Many are labors of love, run by a single lab with little or no dedicated funding and there are many challenges to building and maintaining them. This perspective arose from a meeting of several specialist protein resources and major reference databases held at the Wellcome Trust Genome Campus (Cambridge, UK) on August 11 and 12, 2014. During this meeting some common key challenges involved in creating and maintaining such resources were discussed, along with various approaches to address them. In laying out these challenges, we aim to inform users about how these issues impact our resources and illustrate ways in which our working together could enhance their accuracy, currency, and overall value. Proteins 2015; 83:1005–1013. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. PMID:25820941

  9. Novel microscale approaches for easy, rapid determination of protein stability in academic and commercial settings.

    PubMed

    Alexander, Crispin G; Wanner, Randy; Johnson, Christopher M; Breitsprecher, Dennis; Winter, Gerhard; Duhr, Stefan; Baaske, Philipp; Ferguson, Neil

    2014-12-01

    Chemical denaturant titrations can be used to accurately determine protein stability. However, data acquisition is typically labour intensive, has low throughput and is difficult to automate. These factors, combined with high protein consumption, have limited the adoption of chemical denaturant titrations in commercial settings. Thermal denaturation assays can be automated, sometimes with very high throughput. However, thermal denaturation assays are incompatible with proteins that aggregate at high temperatures and large extrapolation of stability parameters to physiological temperatures can introduce significant uncertainties. We used capillary-based instruments to measure chemical denaturant titrations by intrinsic fluorescence and microscale thermophoresis. This allowed higher throughput, consumed several hundred-fold less protein than conventional, cuvette-based methods yet maintained the high quality of the conventional approaches. We also established efficient strategies for automated, direct determination of protein stability at a range of temperatures via chemical denaturation, which has utility for characterising stability for proteins that are difficult to purify in high yield. This approach may also have merit for proteins that irreversibly denature or aggregate in classical thermal denaturation assays. We also developed procedures for affinity ranking of protein-ligand interactions from ligand-induced changes in chemical denaturation data, and proved the principle for this by correctly ranking the affinity of previously unreported peptide-PDZ domain interactions. The increased throughput, automation and low protein consumption of protein stability determinations afforded by using capillary-based methods to measure denaturant titrations, can help to revolutionise protein research. We believe that the strategies reported are likely to find wide applications in academia, biotherapeutic formulation and drug discovery programmes.

  10. Small-Molecule Stabilization of the 14-3-3/Gab2 Protein-Protein Interaction (PPI) Interface.

    PubMed

    Bier, David; Bartel, Maria; Sies, Katharina; Halbach, Sebastian; Higuchi, Yusuke; Haranosono, Yu; Brummer, Tilman; Kato, Nobuo; Ottmann, Christian

    2016-04-19

    Small-molecule modulation of protein-protein interactions (PPIs) is one of the most promising new areas in drug discovery. In the vast majority of cases only inhibition or disruption of PPIs is realized, whereas the complementary strategy of targeted stabilization of PPIs is clearly under-represented. Here, we report the example of a semi-synthetic natural product derivative--ISIR-005--that stabilizes the cancer-relevant interaction of the adaptor protein 14-3-3 and Gab2. The crystal structure of ISIR-005 in complex with 14-3-3 and the binding motif of Gab2 comprising two phosphorylation sites (Gab2pS210pT391) showed how the stabilizing molecule binds to the rim-of-the-interface of the protein complex. Only in the direct vicinity of 14-3-3/Gab2pT391 site is a pre-formed pocket occupied by ISIR-005; binding of the Gab2pS210 motif to 14-3-3 does not create an interface pocket suitable for the molecule. Accordingly, ISIR-005 only stabilizes the binding of the Gab2pT391 but not the Gab2pS210 site. This study represents structural and biochemical proof of the druggability of the 14-3-3/Gab2 PPI interface with important implications for the development of PPI stabilizers. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. The contribution of polar group burial to protein stability is strongly context-dependent.

    PubMed

    Takano, Kazufumi; Scholtz, J Martin; Sacchettini, James C; Pace, C Nick

    2003-08-22

    We previously suggested that proteins gain more stability from the burial and hydrogen bonding of polar groups than from the burial of nonpolar groups (Pace, C. N. (2001) Biochemistry 40, 310-313). To study this further, we prepared eight Thr-to-Val mutants of RNase Sa, four in which the Thr side chain is hydrogen-bonded and four in which it is not. We measured the stability of these mutants by analyzing their thermal denaturation curves. The four hydrogen-bonded Thr side chains contribute 1.3 +/- 0.9 kcal/mol to the stability; those that are not still contribute 0.4 +/- 0.9 kcal/mol to the stability. For 40 Thr-to-Val mutants of 11 proteins, the average decrease in stability is 1.0 +/- 1.0 kcal/mol when the Thr side chain is hydrogen-bonded and 0.0 +/- 0.5 kcal/mol when it is not. This is clear evidence that hydrogen bonds contribute favorably to protein stability. In addition, we prepared four Val-to-Thr mutants of RNase Sa, measured their stability, and determined their crystal structures. In all cases, the mutants are less stable than the wild-type protein, with the decreases in stability ranging from 0.5 to 4.4 kcal/mol. For 41 Val-to-Thr mutants of 11 proteins, the average decrease in stability is 1.8 +/- 1.3 kcal/mol and is unfavorable for 40 of 41 mutants. This shows that placing an [bond]OH group at a site designed for a [bond]CH3 group is very unfavorable. So, [bond]OH groups can contribute favorably to protein stability, even if they are not hydrogen-bonded, if the site was selected for an [bond]OH group, but they will make an unfavorable contribution to stability, even if they are hydrogen-bonded, when they are placed at a site selected for a [bond]CH3 group. The contribution that polar groups make to protein stability depends strongly on their environment.

  12. Identifying Protein Stabilizing Ligands Using GroEL

    PubMed Central

    Naik, Subhashchandra; Haque, Inamul; Degner, Nick; Kornilayev, Boris; Bomhoff, Gregory; Hodges, Jacob; Khorassani, Ara-Azad; Katayama, Hiroo; Morris, Jill; Kelly, Jeffery; Seed, John; Fisher, Mark T.

    2010-01-01

    Over the past five years, it has become increasingly apparent to researchers that the initial promise and excitement of using gene replacement therapies to ameliorate folding diseases are still far from being broadly or easily applicable. Because a large number of human diseases are protein folding diseases (~30 to 50%), many researchers now realize that more directed approaches to target and reverse the fundamental misfolding reactions preceding disease are highly feasible and offer the potential of developing more targeted drug therapies. This is also true with a large number of so called “orphan protein folding diseases”. The development of a broad-based general screening array method using the chaperonin as a detection platform will enable us to screen large chemical combinatorial libraries for specific ligands against the elusive transient, primary reactions that often lead to protein misfolding. This development will provide a highly desirable tool for the pharmaceutical, academic and medical professions. PMID:19802819

  13. Deficiency of skeletal membrane protein band 4.1 in homozygous hereditary elliptocytosis. Implications for erythrocyte membrane stability.

    PubMed Central

    Tchernia, G; Mohandas, N; Shohet, S B

    1981-01-01

    Erythrocytes from three patients with severe hemolytic anemia, marked erythrocyte fragmentation, and elliptocytic poikilocytosis, were studied in terms of both their membrane protein composition and their mechanical characteristics. Erythrocytes from the patients' parents and one minimally affected and one normal sibling were also studied. Morphologic observations implied that the severely affected patients suffered from homozygous hereditary elliptocytosis because erythrocytes of both parents and the one minimally affected sibling showed moderate elliptocytosis on smear, whereas those of an unaffected sibling had normal morphology. The parallel findings of markedly reduced levels of band 4.1 in the erythrocyte membrane proteins of the patients and an intermediate reduction in the cells of the parents and the putative heterozygous sibling, suggest that the elliptocytic shape of the cells was related to the reduced levels of band 4.1. Additional studies showed marked abnormalities in cellular deformability and membrane fragility in the erythrocytes from the homozygous patients. Importantly, these changes were also closely proportional to the reduced levels of band 4.1, suggesting a central role for this protein in the maintenance of normal membrane stability and normal cell shape. It seems likely that this role for band 4.1 is intimately related to its known biochemical connection to the "membrane skeleton" through its linkage with spectrin and actin. Images PMID:6894932

  14. Effect of short-term low-protein diet supplemented with keto acids on hyperphosphatemia in maintenance hemodialysis patients.

    PubMed

    Li, Haiming; Long, Quan; Shao, Chunhai; Fan, Hong; Yuan, Li; Huang, Bihong; Gu, Yong; Lin, Shanyan; Hao, Chuanming; Chen, Jing

    2011-01-01

    To evaluate the effects of short-term restriction of dietary protein intake (DPI) supplemented with keto acids on hyperphosphatemia in maintenance hemodialysis (MHD) patients. Forty MHD patients with uncontrolled hyperphosphatemia were randomized to either low DPI with keto acid-supplemented (sLP) or normal DPI (NP) group for 8 weeks. After 8 weeks, the sLP group was shifted to NP for another 8 weeks. Low-protein diet (LPD) was individualized with total caloric intake 30-35 kcal/kg/day, protein intake of 0.8 g/kg/day and phosphate intake of 500 mg/day. Keto acids were supplied in a dosage of 12 pills per day. Calcium phosphorous metabolism index and nutritional index (serum albumin, total protein, somatometric measurements, 3-day diaries and Mini-Nutritional Assessment score) were recorded. C-reactive protein, CO(2) combining power and Kt/V were measured to evaluate the inflammation, metabolic acidosis and dialysis adequacy, respectively. Serum phosphorus level and calcium-phosphate product were significantly decreased at the end of the first 8 weeks in the sLP group compared to the basal value and the NP group (p < 0.001). No difference was observed in C-reactive protein, Kt/V and nutritional index, while CO(2) combining power was significantly higher at week 8 in the sLP group (p < 0.001). Short-term restriction of DPI supplemented with keto acids could decrease hyperphosphatemia and calcium-phosphate product, while keeping stable nutritional status among MHD patients. Copyright © 2010 S. Karger AG, Basel.

