Stabilization of Proteins by Polymer Conjugation via ATRP

DTIC Science & Technology

2008-08-31

to increase their solubility and utility in organic solvents and to increase their stability in body. Protein-initiated ATRP would enable us to... Solvent solubilization, therapeutic proteins, hydrophilic polymers, protein stabilization Lance Mabus, Jason Berberich, Bhalchandra Lele, Virginia Depp... solvents and to increase their stability in body. Protein-initiated ATRP would enable us to overcome many problems in conventional technology that

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.

Thermal stability and gel quality of myofibrillar protein as affected by soy protein isolates subjected to an acidic pH and mild heating.

PubMed

Niu, Haili; Xia, Xiufang; Wang, Chao; Kong, Baohua; Liu, Qian

2018-03-01

Thermal stability and gel quality of myofibrillar protein were evaluated with regard to the addition of native soy protein isolates (SPI) and SPI subjected to acidic pH and mild heating (modified SPI). Compared with the control, the addition of modified SPI increased the compression force of the protein gel and decreased water loss (P<0.05). Differential scanning calorimetry results showed that an addition of 0.75% native SPI decreased the first transition temperature (P<0.05), and addition of 0.5% and 0.75% modified SPI exhibited no appreciable changes on it (P>0.05), indicating that a higher concentration of modified SPI would not damage the protein thermal stability. Moreover, the addition of modified SPI enhanced hydrogen bonding and disulphide linkages. Atomic force microscopy analysis revealed that the addition of modified SPI decreased the roughness of the mixed myofibrillar protein gels. Overall, modified SPI has the potential to improve myofibrillar protein gel texture and water holding capacity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Flavor and stability of milk proteins.

PubMed

Smith, T J; Campbell, R E; Jo, Y; Drake, M A

2016-06-01

MPC decreased and furosine concentration increased with storage time and temperature. Solubility of MPC 80 was reduced more than that of MPC 45, but time and temperature adversely affected solubility of both proteins, with storage temperature having the greatest effect. Flavor and shelf stability of milk proteins provide a foundation of knowledge to improve the flavor and shelf-life of milk proteins. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Beyond Anchoring: the Expanding Role of the Hendra Virus Fusion Protein Transmembrane Domain in Protein Folding, Stability, and Function

PubMed Central

Smith, Everett Clinton; Culler, Megan R.; Hellman, Lance M.; Fried, Michael G.; Creamer, Trevor P.

2012-01-01

While work with viral fusion proteins has demonstrated that the transmembrane domain (TMD) can affect protein folding, stability, and membrane fusion promotion, the mechanism(s) remains poorly understood. TMDs could play a role in fusion promotion through direct TMD-TMD interactions, and we have recently shown that isolated TMDs from three paramyxovirus fusion (F) proteins interact as trimers using sedimentation equilibrium (SE) analysis (E. C. Smith, et al., submitted for publication). Immediately N-terminal to the TMD is heptad repeat B (HRB), which plays critical roles in fusion. Interestingly, addition of HRB decreased the stability of the trimeric TMD-TMD interactions. This result, combined with previous findings that HRB forms a trimeric coiled coil in the prefusion form of the whole protein though HRB peptides fail to stably associate in isolation, suggests that the trimeric TMD-TMD interactions work in concert with elements in the F ectodomain head to stabilize a weak HRB interaction. Thus, changes in TMD-TMD interactions could be important in regulating F triggering and refolding. Alanine insertions between the TMD and HRB demonstrated that spacing between these two regions is important for protein stability while not affecting TMD-TMD interactions. Additional mutagenesis of the C-terminal end of the TMD suggests that β-branched residues within the TMD play a role in membrane fusion, potentially through modulation of TMD-TMD interactions. Our results support a model whereby the C-terminal end of the Hendra virus F TMD is an important regulator of TMD-TMD interactions and show that these interactions help hold HRB in place prior to the triggering of membrane fusion. PMID:22238302

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.

Impact of alcohols on the formation and stability of protein-stabilized nanoemulsions.

PubMed

Zeeb, Benjamin; Herz, Eva; McClements, David Julian; Weiss, Jochen

2014-11-01

Nanoemulsions are increasingly being used for encapsulation, protection, and delivery of bioactive lipids, however, their formation from natural emulsifiers is still challenging. We investigated the impact of alcohol on the formation and stability of protein-stabilized oil-in-water nanoemulsions prepared by high-pressure homogenization. The influence of different alcohols (ethanol, 1-propanol, and 1-butanol) at various concentrations (0-25% w/w) on the formation and stability of emulsions stabilized by sodium caseinate, whey protein isolate, and fish gelatin was investigated. The mean particle diameter decreased with increasing alcohol concentrations from 0 to 10%w/w, but extensive droplet aggregation occurred at higher levels. This phenomenon was attributed to enhanced protein-protein interactions between the adsorbed emulsifier molecules in the presence of alcohol leading to droplet flocculation. The smallest droplets (d<100nm) were obtained when 10%w/w 1-butanol was added to sodium caseinate-stabilized nanoemulsions, but relatively small droplets (d<150nm) could also be obtained in the presence of a food-grade alcohol (ethanol). This study demonstrated that alcohol addition might be a useful tool for producing protein-stabilized nanoemulsions suitable for use as delivery systems of lipophilic bioactive agents. Copyright © 2014 Elsevier Inc. All rights reserved.

Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases

PubMed Central

Felsovalyi, Flora; Patel, Tushar; Mangiagalli, Paolo; Kumar, Sanat K; Banta, Scott

2012-01-01

Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior. PMID:22619179

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.

Recent advances in the applications of ionic liquids in protein stability and activity: a review.

PubMed

Patel, Rajan; Kumari, Meena; Khan, Abbul Bashar

2014-04-01

Room temperatures ionic liquids are considered as miraculous solvents for biological system. Due to their inimitable properties and large variety of applications, they have been widely used in enzyme catalysis and protein stability and separation. The related information present in the current review is helpful to the researchers working in the field of biotechnology and biochemistry to design or choose an ionic liquid that can serve as a noble and selective solvent for any particular enzymatic reaction, protein preservation and other protein based applications. We have extensively analyzed the methods used for studying the protein-IL interaction which is useful in providing information about structural and conformational dynamics of protein. This can be helpful to develop and understanding about the effect of ionic liquids on stability and activity of proteins. In addition, the affect of physico-chemical properties of ionic liquids, viz. hydrogen bond capacity and hydrophobicity on protein stability are discussed.

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.

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.

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.

Stability of α-tocopherol in freeze-dried sugar-protein-oil emulsion solids as affected by water plasticization and sugar crystallization.

PubMed

Zhou, Yankun; Roos, Yrjö H

2012-08-01

Water plasticization of sugar-protein encapsulants may cause structural changes and decrease the stability of encapsulated compounds during storage. The retention of α-tocopherol in freeze-dried lactose-milk protein-oil, lactose-soy protein-oil, trehalose-milk protein-oil, and trehalose-soy protein-oil systems at various water activities (a(w)) and in the presence of sugar crystallization was studied. Water sorption was determined gravimetrically. Glass transition and sugar crystallization were studied using differential scanning calorimetry and the retention of α-tocopherol spectrophotometrically. The loss of α-tocopherol followed lipid oxidation, but the greatest stability was found at 0 a(w) presumably because of α-tocopherol immobilization at interfaces and consequent reduction in antioxidant activity. A considerable loss of α-tocopherol coincided with sugar crystallization. The results showed that glassy matrices may protect encapsulated α-tocopherol; however, its role as an antioxidant at increasing aw accelerated its loss. Sugar crystallization excluded the oil-containing α-tocopherol from the protecting matrices and exposed it to surroundings, which decreased the stability of α-tocopherol.

Trimeric transmembrane domain interactions in paramyxovirus fusion proteins: roles in protein folding, stability, and function.

PubMed

Smith, Everett Clinton; Smith, Stacy E; Carter, James R; Webb, Stacy R; Gibson, Kathleen M; Hellman, Lance M; Fried, Michael G; Dutch, Rebecca Ellis

2013-12-13

Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion.

Stability and the Evolvability of Function in a Model Protein

PubMed Central

Bloom, Jesse D.; Wilke, Claus O.; Arnold, Frances H.; Adami, Christoph

2004-01-01

Functional proteins must fold with some minimal stability to a structure that can perform a biochemical task. Here we use a simple model to investigate the relationship between the stability requirement and the capacity of a protein to evolve the function of binding to a ligand. Although our model contains no built-in tradeoff between stability and function, proteins evolved function more efficiently when the stability requirement was relaxed. Proteins with both high stability and high function evolved more efficiently when the stability requirement was gradually increased than when there was constant selection for high stability. These results show that in our model, the evolution of function is enhanced by allowing proteins to explore sequences corresponding to marginally stable structures, and that it is easier to improve stability while maintaining high function than to improve function while maintaining high stability. Our model also demonstrates that even in the absence of a fundamental biophysical tradeoff between stability and function, the speed with which function can evolve is limited by the stability requirement imposed on the protein. PMID:15111394

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

Nanostructured Mineral Coatings Stabilize Proteins for Therapeutic Delivery.