  15. G protein-coupled receptors in stem cell maintenance and somatic reprogramming to pluripotent or cancer stem cells.

    PubMed

    Choi, Hye Yeon; Saha, Subbroto Kumar; Kim, Kyeongseok; Kim, Sangsu; Yang, Gwang-Mo; Kim, BongWoo; Kim, Jin-hoi; Cho, Ssang-Goo

    2015-02-01

    G protein-coupled receptors (GPCRs) are a large class of transmembrane receptors categorized into five distinct families: rhodopsin, secretin, adhesion, glutamate, and frizzled. They bind and regulate 80% of all hormones and account for 20-50% of the pharmaceuticals currently on the market. Hundreds of GPCRs integrate and coordinate the functions of individual cells, mediating signaling between various organs. GPCRs are crucial players in tumor progression, adipogenesis, and inflammation. Several studies have also confirmed their central roles in embryonic development and stem cell maintenance. Recently, GPCRs have emerged as key players in the regulation of cell survival, proliferation, migration, and self-renewal in pluripotent (PSCs) and cancer stem cells (CSCs). Our study and other reports have revealed that the expression of many GPCRs is modulated during the generation of induced PSCs (iPSCs) or CSCs as well as during CSC sphere formation. These GPCRs may have crucial roles in the regulation of selfrenewal and other biological properties of iPSCs and CSCs. This review addresses the current understanding of the role of GPCRs in stem cell maintenance and somatic reprogramming to PSCs or CSCs.

  16. G protein-coupled receptors in stem cell maintenance and somatic reprogramming to pluripotent or cancer stem cells

    PubMed Central

    Choi, Hye Yeon; Saha, Subbroto Kumar; Kim, Kyeongseok; Kim, Sangsu; Yang, Gwang-Mo; Kim, BongWoo; Kim, Jin-hoi; Cho, Ssang-Goo

    2015-01-01

    G protein-coupled receptors (GPCRs) are a large class of transmembrane receptors categorized into five distinct families: rhodopsin, secretin, adhesion, glutamate, and frizzled. They bind and regulate 80% of all hormones and account for 20-50% of the pharmaceuticals currently on the market. Hundreds of GPCRs integrate and coordinate the functions of individual cells, mediating signaling between various organs. GPCRs are crucial players in tumor progression, adipogenesis, and inflammation. Several studies have also confirmed their central roles in embryonic development and stem cell maintenance. Recently, GPCRs have emerged as key players in the regulation of cell survival, proliferation, migration, and self-renewal in pluripotent (PSCs) and cancer stem cells (CSCs). Our study and other reports have revealed that the expression of many GPCRs is modulated during the generation of induced PSCs (iPSCs) or CSCs as well as during CSC sphere formation. These GPCRs may have crucial roles in the regulation of selfrenewal and other biological properties of iPSCs and CSCs. This review addresses the current understanding of the role of GPCRs in stem cell maintenance and somatic reprogramming to PSCs or CSCs. [BMB Reports 2015; 48(2): 68-80] PMID:25413305

  17. The influence of the maintenance of terraced areas on slope stability during the November 2014 flood event in Liguria (northwestern Italy)

    NASA Astrophysics Data System (ADS)

    Giordan, Daniele; Poggi, Flavio; Baldo, Marco; Cignetti, Martina

    2016-04-01

    Terraced environments are a widespread feature of the coastal settlement of eastern Liguria (northwestern Italy) and they constitute a well-known favorable role in slope stability. In this region, starting from the twentieth century, the progressive abandonment of agriculture determines a progressively increasing lack of maintenance of the terraces, consequently raising the level of slope instability. Moreover, it should be taken into account not only the level of terraces maintenance, but also their interaction with several factors as i) geological and geomorphological conditions, ii) soil properties, iii) hydrological and hydrogeological conditions, and iv) land use, causing an increase in landslides occurrence. The definition of managed terraces effects on slope stability and their response to external stress like a flood event is rather complicated; a possible approach is a statistical analysis of the effects of a flood event over a large terraced area, distinguishing the maintained sectors from the abandoned ones. After the November 2014 flood event, which affected several sectors of the Liguria region, where a high number of shallow landslides were triggered, an airborne LiDAR survey of the damaged area was carried out. In particular, a high resolution Digital Terrain Model (DTM) resampled to a lower density (1 square meter grid spacing) and a photogrammetric coverage of the area was performed, in order to create a landslide map of the flood event. The surveyed area covered more than 380 square kilometers, and over 1600 shallow landslides triggered by the flood event were identified and inventoried. The high resolution DTM allowed the identification of terraced areas also in wooded sectors and a sharp mapping of the extension of terraced slopes in this portion of Liguria region. By considering: i) the terraced areas recognized through DTM analysis, ii) the mapped landslides, and iii) the land use classification, a correlation between the presence of terraces

  18. Mathematics, thermodynamics, and modeling to address ten common misconceptions about protein structure, folding, and stability.

    PubMed

    Robic, Srebrenka

    2010-01-01

    To fully understand the roles proteins play in cellular processes, students need to grasp complex ideas about protein structure, folding, and stability. Our current understanding of these topics is based on mathematical models and experimental data. However, protein structure, folding, and stability are often introduced as descriptive, qualitative phenomena in undergraduate classes. In the process of learning about these topics, students often form incorrect ideas. For example, by learning about protein folding in the context of protein synthesis, students may come to an incorrect conclusion that once synthesized on the ribosome, a protein spends its entire cellular life time in its fully folded native confirmation. This is clearly not true; proteins are dynamic structures that undergo both local fluctuations and global unfolding events. To prevent and address such misconceptions, basic concepts of protein science can be introduced in the context of simple mathematical models and hands-on explorations of publicly available data sets. Ten common misconceptions about proteins are presented, along with suggestions for using equations, models, sequence, structure, and thermodynamic data to help students gain a deeper understanding of basic concepts relating to protein structure, folding, and stability.

  19. Mathematics, Thermodynamics, and Modeling to Address Ten Common Misconceptions about Protein Structure, Folding, and Stability

    PubMed Central

    2010-01-01

    To fully understand the roles proteins play in cellular processes, students need to grasp complex ideas about protein structure, folding, and stability. Our current understanding of these topics is based on mathematical models and experimental data. However, protein structure, folding, and stability are often introduced as descriptive, qualitative phenomena in undergraduate classes. In the process of learning about these topics, students often form incorrect ideas. For example, by learning about protein folding in the context of protein synthesis, students may come to an incorrect conclusion that once synthesized on the ribosome, a protein spends its entire cellular life time in its fully folded native confirmation. This is clearly not true; proteins are dynamic structures that undergo both local fluctuations and global unfolding events. To prevent and address such misconceptions, basic concepts of protein science can be introduced in the context of simple mathematical models and hands-on explorations of publicly available data sets. Ten common misconceptions about proteins are presented, along with suggestions for using equations, models, sequence, structure, and thermodynamic data to help students gain a deeper understanding of basic concepts relating to protein structure, folding, and stability. PMID:20810950

  20. Characterization of the Stability and Bio-functionality of Tethered Proteins on Bioengineered Scaffolds

    PubMed Central

    Wang, Ting-Yi; Bruggeman, Kiara A. F.; Sheean, Rebecca K.; Turner, Bradley J.; Nisbet, David R.; Parish, Clare L.

    2014-01-01

    Various engineering applications have been utilized to deliver molecules and compounds in both innate and biological settings. In the context of biological applications, the timely delivery of molecules can be critical for cellular and organ function. As such, previous studies have demonstrated the superiority of long-term protein delivery, by way of protein tethering onto bioengineered scaffolds, compared with conventional delivery of soluble protein in vitro and in vivo. Despite such benefits little knowledge exists regarding the stability, release kinetics, longevity, activation of intracellular pathway, and functionality of these proteins over time. By way of example, here we examined the stability, degradation and functionality of a protein, glial-derived neurotrophic factor (GDNF), which is known to influence neuronal survival, differentiation, and neurite morphogenesis. Enzyme-linked immunosorbent assays (ELISA) revealed that GDNF, covalently tethered onto polycaprolactone (PCL) electrospun nanofibrous scaffolds, remained present on the scaffold surface for 120 days, with no evidence of protein leaching or degradation. The tethered GDNF protein remained functional and capable of activating downstream signaling cascades, as revealed by its capacity to phosphorylate intracellular Erk in a neural cell line. Furthermore, immobilization of GDNF protein promoted cell survival and differentiation in culture at both 3 and 7 days, further validating prolonged functionality of the protein, well beyond the minutes to hours timeframe observed for soluble proteins under the same culture conditions. This study provides important evidence of the stability and functionality kinetics of tethered molecules. PMID:24700461

  1. Characterization of milk proteins-lutein complexes and the impact on lutein chemical stability.

    PubMed

    Yi, Jiang; Fan, Yuting; Yokoyama, Wallace; Zhang, Yuzhu; Zhao, Liqing

    2016-06-01

    In this study, the interaction of WPI (whey protein isolate) and SC (sodium caseinate) with hydrophobic lutein was investigated through UV-vis spectroscopy and circular dichroism (CD) as well as fluorescence. The effects on lutein's chemical stability were also examined. The decrease of turbidity of lutein suggested that lutein's aqueous solubility was improved after binding with milk proteins. CD analysis indicated lutein had little impact on the secondary structures of both proteins. Different preparation methods have significant impacts on the binding constant. Fluorescence results indicated that WPI and SC interact with lutein by hydrophobic contacts. Milk proteins have protective effects on lutein against oxidation and decomposition, and SC showed better capability in protecting lutein from oxidation than WPI during 16 days storage. The lutein's chemical stability was increased with increasing of proteins concentration. The results indicated that milk proteins may act as effective carriers for lipophilic nutraceuticals.

  2. Coexistence of flexibility and stability of proteins: an equation of state.

    PubMed

    de Leeuw, Marina; Reuveni, Shlomi; Klafter, Joseph; Granek, Rony

    2009-10-09

    We consider a recently suggested "equation of state" for natively folded proteins, and verify its validity for a set of about 5800 proteins. The equation is based on a fractal viewpoint of proteins, on a generalization of the Landau-Peierls instability, and on a marginal stability criterion. The latter allows for coexistence of stability and flexibility of proteins, which is required for their proper function. The equation of state relates the protein fractal dimension d(f), its spectral dimension d(s), and the number of amino acids N. Using structural data from the protein data bank (PDB) and the Gaussian network model (GNM), we compute d(f) and d(s) for the entire set and demonstrate that the equation of state is well obeyed. Addressing the fractal properties and making use of the equation of state may help to engineer biologically inspired catalysts.

  3. Mtr4-like protein coordinates nuclear RNA processing for heterochromatin assembly and for telomere maintenance

    PubMed Central

    Lee, Nathan N.; Chalamcharla, Venkata R.; Reyes-Turcu, Francisca; Mehta, Sameet; Zofall, Martin; Balachandran, Vanivilasini; Dhakshnamoorthy, Jothy; Taneja, Nitika; Yamanaka, Soichiro; Zhou, Ming; Grewal, Shiv I. S.

    2013-01-01

    SUMMARY The regulation of protein-coding and noncoding RNAs is linked to nuclear processes including chromatin modifications and gene silencing. However, the mechanisms that distinguish RNAs and mediate their functions are poorly understood. We describe a nuclear RNA processing network in fission yeast with a core module comprising the Mtr4-like protein, Mtl1, and the zinc finger protein, Red1. The Mtl1-Red1 core promotes degradation of mRNAs and noncoding RNAs, and associates with different proteins to assemble heterochromatin via distinct mechanisms. Mtl1 also forms Red1-independent interactions with evolutionarily conserved proteins named Nrl1 and Ctr1, which associate with splicing factors. Whereas Nrl1 targets transcripts with cryptic introns to form heterochromatin at developmental genes and retrotransposons, Ctr1 functions in processing intron-containing telomerase RNA. Together with our discovery of widespread cryptic introns, including in noncoding RNAs, these findings reveal unique cellular strategies for recognizing regulatory RNAs and coordinating their functions in response to developmental and environmental cues. PMID:24210919

  4. A miniature protein stabilized by a cation-π interaction network core

    PubMed Central

    Craven, Timothy W.; Cho, Min-Kyu; Traaseth, Nathaniel J.; Bonneau, Richard; Kirshenbaum, Kent

    2016-01-01

    The design of folded miniature proteins is predicated on establishing non-covalent interactions that direct the self-assembly of discrete thermo-stable tertiary structures. In this work, we describe how a network of cation-π interactions present in proteins containing “WSXWS motifs” can be emulated to stabilize the core of a miniature protein. This 19-residue protein sequence recapitulates a set of interdigitated arginine and tryptophan residues that stabilize a distinctive β-strand:loop:PPII-helix topology. Validation of the compact fold determined by NMR was carried out by mutagenesis of the cation-π network and by comparison to the corresponding disulfide-bridged structure. These results support the involvement of a coordinated set of cation-π interactions that stabilize the tertiary structure. PMID:26812069

  5. Lentil and chickpea protein-stabilized emulsions: optimization of emulsion formulation.

    PubMed

    Can Karaca, Asli; Nickerson, Michael T; Low, Nicholas H

    2011-12-28

    Chickpea and lentil protein-stabilized emulsions were optimized with regard to pH (3.0-8.0), protein concentration (1.1-4.1% w/w), and oil content (20-40%) for their ability to form and stabilize oil-in-water emulsions using response surface methodology. Specifically, creaming stability, droplet size, and droplet charge were assessed. Optimum conditions for minimal creaming (no serum separation after 24 h), small droplet size (<2 μm), and high net droplet charge (absolute value of ZP > 40 mV) were identified as 4.1% protein, 40% oil, and pH 3.0 or 8.0, regardless of the plant protein used for emulsion preparation.

  6. New insights into structural determinants of prion protein folding and stability.

    PubMed

    Benetti, Federico; Legname, Giuseppe

    2015-01-01

    Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a β-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a β-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability.

  7. Biochemical characterization of the minichromosome maintenance (MCM) protein of the crenarchaeote Aeropyrum pernix and its interactions with the origin recognition complex (ORC) proteins.

    PubMed

    Atanassova, Neli; Grainge, Ian

    2008-12-16

    Replication in archaea is carried out by proteins that are homologues of eukaryotic counterparts. However, the archaeal systems tend to be much simpler with fewer different genes encoding the core functions than in eukaryotic counterparts. In many archaea, there is a single minichromosome maintenance (MCM) homologue, presumed to be the replicative helicase and between one and three origin recognition complex (ORC) homologues involved in binding to the replication origins. Here we describe the cloning and characterization of the MCM protein from the crenarchaeote Aeropyrum pernix. Like other eukaryotic and archaeal MCM proteins, it is found to be an ATP-dependent DNA helicase, and the putative active site residues involved in ATP binding and hydrolysis are confirmed by mutation. Deletion of the N-terminal 256 amino acids yielded a protein with higher ATPase activity in the absence of DNA and retained robust helicase activity. Interactions with the ORC proteins of A. pernix were examined, and it was found that both ORC homologues could inhibit the helicase activity of MCM. Further it was found that ORC2 could autophosphorylate in the presence of ATP and more remarkably could phosphorylate MCM in a species-specific manner.

  8. Evaluation of Minichromosome Maintenance Protein 7 and c-KIT as Prognostic Markers in Feline Cutaneous Mast Cell Tumours.

    PubMed

    Dobromylskyj, M J; Rasotto, R; Melville, K; Smith, K C; Berlato, D

    2015-11-01

    Mast cell tumours (MCTs) are a common skin tumour in cats, but there is currently no histological grading system or reliable prognostic marker for this species (unlike the situation for dogs). This study utilized a set of 71 feline cutaneous MCTs with known clinical outcomes to assess the potential of various prognostic markers, including the cellular proliferation marker minichromosome maintenance protein (MCM)-7, mitotic index and various KIT labelling characteristics, including KIT positivity, KIT labelling pattern and KIT immunoreactivity score (IS). Of the factors studied, the mitotic index and the KIT labelling pattern were the only features associated significantly with survival times, while the proliferation marker MCM7 and the KIT IS were not. The study also highlights the variability of KIT labelling characteristics between tumours, which may prevent use of this marker as a diagnostic and prognostic tool.

  9. Maintenance energy requirements of beef cows and relationship with cow and calf performance, metabolic hormones, and functional proteins.

    PubMed

    Cooper-Prado, M J; Long, N M; Davis, M P; Wright, E C; Madden, R D; Dilwith, J W; Bailey, C L; Spicer, L J; Wettemann, R P

    2014-08-01

    Gestating Angus, nonlactating, spring-calving cows were used to determine variation in maintenance energy requirements (MR); to evaluate the relationship among MR and cow and calf performance, plasma concentrations of IGF-I, T4, glucose, insulin, and ruminal temperature; and to describe the LM proteome and evaluate protein abundance in cows with different MR. Cows (4 to 7 yr of age) with a BCS of 5.0 ± 0.2 and BW of 582 ± 37 kg in the second to third trimester of gestation were studied in 3 trials (trial 1, n = 23; trial 2, n = 32; trial 3, n = 38). Cows were individually fed a complete diet in amounts to meet predicted MR (Level 1 Model of NRC), and feed intake was adjusted weekly until constant BW was achieved for at least 21 d (maintenance). Cows were classified on the basis of MR as low (>0.5 SD less than mean, LMR), moderate (±0.5 SD of mean, MMR), or high (>0.5 SD more than mean, HMR) MR. Blood samples were taken at maintenance and at 2 mo postpartum in trial 2. Muscle biopsies were taken from LMR and HMR after cows consumed actual MR for 28 d (trial 2) or 21 d (trial 3). Proteins from LM were separated by 2-dimensional difference gel electrophoresis and were identified, and abundance was quantified and compared. The greatest differences in MR between cows were 29%, 24%, and 25% in trials 1, 2, and 3, respectively. Daily MR (NEm, kcal·BW(-0.75)·d(-1)) averaged 89.2 ± 6.3, 93.0 ± 4.9, and 90.4 ± 4.6 in trials 1, 2, and 3, respectively. Postpartum BW and BCS, calf birth and weaning weights, postpartum luteal activity, and ruminal temperature were not influenced by MR of the cows. Concentrations of IGF-I were greater (P = 0.001) in plasma of MMR compared with LMR cows consuming predicted MR diets, and MR was negatively correlated with concentrations of IGF-I in plasma (r = -0.38; P = 0.05) at 2 mo postpartum. A total of 103 proteins were isolated from LM; 52 gene products were identified. Abundance of specific proteins in the LM was not influenced (P > 0

  10. Structure of a double hexamer of the Pyrococcus furiosus minichromosome maintenance protein N-terminal domain

    SciTech Connect

    Meagher, Martin; Enemark, Eric J.

    2016-06-22

    The crystal structure of the N-terminal domain of thePyrococcus furiosusminichromosome maintenance (MCM) protein as a double hexamer is described. The MCM complex is a ring-shaped helicase that unwinds DNA at the replication fork of eukaryotes and archaea. Prior to replication initiation, the MCM complex assembles as an inactive double hexamer at specific sites of DNA. The presented structure is highly consistent with previous MCM double-hexamer structures and shows two MCM hexamers with a head-to-head interaction mediated by the N-terminal domain. Minor differences include a diminished head-to-head interaction and a slightly reduced inter-hexamer rotation.

  11. Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian Schmidtea mediterranea.

    PubMed

    Bansal, Dhiru; Kulkarni, Jahnavi; Nadahalli, Kavana; Lakshmanan, Vairavan; Krishna, Srikar; Sasidharan, Vidyanand; Geo, Jini; Dilipkumar, Shilpa; Pasricha, Renu; Gulyani, Akash; Raghavan, Srikala; Palakodeti, Dasaradhi

    2017-09-01

    Identifying key cellular events that facilitate stem cell function and tissue organization is crucial for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic poly A-binding protein, SMED-PABPC2. Knockdown of smed-pabpc2 leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing, resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggests that epidermal lineage transcripts, including zfp-1, are translationally regulated by SMED-PABPC2. Together, our results uncover a novel role for SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing and subsequent processes for regeneration. © 2017. Published by The Company of Biologists Ltd.

  12. Evolutionary Dynamics on Protein Bi-stability Landscapes can Potentially Resolve Adaptive Conflicts

    PubMed Central

    Sikosek, Tobias; Bornberg-Bauer, Erich; Chan, Hue Sun

    2012-01-01

    Experimental studies have shown that some proteins exist in two alternative native-state conformations. It has been proposed that such bi-stable proteins can potentially function as evolutionary bridges at the interface between two neutral networks of protein sequences that fold uniquely into the two different native conformations. Under adaptive conflict scenarios, bi-stable proteins may be of particular advantage if they simultaneously provide two beneficial biological functions. However, computational models that simulate protein structure evolution do not yet recognize the importance of bi-stability. Here we use a biophysical model to analyze sequence space to identify bi-stable or multi-stable proteins with two or more equally stable native-state structures. The inclusion of such proteins enhances phenotype connectivity between neutral networks in sequence space. Consideration of the sequence space neighborhood of bridge proteins revealed that bi-stability decreases gradually with each mutation that takes the sequence further away from an exactly bi-stable protein. With relaxed selection pressures, we found that bi-stable proteins in our model are highly successful under simulated adaptive conflict. Inspired by these model predictions, we developed a method to identify real proteins in the PDB with bridge-like properties, and have verified a clear bi-stability gradient for a series of mutants studied by Alexander et al. (Proc Nat Acad Sci USA 2009, 106:21149–21154) that connect two sequences that fold uniquely into two different native structures via a bridge-like intermediate mutant sequence. Based on these findings, new testable predictions for future studies on protein bi-stability and evolution are discussed. PMID:23028272

  13. Effects of selective exercise for the deep abdominal muscles and lumbar stabilization exercise on the thickness of the transversus abdominis and postural maintenance.

    PubMed

    Lee, Jung-Seok; Kim, Tae-Ho; Kim, Da-Yeon; Shim, Jae-Ho; Lim, Jin-Yong

    2015-02-01

    [Purpose] The purpose of this study was to examine the effects of selective exercise for the deep abdominal muscles (SEDA) and lumbar stabilization exercise (LSE) on the thickness of the transversus abdominis and postural maintenance on an unstable base of support. [Subjects and Methods] The subjects of this study were 20 male and 10 female adults in their 20s without lumbar pain. They were equally and randomly assigned to a SEDA group and a LSE group. The thickness of the transversus abdominis was measured using ultrasound imaging during rest and drawing-in. The thickness of the transversus abdominis was measured when subjects raised their right and left legs while lying on a Swiss ball. [Results] Initially, there were no differences between the two groups. After the intervention, significant differences were observed in all parameters. A significant interaction between group and period was not found for any parameters. [Conclusion] In conclusion, both SEDA and LSE thickened the transversus abdominis, which is a deep abdominal muscle, thereby adjusting posture, and stabilizing the trunk. These exercises increased the thickness of the deep abdominal muscles. They are important exercises for improving the stability of athletes or patients who need postural adjustment.

  14. Expression of chromosomal regional maintenance protein-1 may be associated with subcellular survivin expression in human gastric and colorectal carcinoma

    PubMed Central

    Shintani, Michiko; Tashiro, Akito; Sangawa, Akiko; Yamao, Naoki; Kamoshida, Shingo

    2016-01-01

    Survivin, a member of the inhibitor of apoptosis protein family, is a potential prognostic marker and molecular target for anticancer therapies. Chromosomal regional maintenance protein-1 (CRM-1) mediates the nuclear export of proteins such as survivin. The aims of the present study were to compare the expression and subcellular localization of CRM-1 in human gastric and colorectal carcinomas and to assess the association between CRM-1 and survivin expression in these tumor types. The nuclear and cytoplasmic CRM-1 expression rates in gastric carcinoma were 61% (42/69) and 29% (20/69), respectively, while the nuclear and cytoplasmic CRM-1 expression rates in colorectal carcinoma were 55% (43/78) and 37% (29/78), respectively. Nuclear and cytoplasmic CRM-1 expression was found to be significantly correlated with nuclear and cytoplasmic survivin expression in colorectal carcinoma, but not gastric carcinoma. These results indicate that CRM-1 expression patterns differ between gastric and colorectal carcinomas and thus, we hypothesize that CRM-1-mediated nuclear export of survivin may be deregulated in gastric carcinoma. Therefore, CRM-1 may exhibit different functions in gastric and colorectal carcinoma. PMID:28105170

  15. Protein Kinase D Controls the Integrity of Golgi Apparatus and the Maintenance of Dendritic Arborization in Hippocampal Neurons

    PubMed Central

    Czöndör, Katalin; Ellwanger, Kornelia; Fuchs, Yannick F.; Lutz, Sylke; Gulyás, Márton; Mansuy, Isabelle M.; Hausser, Angelika; Pfizenmaier, Klaus

    2009-01-01

    Protein kinase D (PKD) is known to participate in various cellular functions, including secretory vesicle fission from the Golgi and plasma membrane-directed transport. Here, we report on expression and function of PKD in hippocampal neurons. Expression of an enhanced green fluorescent protein (EGFP)-tagged PKD activity reporter in mouse embryonal hippocampal neurons revealed high endogenous PKD activity at the Golgi complex and in the dendrites, whereas PKD activity was excluded from the axon in parallel with axonal maturation. Expression of fluorescently tagged wild-type PKD1 and constitutively active PKD1S738/742E (caPKD1) in neurons revealed that both proteins were slightly enriched at the trans-Golgi network (TGN) and did not interfere with its thread-like morphology. By contrast, expression of dominant-negative kinase inactive PKD1K612W (kdPKD1) led to the disruption of the neuronal Golgi complex, with kdPKD1 strongly localized to the TGN fragments. Similar findings were obtained from transgenic mice with inducible, neuron-specific expression of kdPKD1-EGFP. As a prominent consequence of kdPKD1 expression, the dendritic tree of transfected neurons was reduced, whereas caPKD1 increased dendritic arborization. Our results thus provide direct evidence that PKD activity is selectively involved in the maintenance of dendritic arborization and Golgi structure of hippocampal neurons. PMID:19211839

  16. Relatively high-protein or 'low-carb' energy-restricted diets for body weight loss and body weight maintenance?

    PubMed

    Soenen, Stijn; Bonomi, Alberto G; Lemmens, Sofie G T; Scholte, Jolande; Thijssen, Myriam A M A; van Berkum, Frank; Westerterp-Plantenga, Margriet S

    2012-10-10

    'Low-carb' diets have been suggested to be effective in body weight (BW) management. However, these diets are relatively high in protein as well. To unravel whether body-weight loss and weight-maintenance depends on the high-protein or the 'low-carb' component of the diet. Body-weight (BW), fat mass (FM), blood- and urine-parameters of 132 participants (age=50 ± 12 yr; BW=107 ± 20 kg; BMI=37 ± 6 kg/m(2); FM=47.5 ± 11.9 kg) were compared after 3 and 12 months between four energy-restricted diets with 33% of energy requirement for the first 3 months, and 67% for the last 9 months: normal-protein normal-carbohydrate (NPNC), normal-protein low-carbohydrate (NPLC); high-protein normal-carbohydrate (HPNC), high-protein low-carbohydrate (HPLC); 24h N-analyses confirmed daily protein intakes for the normal-protein diets of 0.7 ± 0.1 and for the high-protein diets of 1.1 ± 0.2g/kg BW (p<0.01). BW and FM decreased over 3 months (p<0.001): HP (-14.1 ± 4 kg; -11.9 ± 1.7 kg) vs. NP (-11.5 ± 4 kg; -9.3 ± 0.7 kg) (p<0.001); LC (-13.5 ± 4 kg; -11.0 ± 1.2 kg) vs. NC (-12.3 ± 3 kg; -10.3 ± 1.1 kg) (ns). Diet × time interaction showed HPLC (-14.7 ± 5 kg; -11.9 ± 1.6 kg) vs. HPNC (-13.8 ± 3 kg; -11.9 ± 1.8 kg) (ns); NPLC (-12.2 ± 4 kg; -10.0 ± 0.8 kg) vs. NPNC (-10.7 ± 4 kg; -8.6 ± 0.7 kg) (ns); HPLC vs. NPLC (p<0.001); HPNC vs. NPNC (p<0.001). Decreases over 12 months (p<0.001) showed HP (-12.8 ± 4 kg; -9.1 ± 0.8 kg) vs. NP (-8.9 ± 3 kg; -7.7 ± 0.6 kg) (p<0.001); LC (-10.6 ± 4 kg; -8.3 ± 0.7 kg) vs. NC (11.1 ± 3 kg; 9.3 ± 0.7 kg) (ns). Diet × time interaction showed HPLC (-11.6 ± 5 kg ; -8.2 ± 0.7 kg) vs. HPNC (-14.1 ± 4 kg; -10.0 ± 0.9 kg) (ns); NPNC (-8.2 ± 3 kg; -6.7 ± 0.6 kg) vs. NPLC (-9.7 ± 3 kg; -8.5 ± 0.7 kg) (ns); HPLC vs. NPLC (p<0.01); HPNC vs. NPNC (p<0.01). HPNC vs. all other diets reduced diastolic blood pressure more. Relationships between changes in BW, FM, FFM or metabolic parameters and energy percentage of fat in the diet

  17. Documentation of an Imperative To Improve Methods for Predicting Membrane Protein Stability.

    PubMed

    Kroncke, Brett M; Duran, Amanda M; Mendenhall, Jeffrey L; Meiler, Jens; Blume, Jeffrey D; Sanders, Charles R

    2016-09-13

    There is a compelling and growing need to accurately predict the impact of amino acid mutations on protein stability for problems in personalized medicine and other applications. Here the ability of 10 computational tools to accurately predict mutation-induced perturbation of folding stability (ΔΔG) for membrane proteins of known structure was assessed. All methods for predicting ΔΔG values performed significantly worse when applied to membrane proteins than when applied to soluble proteins, yielding estimated concordance, Pearson, and Spearman correlation coefficients of <0.4 for membrane proteins. Rosetta and PROVEAN showed a modest ability to classify mutations as destabilizing (ΔΔG < -0.5 kcal/mol), with a 7 in 10 chance of correctly discriminating a randomly chosen destabilizing variant from a randomly chosen stabilizing variant. However, even this performance is significantly worse than for soluble proteins. This study highlights the need for further development of reliable and reproducible methods for predicting thermodynamic folding stability in membrane proteins.

  18. Prediction of salt effects on protein phase behavior by HIC retention and thermal stability.

    PubMed

    Baumgartner, Kai; Großhans, Steffen; Schütz, Juliane; Suhm, Susanna; Hubbuch, Jürgen

    2016-09-05

    In the biopharmaceutical industry it is mandatory to know and ensure the correct protein phase state as a critical quality attribute in every process step. Unwanted protein precipitation or crystallization can lead to column, pipe or filter blocking. In formulation, the formation of aggregates can even be lethal when injected into the patient. The typical methodology to illustrate protein phase states is the generation of protein phase diagrams. Commonly, protein phase behavior is shown in dependence of protein and precipitant concentration. Despite using high-throughput methods for the generation of phase diagrams, the time necessary to reach equilibrium is the bottleneck. Faster methods to predict protein phase behavior are desirable. In this study, hydrophobic interaction chromatography retention times were correlated to crystal size and form. High-throughput thermal stability measurements (melting and aggregation temperatures), using an Optim(®)2 system, were successfully correlated to glucose isomerase stability. By using hydrophobic interaction chromatography and thermal stability determinations, glucose isomerase conformational and colloidal stability were successfully predicted for different salts in a specific pH range.

  19. Molecular insights into the stabilization of protein-protein interactions with small molecule: The FKBP12-rapamycin-FRB case study

    NASA Astrophysics Data System (ADS)

    Chaurasia, Shilpi; Pieraccini, Stefano; De Gonda, Riccardo; Conti, Simone; Sironi, Maurizio

    2013-11-01

    Targetting protein-protein interactions is a challenging task in drug discovery process. Despite the challenges, several studies provided evidences for the development of small molecules modulating protein-protein interactions. Here we consider a typical case of protein-protein interaction stabilization: the complex between FKBP12 and FRB with rapamycin. We have analyzed the stability of the complex and characterized its interactions at the atomic level by performing free energy calculations and computational alanine scanning. It is shown that rapamycin stabilizes the complex by acting as a bridge between the two proteins; and the complex is stable only in the presence of rapamycin.

  20. Addition of Monovalent Electrolytes to Improve Storage Stability of Freeze-Dried Protein Formulations.

    PubMed

    Goshima, Hiroshika; Forney-Stevens, Kelly M; Liu, Ming; Qian, Ken K; Tyagi, Madhusudan; Cicerone, Marcus T; Pikal, Michael J

    2016-02-01

    This study investigates the effect of low levels of electrolytes on storage stability in freeze-dried sucrose-based protein formulations. Both bovine serum albumin and recombinant human serum albumin were freeze dried with sucrose and alkali halides (LiCl, NaCl, KCl, RbCl, and CsCl) at selected low levels. All formulations were stored at 50 °C and 65 °C up to 2 months and then assayed for protein aggregation. The data demonstrate that low levels of LiCl and NaCl enhance stability. No obvious correlations with either protein secondary structure or global dynamics (structural relaxation time) were found. However, good correlations were found between stability and both free-volume hole size via positron annihilation lifetime spectroscopy (PALS) and fast dynamics by neutron scattering. Volume changes on mixing and the partial molal volume of salt were also studied in an effort to detect decreases in free volume. These data did not support the hypothesis that reduction in free volume was the primary mechanism for salt-induced stabilization. Finally, a positive effect of postlyophilization annealing on stability was demonstrated. In summary, we find that small amounts of LiCl and NaCl significantly stabilize these proteins, which is a result at variance with conventional formulation wisdom. Copyright © 2016 American Pharmacists Association®. All rights reserved.

  1. Effects of actin-binding proteins on the thermal stability of monomeric actin.

    PubMed

    Pivovarova, Anastasia V; Chebotareva, Natalia A; Kremneva, Elena V; Lappalainen, Pekka; Levitsky, Dmitrii I

    2013-01-08

    Differential scanning calorimetry (DSC) was applied to investigate the thermal unfolding of rabbit skeletal muscle G-actin in its complexes with actin-binding proteins, cofilin, twinfilin, and profilin. The results show that the effects of these proteins on the thermal stability of G-actin depend on the nucleotide, ATP or ADP, bound in the nucleotide-binding cleft between actin subdomains 2 and 4. Interestingly, cofilin binding stabilizes both ATP-G-actin and ADP-G-actin, whereas twinfilin increases the thermal stability of the ADP-G-actin but not that of the ATP-G-actin. By contrast, profilin strongly decreases the thermal stability of the ATP-G-actin but has no appreciable effect on the ADP-G-actin. Comparison of these DSC results with literature data reveals a relationship between the effects of actin-binding proteins on the thermal unfolding of G-actin, stabilization or destabilization, and their effects on the rate of nucleotide exchange in the nucleotide-binding cleft, decrease or increase. These results suggest that the thermal stability of G-actin depends, at least partially, on the conformation of the nucleotide-binding cleft: the actin molecule is more stable when the cleft is closed, while an opening of the cleft leads to significant destabilization of G-actin. Thus, DSC studies of the thermal unfolding of G-actin can provide new valuable information about the conformational changes induced by actin-binding proteins in the actin molecule.

  2. Glycosylation May Reduce Protein Thermodynamic Stability by Inducing a Conformational Distortion.

    PubMed

    Gavrilov, Yulian; Shental-Bechor, Dalit; Greenblatt, Harry M; Levy, Yaakov

    2015-09-17

    Glycosylation plays not only a functional role but can also modify the biophysical properties of the modified protein. Usually, natural glycosylation results in protein stabilization; however, in vitro and in silico studies showed that sometimes glycosylation results in thermodynamic destabilization. Here, we applied coarse-grained and all-atom molecular dynamics simulations to understand the mechanism underlying the loss of stability of the MM1 protein by glycosylation. We show that the origin of the destabilization is a conformational distortion of the protein caused by the interaction of the monosaccharide with the protein surface. Though glycosylation creates new short-range glycan-protein interactions that stabilize the conjugated protein, it breaks long-range protein-protein interactions. This has a destabilizing effect because the probability of long- and short-range interactions forming differs between the folded and unfolded states. The destabilization originates not from simple loss of interactions but due to a trade-off between the short- and long-range interactions.

  3. Detecting Selection on Protein Stability through Statistical Mechanical Models of Folding and Evolution

    PubMed Central

    Bastolla, Ugo

    2014-01-01

    The properties of biomolecules depend both on physics and on the evolutionary process that formed them. These two points of view produce a powerful synergism. Physics sets the stage and the constraints that molecular evolution has to obey, and evolutionary theory helps in rationalizing the physical properties of biomolecules, including protein folding thermodynamics. To complete the parallelism, protein thermodynamics is founded on the statistical mechanics in the space of protein structures, and molecular evolution can be viewed as statistical mechanics in the space of protein sequences. In this review, we will integrate both points of view, applying them to detecting selection on the stability of the folded state of proteins. We will start discussing positive design, which strengthens the stability of the folded against the unfolded state of proteins. Positive design justifies why statistical potentials for protein folding can be obtained from the frequencies of structural motifs. Stability against unfolding is easier to achieve for longer proteins. On the contrary, negative design, which consists in destabilizing frequently formed misfolded conformations, is more difficult to achieve for longer proteins. The folding rate can be enhanced by strengthening short-range native interactions, but this requirement contrasts with negative design, and evolution has to trade-off between them. Finally, selection can accelerate functional movements by favoring low frequency normal modes of the dynamics of the native state that strongly correlate with the functional conformation change. PMID:24970217

  4. Co-translational stabilization of insoluble proteins in cell-free expression systems.

    PubMed

    Kai, Lei; Orbán, Erika; Henrich, Erik; Proverbio, Davide; Dötsch, Volker; Bernhard, Frank

    2015-01-01

    Precipitation, aggregation, and inclusion body (IB) formation are frequently observed problems upon overexpression of recombinant proteins. The open accessibility of cell-free reactions allows addressing such critical steps by the addition of protein stabilizers such as chemical chaperones or detergents directly into the expression reactions. This approach could therefore reduce or even prevent initial protein precipitation already in the translation environment. The strategy might be considered to generally improve protein sample quality and to rescue proteins that are difficult to refold from IBs or from aggregated precipitates. We describe a protocol for the co-translational stabilization of difficult proteins by their expression in the presence of supplements such as alcohols, poly-ions, or detergents. We compile potentially useful compounds together with their recommended stock and working concentrations. Examples of screening experiments in order to systematically identify compounds or compound mixtures that stabilize particular proteins of interest are given. The method can primarily be considered for the production of unstable soluble proteins or of membrane proteins containing larger soluble domains.

  5. Effect of gold nanoparticle conjugation on the activity and stability of functional proteins.

    PubMed

    Bailes, Julian; Gazi, Sara; Ivanova, Rositsa; Soloviev, Mikhail

    2012-01-01

    Immobilization of functional proteins such as enzymes on solid surfaces produces a variety of effects ranging from the reversal and strong inhibition to the enhancement of protein stability and function. Such effects are protein-dependent and are affected by the physical and chemical properties of the surfaces. Functional consequences of protein immobilization on the surface of gold nanoparticles (AuNPs) are protein-dependent and require thorough investigation using suitable functional tests. However, traditional approaches to making control samples, i.e., immobilized protein vs. protein in solution in absence of any nanoparticles do not provide sufficiently identical reaction conditions and complicate interpretation of the results. This report provides advice and methods for preparing AuNP-conjugated preparations generally suitable for studying the effects of immobilization on the activity and stability of different functional proteins. We use bovine catalase to illustrate our approach, but the methods are easily adaptable to any other enzyme or protein. The AuNP-immobilized enzyme showed increased stability at elevated temperatures compared to the same enzyme in solution.

  6. p73 expression is regulated by ribosomal protein RPL26 through mRNA translation and protein stability

    PubMed Central

    Yan, Wensheng; Chen, Xinbin

    2016-01-01

    p73, a p53 family tumor suppressor, is regulated by multiple mechanisms, including transcription and mRNA and protein stability. However, whether p73 expression is regulated via mRNA translation has not been explored. To test this, we examined whether ribosomal protein 26 (RPL26) plays a role in p73 expression. Here, we showed that p73 expression is controlled by RPL26 via protein stability and mRNA translation. To examine whether MDM2 mediates RPL26 to regulate p73 protein stability, we generated multiple MDM2-knockout cell lines by CRISPR-cas9. We found that in the absence of MDM2, the half-life of p73 protein is markedly increased. Interestingly, we also found that RPL26 is still capable of regulating p73 expression, albeit to a lesser extent, in MDM2-KO cells compared to that in isogenic control cells, suggesting that RPL26 regulates p73 expression via multiple mechanisms. Indeed, we found that RPL26 is necessary for efficient assembly of polysomes on p73 mRNA and de novo synthesis of p73 protein. Consistently, we found that RPL26 directly binds to p73 3′ untranslated region (3′UTR) and that RPL26 is necessary for efficient expression of an eGFP reporter that carries p73 3′UTR. We also found that RPL26 interacts with cap-binding protein eIF4E and enhances the association of eIF4E with p73 mRNA, leading to increased p73 mRNA translation. Finally, we showed that knockdown of RPL26 promotes, whereas ectopic expression of RPL26 inhibits, cell growth in a TAp73-dependent manner. Together, our data indicate that RPL26 regulates p73 expression via two distinct mechanisms: protein stability and mRNA translation. PMID:27825141

  7. Improving Protein Stability and Controlling Protein Release by Adding Poly (Cyclohexane-1, 4-Diyl Acetone Dimethylene Ketal) to PLGA Microspheres.

    PubMed

    Wang, Chenhui; Yu, Changhui; Yu, Kongtong; Teng, Lesheng; Liu, Jiaxin; Wang, Xuesong; Sun, Fengying; Li, Youxin

    2015-01-01

    The use of biodegradable polymers such as PLGA to encapsulate therapeutic proteins for their controlled release has received tremendous interest. However, an acidic environment caused by PLGA degradation productions leads to protein incomplete release and chemical degradation. The aim of this study was to develop novel PCADK/PLGA microspheres to improve protein stability and release behavior. Bovine serum albumin (BSA) incubated in PCADK and PLGA degradation products was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC-HPLC), circular dichroism (CD) and fluorescence spectroscopy. Blended microspheres of PCADK/PLGA were prepared in different ratios and the release behaviors of the microspheres and the protein stability were then measured. The degradation properties of the microspheres and the pH inside the microspheres were systematically investigated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) to examine the mechanism of autocatalytic degradation and protein stability. BSA was more stable in the presence of PCADK monomers than it was in the presence of PLGA monomers, revealing that PCADK is highly compatible with this protein. PCADK/PLGA microspheres were successfully prepared, and 2/8 was determined to be the optimal ratio. Further, 43% of the BSA formed water-insoluble aggregates in the presence of PCADK/PLGA microspheres, compared with 57% for the PLGA microspheres, demonstrating that the BSA encapsulated in PCADK/PLGA blended microspheres was more stable than in PLGA microspheres. The PCADK/PLGA blended microspheres improved protein stability and release behavior, providing a promising protein drug delivery system.

  8. Maintenance of a Protein Structure in the Dynamic Evolution of TIMPs over 600 Million Years

    PubMed Central

    Nicosia, Aldo; Maggio, Teresa; Costa, Salvatore; Salamone, Monica; Tagliavia, Marcello; Mazzola, Salvatore; Gianguzza, Fabrizio; Cuttitta, Angela

    2016-01-01

    Deciphering the events leading to protein evolution represents a challenge, especially for protein families showing complex evolutionary history. Among them, TIMPs represent an ancient eukaryotic protein family widely distributed in the animal kingdom. They are known to control the turnover of the extracellular matrix and are considered to arise early during metazoan evolution, arguably tuning essential features of tissue and epithelial organization. To probe the structure and molecular evolution of TIMPs within metazoans, we report the mining and structural characterization of a large data set of TIMPs over approximately 600 Myr. The TIMPs repertoire was explored starting from the Cnidaria phylum, coeval with the origins of connective tissue, to great apes and humans. Despite dramatic sequence differences compared with highest metazoans, the ancestral proteins displayed the canonical TIMP fold. Only small structural changes, represented by an α-helix located in the N-domain, have occurred over the evolution. Both the occurrence of such secondary structure elements and the relative solvent accessibility of the corresponding residues in the three-dimensional structures raises the possibility that these sites represent unconserved element prone to accept variations. PMID:26957029

  9. Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication

    SciTech Connect

    Kanginakudru, Sriramana; DeSmet, Marsha; Thomas, Yanique; Morgan, Iain M.; Androphy, Elliot J.

    2015-04-15

    The evolutionarily conserved DNA topoisomerase II beta-binding protein 1 (TopBP1) functions in DNA replication, DNA damage response, and cell survival. We analyzed the role of TopBP1 in human and bovine papillomavirus genome replication. Consistent with prior reports, TopBP1 co-localized in discrete nuclear foci and was in complex with papillomavirus E2 protein. Similar to E2, TopBP1 is recruited to the region of the viral origin of replication during G1/S and early S phase. TopBP1 knockdown increased, while over-expression decreased transient virus replication, without affecting cell cycle. Similarly, using cell lines harboring HPV-16 or HPV-31 genome, TopBP1 knockdown increased while over-expression reduced viral copy number relative to genomic DNA. We propose a model in which TopBP1 serves dual roles in viral replication: it is essential for initiation of replication yet it restricts viral copy number. - Highlights: • Protein interaction study confirmed In-situ interaction between TopBP1 and E2. • TopBP1 present at papillomavirus ori in G1/S and early S phase of cell cycle. • TopBP1 knockdown increased, over-expression reduced virus replication. • TopBP1 protein level change did not influence cell survival or cell cycle. • TopBP1 displaced from papillomavirus ori after initiation of replication.

  10. Key challenges for the creation and maintenance of specialist protein resources.

    PubMed

    Holliday, Gemma L; Bairoch, Amos; Bagos, Pantelis G; Chatonnet, Arnaud; Craik, David J; Finn, Robert D; Henrissat, Bernard; Landsman, David; Manning, Gerard; Nagano, Nozomi; O'Donovan, Claire; Pruitt, Kim D; Rawlings, Neil D; Saier, Milton; Sowdhamini, Ramanathan; Spedding, Michael; Srinivasan, Narayanaswamy; Vriend, Gert; Babbitt, Patricia C; Bateman, Alex

    2015-06-01

    As the volume of data relating to proteins increases, researchers rely more and more on the analysis of published data, thus increasing the importance of good access to these data that vary from the supplemental material of individual articles, all the way to major reference databases with professional staff and long-term funding. Specialist protein resources fill an important middle ground, providing interactive web interfaces to their databases for a focused topic or family of proteins, using specialized approaches that are not feasible in the major reference databases. Many are labors of love, run by a single lab with little or no dedicated funding and there are many challenges to building and maintaining them. This perspective arose from a meeting of several specialist protein resources and major reference databases held at the Wellcome Trust Genome Campus (Cambridge, UK) on August 11 and 12, 2014. During this meeting some common key challenges involved in creating and maintaining such resources were discussed, along with various approaches to address them. In laying out these challenges, we aim to inform users about how these issues impact our resources and illustrate ways in which our working together could enhance their accuracy, currency, and overall value.

  11. Different phenotypes in vivo are associated with ATPase motif mutations in Schizosaccharomyces pombe minichromosome maintenance proteins.

    PubMed Central

    Gómez, Eliana B; Catlett, Michael G; Forsburg, Susan L

    2002-01-01

    The six conserved MCM proteins are essential for normal DNA replication. They share a central core of homology that contains sequences related to DNA-dependent and AAA(+) ATPases. It has been suggested that the MCMs form a replicative helicase because a hexameric subcomplex formed by MCM4, -6, and -7 proteins has in vitro DNA helicase activity. To test whether ATPase and helicase activities are required for MCM protein function in vivo, we mutated conserved residues in the Walker A and Walker B motifs of MCM4, -6, and -7 and determined that equivalent mutations in these three proteins have different in vivo effects in fission yeast. Some mutations reported to abolish the in vitro helicase activity of the mouse MCM4/6/7 subcomplex do not affect the in vivo function of fission yeast MCM complex. Mutations of consensus CDK sites in Mcm4p and Mcm7p also have no phenotypic consequences. Co-immunoprecipitation analyses and in situ chromatin-binding experiments were used to study the ability of the mutant Mcm4ps to associate with the other MCMs, localize to the nucleus, and bind to chromatin. We conclude that the role of ATP binding and hydrolysis is different for different MCM subunits. PMID:11973289

  12. Protein Stability and Dynamics Modulation: The Case of Human Frataxin

    PubMed Central

    Gallo, Mariana; Salvay, Andres G.; Ferreiro, Diego U.; Santos, Javier

    2012-01-01

    Frataxin (FXN) is an α/β protein that plays an essential role in iron homeostasis. Apparently, the function of human FXN (hFXN) depends on the cooperative formation of crucial interactions between helix α1, helix α2, and the C-terminal region (CTR) of the protein. In this work we quantitatively explore these relationships using a purified recombinant fragment hFXN90–195. This variant shows the hydrodynamic behavior expected for a monomeric globular domain. Circular dichroism, fluorescence, and NMR spectroscopies show that hFXN90–195 presents native-like secondary and tertiary structure. However, chemical and temperature induced denaturation show that CTR truncation significantly destabilizes the overall hFXN fold. Accordingly, limited proteolysis experiments suggest that the native-state dynamics of hFXN90–195 and hFXN90–210 are indeed different, being the former form much more sensitive to the protease at specific sites. The overall folding dynamics of hFXN fold was further explored with structure-based protein folding simulations. These suggest that the native ensemble of hFXN can be decomposed in at least two substates, one with consolidation of the CTR and the other without consolidation of the CTR. Explicit-solvent all atom simulations identify some of the proteolytic target sites as flexible regions of the protein. We propose that the local unfolding of CTR may be a critical step for the global unfolding of hFXN, and that modulation of the CTR interactions may strongly affect hFXN physiological function. PMID:23049850

  13. Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability

    PubMed Central

    Tahara, Maino; Bürckert, Jean-Philippe; Kanou, Kazuhiko; Maenaka, Katsumi; Muller, Claude P.; Takeda, Makoto

    2016-01-01

    Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors. PMID:27490564

  14. Determinants of the serum concentrations of low molecular weight proteins in patients on maintenance hemodialysis.

    PubMed

    Kabanda, A; Jadoul, M; Pochet, J M; Lauwerys, R; van Ypersele de Strihou, C; Bernard, A

    1994-06-01

    Factors influencing the serum concentrations of low molecular weight proteins (LMWP) during long-term hemodialysis were studied in 112 patients undergoing dialysis for an average of 61.1 months (range 1 to 243). These patients were treated with AN69, cellulose acetate, cuprophan or polysulfone membranes. The following proteins were measured in serum before and after a four hour dialysis session: cystatin C (CYST C), beta 2-microglobulin (beta 2 m), Clara cell protein (CC16) and retinol-binding protein (RBP). Predialysis levels of the four proteins were markedly elevated. In simple regression analysis, pre-dialysis serum concentrations of beta 2 m and CC16 weakly correlated with the duration of dialysis treatment, but these relations completely disappeared when a stepwise regression analysis was performed using as predictors age, sex, residual diuresis, body weight loss (BWL), duration of hemodialysis and the type or ultrafiltration coefficient (UFC) of the membranes. The only significant determinants which emerged from this analysis were the residual diuresis and age which negatively correlated with CYST C, beta 2m and CC16 (residual diuresis only), and sex which influenced CYST C. During the dialysis session, the microproteins underwent changes that were related to their molecular radius, the membrane UFC and the BWL. After adjustment for the latter, high flux membranes (UFC > or = 15 ml/h.m2.mm Hg) allowed up to 50% of CYST C and 25% of beta 2m to be removed. No significant elimination of CC16 and RBP was evident. On the basis of these results, we estimated the effective pore radius of high flux membranes between 1.5 and 1.7 nm and that of low flux membranes as below 1.5 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. SIRT6 stabilizes DNA-dependent Protein Kinase at chromatin for DNA double-strand break repair

    PubMed Central

    McCord, Ronald A.; Michishita, Eriko; Hong, Tao; Berber, Elisabeth; Boxer, Lisa D.; Kusumoto, Rika; Guan, Shenheng; Shi, Xiaobing; Gozani, Or; Burlingame, Alma L.; Bohr, Vilhelm A.; Chua, Katrin F.

    2009-01-01

    The Sir2 chromatin regulatory factor links maintenance of genomic stability to life span extension in yeast. The mammalian Sir2 family member SIRT6 has been proposed to have analogous functions, because SIRT6-deficiency leads to shortened life span and an aging-like degenerative phenotype in mice, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA-dependent protein kinase) and promotes DNA DSB repair. In response to DSBs, SIRT6 associates dynamically with chromatin and is necessary for an acute decrease in global cellular acetylation levels on histone H3 Lysine 9. Moreover, SIRT6 is required for mobilization of the DNA-PK catalytic subunit (DNA-PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor with chromatin impacts on the efficiency of repair, and establish a link between chromatin regulation, DNA repair, and a mammalian Sir2 factor. PMID:20157594

  16. Regulation of Paramyxovirus Fusion Activation: the Hemagglutinin-Neuraminidase Protein Stabilizes the Fusion Protein in a Pretriggered State

    PubMed Central

    Salah, Zuhair W.; Gui, Long; DeVito, Ilaria; Jurgens, Eric M.; Lu, Hong; Yokoyama, Christine C.; Palermo, Laura M.; Lee, Kelly K.

    2012-01-01

    The hemagglutinin (HA)-neuraminidase protein (HN) of paramyxoviruses carries out three discrete activities, each of which affects the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. Binding of HN to its sialic acid receptor on a target cell triggers its activation of the fusion protein (F), which then inserts into the target cell and mediates the membrane fusion that initiates infection. We provide new evidence for a fourth function of HN: stabilization of the F protein in its pretriggered state before activation. Influenza virus hemagglutinin protein (uncleaved HA) was used as a nonspecific binding protein to tether F-expressing cells to target cells, and heat was used to activate F, indicating that the prefusion state of F can be triggered to initiate structural rearrangement and fusion by temperature. HN expression along with uncleaved HA and F enhances the F activation if HN is permitted to engage the receptor. However, if HN is prevented from engaging the receptor by the use of a small compound, temperature-induced F activation is curtailed. The results indicate that HN helps stabilize the prefusion state of F, and analysis of a stalk domain mutant HN reveals that the stalk domain of HN mediates the F-stabilization effect. PMID:22993149

  17. Biophysical stability of hyFc fusion protein with regards to buffers and various excipients.

    PubMed

    Lim, Jun Yeul; Kim, Nam Ah; Lim, Dae Gon; Eun, Chang-yong; Choi, Donghoon; Jeong, Seong Hoon

    2016-05-01

    A novel non-cytolytic hybrid Fc (hyFc) with an intact Ig structure without any mutation in the hyFc region, was developed to construct a long-acting agonistic protein. The stability of interleukin-7 (IL-7) fused with the hyFc (GXN-04) was evaluated to develop early formulations. Various biophysical methods were utilized and three different buffer systems with various pH ranges were investigated including histidine-acetate, sodium citrate, and tris buffers. Various excipients were incorporated into the systems to obtain optimum protein stability. Two evident thermal transitions were observed with the unfolding of IL-7 and hyFc. The Tm and ΔH increased with pH, suggesting increased conformational stability. Increased Z-average size with PDI and decreased zeta potential with pH increase, with the exception of tris buffer, showed aggregation issues. Moreover, tris buffer at higher pH showed aggregation peaks from DLS. Non-ionic surfactants were effective against agitation by outcompeting protein molecules for hydrophobic surfaces. Sucrose and sorbitol accelerated protein aggregation during agitation, but exhibited a protective effect against oxidation, with preferential exclusion favoring the compact states of GXN-04. The stability of GXN-04 was varied by basal buffers and excipients, hence the buffers and excipients need to be evaluated carefully to achieve the maximum stability of proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Improved insights into protein thermal stability: from the molecular to the structurome scale.

    PubMed

    Pucci, Fabrizio; Rooman, Marianne

    2016-11-13

    Despite the intense efforts of the last decades to understand the thermal stability of proteins, the mechanisms responsible for its modulation still remain debated. In this investigation, we tackle this issue by showing how a multiscale perspective can yield new insights. With the help of temperature-dependent statistical potentials, we analysed some amino acid interactions at the molecular level, which are suggested to be relevant for the enhancement of thermal resistance. We then investigated the thermal stability at the protein level by quantifying its modification upon amino acid substitutions. Finally, a large scale analysis of protein stability-at the structurome level-contributed to the clarification of the relation between stability and natural evolution, thereby showing that the mutational profile of proteins differs according to their thermal properties. Some considerations on how the multiscale approach could help in unravelling the protein stability mechanisms are briefly discussed.This article is part of the themed issue 'Multiscale modelling at the physics-chemistry-biology interface'. © 2016 The Author(s).

  19. Influence of Miscibility of Protein-Sugar Lyophilizates on Their Storage Stability.

    PubMed

    Mensink, Maarten A; Nethercott, Matthew J; Hinrichs, Wouter L J; van der Voort Maarschalk, Kees; Frijlink, Henderik W; Munson, Eric J; Pikal, Michael J

    2016-09-01

    For sugars to act as successful stabilizers of proteins during lyophilization and subsequent storage, they need to have several characteristics. One of them is that they need to be able to form interactions with the protein and for that miscibility is essential. To evaluate the influence of protein-sugar miscibility on protein storage stability, model protein IgG was lyophilized in the presence of various sugars of different molecular weight. By comparing solid-state nuclear magnetic resonance spectroscopy relaxation times of both protein and sugar on two different timescales, i.e., (1)H T1 and (1)H T1ρ, miscibility of the two components was established on a 2-5- and a 20-50-nm length scale, respectively, and related to protein storage stability. Smaller sugars showed better miscibility with IgG, and the tendency of IgG to aggregate during storage was lower for smaller sugars. The largest sugar performed worst and was phase separated on both length scales. Additionally, shorter protein (1)H T1 relaxation times correlated with higher aggregation rates during storage. The enzyme-linked immunosorbent assay (ELISA) assay showed overlapping effects of aggregation and chemical degradation and did not correspond as well with the miscibility. Because of the small scale at which miscibility was determined (2-5 nm) and the size of the protein domains (∼2.5 × 2.5 × 5 nm), the miscibility data give an indirect measure of interaction between protein and sugar. This reduced interaction could be the result of steric hindrance, providing a possible explanation as to why smaller sugars show better miscibility and storage stability with the protein.

  20. Studying the role of cooperative hydration in stabilizing folded protein states.

    PubMed

    Huggins, David J

    2016-12-01

    Understanding and modelling protein folding remains a key scientific and engineering challenge. Two key questions in protein folding are (1) why many proteins adopt a folded state and (2) how these proteins transition from the random coil ensemble to a folded state. In this paper we employ molecular dynamics simulations to address the first of these questions. Computational methods are well-placed to address this issue due to their ability to analyze systems at atomic-level resolution. Traditionally, the stability of folded proteins has been ascribed to the balance of two types of intermolecular interactions: hydrogen-bonding interactions and hydrophobic contacts. In this study, we explore a third type of intermolecular interaction: cooperative hydration of protein surface residues. To achieve this, we consider multiple independent simulations of the villin headpiece domain to quantify the contributions of different interactions to the energy of the native and fully extended states. In addition, we consider whether these findings are robust with respect to the protein forcefield, the water model, and the presence of salt. In all cases, we identify many cooperatively hydrated interactions that are transient but energetically favor the native state. Whilst further work on additional protein structures, forcefields, and water models is necessary, these results suggest a role for cooperative hydration in protein folding that should be explored further. Rational design of cooperative hydration on the protein surface could be a viable strategy for increasing protein stability. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. Formation and stability of secondary structures in globular proteins

    NASA Astrophysics Data System (ADS)

    Bascle, J.; Garel, T.; Orland, H.

    1993-02-01

    We study two models for the formation and packing of helices and sheets in globular (compact) proteins. These models, based on weighted Hamiltonian paths on a regular lattice both exhibit a first order transition between a compact high temperature phase, with no extended secondary structures, and a quasi-frozen compact phase, with secondary structures invading the whole lattice. The quasi-frozen phase with very weak temperature dependence, is identified as the native phase of proteins, whereas the high-temperature phase may be relevant to the so-called molten globule state of proteins. Nous étudions deux modèles pour la formation et l'empilement d'hélices ou de feuillets dans la phase globulaire (compacte) des protéines. ces modèles, fondés sur des chemins hamiltoniens pondérés sur réseau, possèdent une transition de phase du premier ordre, entre (i) une phase haute température compacte, avec structures secondaires non étendues, et (ii) une phase compacte quasi-gelée, où les structures secondaires envahissent tout le réseau. La phase quasi-gelée, qui a une dépendance en température très faible, est identifiée à la phase native des protéines; la phase haute température est peut-être reliée à la phase native “globule fondu” (molten globule) des protéines.

  2. CEP152 is a genome maintenance protein disrupted in Seckel syndrome.

    PubMed

    Kalay, Ersan; Yigit, Gökhan; Aslan, Yakup; Brown, Karen E; Pohl, Esther; Bicknell, Louise S; Kayserili, Hülya; Li, Yun; Tüysüz, Beyhan; Nürnberg, Gudrun; Kiess, Wieland; Koegl, Manfred; Baessmann, Ingelore; Buruk, Kurtulus; Toraman, Bayram; Kayipmaz, Saadettin; Kul, Sibel; Ikbal, Mevlit; Turner, Daniel J; Taylor, Martin S; Aerts, Jan; Scott, Carol; Milstein, Karen; Dollfus, Helene; Wieczorek, Dagmar; Brunner, Han G; Hurles, Matthew; Jackson, Andrew P; Rauch, Anita; Nürnberg, Peter; Karagüzel, Ahmet; Wollnik, Bernd

    2011-01-01

    Functional impairment of DNA damage response pathways leads to increased genomic instability. Here we describe the centrosomal protein CEP152 as a new regulator of genomic integrity and cellular response to DNA damage. Using homozygosity mapping and exome sequencing, we identified CEP152 mutations in Seckel syndrome and showed that impaired CEP152 function leads to accumulation of genomic defects resulting from replicative stress through enhanced activation of ATM signaling and increased H2AX phosphorylation.

  3. CEP152 is a genome maintenance protein disrupted in Seckel syndrome

    PubMed Central

    Kalay, Ersan; Yigit, Gökhan; Aslan, Yakup; Brown, Karen E; Pohl, Esther; Bicknell, Louise S; Kayserili, Hülya; Li, Yun; Tüysüz, Beyhan; Nürnberg, Gudrun; Kiess, Wieland; Koegl, Manfred; Baessmann, Ingelore; Buruk, Kurtulus; Toraman, Bayram; Kayipmaz, Saadettin; Kul, Sibel; Ikbal, Mevlit; Turner, Daniel J; Taylor, Martin S; Aerts, Jan; Scott, Carol; Milstein, Karen; Dollfus, Helene; Wieczorek, Dagmar; Brunner, Han G; Hurles, Matthew; Jackson, Andrew P; Rauch, Anita; Nürnberg, Peter; Karagüzel, Ahmet; Wollnik, Bernd

    2012-01-01

    Functional impairment of DNA damage response pathways leads to increased genomic instability. Here we describe the centrosomal protein CEP152 as a new regulator of genomic integrity and cellular response to DNA damage. Using homozygosity mapping and exome sequencing, we identified CEP152 mutations in Seckel syndrome and showed that impaired CEP152 function leads to accumulation of genomic defects resulting from replicative stress through enhanced activation of ATM signaling and increased H2AX phosphorylation. PMID:21131973

  4. In vivo requirement of protein prenylation for maintenance of retinal cytoarchitecture and photoreceptor structure

    PubMed Central

    1995-01-01

    Recent studies have demonstrated that inhibition of mevalonate synthesis in cultured cells leads to altered cell morphology due to inhibition of protein prenylation. To investigate the effects in vivo of mevalonate deprivation in nondividing, terminally differentiated neural cells, we have analyzed the effects on retinal tissue of intravitreal injection of lovastatin, a potent inhibitor of the mevalonate-producing enzyme, HMG-CoA reductase. A single injection of lovastatin (0.25 mumol) produced profound dysplastic-like changes in adult rat retinas primarily involving the photoreceptor layer. Within 2 d after injection, photoreceptor nuclei migrated in a circular pattern resulting in the formation of rosette-like structures by 4 d. Also during this period, photoreceptor inner and outer segment degeneration was evident. By 21 d, intact photoreceptor nuclei with remnants of inner and outer segments were dispersed throughout all retinal layers. To investigate the biochemical specificity of the lovastatin-induced alterations, and to distinguish the relative importance of the various branches of the mevalonate pathway, the incorporation of [3H]acetate into retinal lipids was examined in the presence and absence of metabolic inhibitors. HPLC analysis of lovastatin-treated retinas revealed a dramatic reduction in the incorporation of intravitreally injected [3H]acetate into nonsaponifiable lipids, compared with controls. In contrast, intravitreal injection of NB-598, a specific inhibitor of squalene epoxidase, eliminated the conversion of newly synthesized squalene to sterols without obvious pathology. Hence, involvement to the sterol branch of isoprenoid metabolism in the lovastatin-induced morphologic disruption was obviated. Intravitreal injection of 0.27 mumol of N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), an inhibitor of carboxyl methyltransferase activity and prenylated protein function, produced morphologic changes that were virtually indistinguishable from

  5. Differential scanning calorimetry to quantify the stability of protein cages.

    PubMed

    Zhang, Yu; Ardejani, Maziar S

    2015-01-01

    Differential scanning calorimetry (DSC) is an experimental technique through which the differences in amount of heat required to maintain equal temperature between a sample and a reference cell are measured at various temperatures. The quantified heat relates to the differences in apparent heat capacity of the analytes. The data from DSC studies will thereby provide direct information about the energetics of thermally induced processes in the sample. Here we present a detailed protocol to quantify the thermostability of protein cage, bacterioferritin (BFR), using differential scanning calorimetry.

  6. Glycans as biofunctional ligands for gold nanorods: stability and targeting in protein-rich media.

    PubMed

    García, Isabel; Sánchez-Iglesias, Ana; Henriksen-Lacey, Malou; Grzelczak, Marek; Penadés, Soledad; Liz-Marzán, Luis M

    2015-03-18

    Poly(ethylene glycol) (PEG) has become the gold standard for stabilization of plasmonic nanoparticles (NPs) in biofluids, because it prevents aggregation while minimizing unspecific interactions with proteins. Application of Au NPs in biological environments requires the use of ligands that can target selected receptors, even in the presence of protein-rich media. We demonstrate here the stabilizing effect of low-molecular-weight glycans on both spherical and rod-like plasmonic NPs under physiological conditions, as bench-marked against the well-established PEG ligands. Glycan-coated NPs are resistant to adsorption of proteins from serum-containing media and avoid phagocytosis by macrophage-like cells, but retain selectivity toward carbohydrate-binding proteins in protein-rich biological media. These results open the way toward the design of efficient therapeutic/diagnostic glycan-decorated plasmonic nanotools for specific biological applications.

  7. Connecting two proteins using a fusion alpha helix stabilized by a chemical cross linker

    NASA Astrophysics Data System (ADS)

    Jeong, Woo Hyeon; Lee, Haerim; Song, Dong Hyun; Eom, Jae-Hoon; Kim, Sun Chang; Lee, Hee-Seung; Lee, Hayyoung; Lee, Jie-Oh

    2016-03-01

    Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Most of the cross linkers available have flexible spacers. Because of this, the linked hybrids have significant structural flexibility and the relative structure between their two components is largely unpredictable. Here we describe a method of connecting two proteins via a `fusion α helix' formed by joining two pre-existing helices into a single extended helix. Because simple ligation of two helices does not guarantee the formation of a continuous helix, we used EY-CBS, a synthetic cross linker that has been shown to react selectively with cysteines in α-helices, to stabilize the connecting helix. Formation and stabilization of the fusion helix was confirmed by determining the crystal structures of the fusion proteins with and without bound EY-CBS. Our method should be widely applicable for linking protein building blocks to generate predictable structures.

  8. Use of whey protein soluble aggregates for thermal stability-a hypothesis paper.

    PubMed

    Ryan, Kelsey N; Zhong, Qixin; Foegeding, Edward A

    2013-08-01

    Forming whey proteins into soluble aggregates is a modification shown to improve or expand the applications in foaming, emulsification, gelation, film-formation, and encapsulation. Whey protein soluble aggregates are defined as aggregates that are intermediates between monomer proteins and an insoluble gel network or precipitate. The conditions under which whey proteins denature and aggregate have been extensively studied and can be used as guiding principles of producing soluble aggregates. These conditions are reviewed for pH, ion type and concentration, cosolutes, and protein concentration, along with heating temperature and duration. Combinations of these conditions can be used to design soluble aggregates with desired physicochemical properties including surface charge, surface hydrophobicity, size, and shape. These properties in turn can be used to obtain target macroscopic properties, such as viscosity, clarity, and stability, of the final product. A proposed approach to designing soluble aggregates with improved thermal stability for beverage applications is presented.

  9. Preparation of iron bound succinylated milk protein concentrate and evaluation of its stability.

    PubMed

    Shilpashree, B G; Arora, Sumit; Sharma, Vivek; Bajaj, Rajesh Kumar; Tomar, S K

    2016-04-01

    Major problems associated with the fortification of soluble iron salts include chemical reactivity and incompatibility with other components. Milk protein concentrate (MPC) are able to bind significant amount of iron due to the presence of both casein and whey protein. MPC in its native state possess very poor solubility, therefore, succinylated derivatives of MPC (succ. MPC) were also used for the preparation of protein-iron complex. Preparation of the complex involved centrifugation (to remove insoluble iron), ultrafiltration (to remove unbound iron) and lyophilisation (to attain in dry form). Iron binding ability of MPC enhanced significantly (P<0.05) upon succinylation. Stability of bound iron from both varieties of complexes was monitored und