PubMed

Yu, Xiaohua; Biedrzycki, Adam H; Khalil, Andrew S; Hess, Dalton; Umhoefer, Jennifer M; Markel, Mark D; Murphy, William L

2017-09-01

Proteins tend to lose their biological activity due to their fragile structural conformation during formulation, storage, and delivery. Thus, the inability to stabilize proteins in controlled-release systems represents a major obstacle in drug delivery. Here, a bone mineral inspired protein stabilization strategy is presented, which uses nanostructured mineral coatings on medical devices. Proteins bound within the nanostructured coatings demonstrate enhanced stability against extreme external stressors, including organic solvents, proteases, and ethylene oxide gas sterilization. The protein stabilization effect is attributed to the maintenance of protein conformational structure, which is closely related to the nanoscale feature sizes of the mineral coatings. Basic fibroblast growth factor (bFGF) released from a nanostructured mineral coating maintains its biological activity for weeks during release, while it maintains activity for less than 7 d during release from commonly used polymeric microspheres. Delivery of the growth factors bFGF and vascular endothelial growth factor using a mineral coated surgical suture significantly improves functional Achilles tendon healing in a rabbit model, resulting in increased vascularization, more mature collagen fiber organization, and a two fold improvement in mechanical properties. The findings of this study demonstrate that biomimetic interactions between proteins and nanostructured minerals provide a new, broadly applicable mechanism to stabilize proteins in the context of drug delivery and regenerative medicine. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Engineering proteins with tunable thermodynamic and kinetic stabilities.

PubMed

Pey, Angel L; Rodriguez-Larrea, David; Bomke, Susanne; Dammers, Susanne; Godoy-Ruiz, Raquel; Garcia-Mira, Maria M; Sanchez-Ruiz, Jose M

2008-04-01

It is widely recognized that enhancement of protein stability is an important biotechnological goal. However, some applications at least, could actually benefit from stability being strongly dependent on a suitable environment variable, in such a way that enhanced stability or decreased stability could be realized as required. In therapeutic applications, for instance, a long shelf-life under storage conditions may be convenient, but a sufficiently fast degradation of the protein after it has performed the planned molecular task in vivo may avoid side effects and toxicity. Undesirable effects associated to high stability are also likely to occur in food-industry applications. Clearly, one fundamental factor involved here is the kinetic stability of the protein, which relates to the time-scale of the irreversible denaturation processes and which is determined to some significant extent by the free-energy barrier for unfolding (the barrier that "separates" the native state from the highly-susceptible-to-irreversible-alterations nonnative states). With an appropriate experimental model, we show that strong environment-dependencies of the thermodynamic and kinetic stabilities can be achieved using robust protein engineering. We use sequence-alignment analysis and simple computational electrostatics to design stabilizing and destabilizing mutations, the latter introducing interactions between like charges which are screened out at high salt. Our design procedures lead naturally to mutating regions which are mostly unstructured in the transition state for unfolding. As a result, the large salt effect on the thermodynamic stability of our consensus plus charge-reversal variant translates into dramatic changes in the time-scale associated to the unfolding barrier: from the order of years at high salt to the order of days at low salt. Certainly, large changes in salt concentration are not expected to occur in biological systems in vivo. Hence, proteins with strong salt

Exploring Protein Stability by Comparative Molecular Dynamics Simulations of Homologous Hyperthermophilic, Mesophilic, and Psychrophilic Proteins.

PubMed

Khan, Sara; Farooq, Umar; Kurnikova, Maria

2016-11-28

In the present studies, we analyzed the influence of temperature on the stability and dynamics of the α subunit of tryptophan synthase (TRPS) from hyperthermophilic, mesophilic, and psychrophilic homologues at different temperatures by molecular dynamics simulations. Employing different indicators such as root-mean-square deviations, root-mean-square fluctuations, principal component analysis, and free energy landscapes, this study manifests the diverse behavior of these homologues with changes in temperature. Especially, an enhancement in the collective motions, classified as representative motions, is observed at high temperature. Similarly, the criterion for the selection of electrostatic interactions in terms of their life span (duty cycle) has indeed helped in identifying the short- and long-lived electrostatic interactions and how they affect the protein's overall stability at different temperatures. Rigidity and flexibility patterns of the homologous proteins are examined using FIRST software along with the calculation of duty cycles with various threshold limits at different temperatures. Rigid cluster decomposition in TRPS of psychrophilic, mesophilic, and hyperthermophilic origin identifies the flexible and rigid regions in the protein. Early loss of rigidity is observed in mesophilic TRPS via loss of contact between the major fragments of the protein compared with the other homologues. In spite of the high similarity of their three-dimensional structures, the overall responses of the three proteins to varying temperatures are significantly different.

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.

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.

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

STRUM: structure-based prediction of protein stability changes upon single-point mutation

PubMed Central

Quan, Lijun; Lv, Qiang; Zhang, Yang

2016-01-01

Motivation: Mutations in human genome are mainly through single nucleotide polymorphism, some of which can affect stability and function of proteins, causing human diseases. Several methods have been proposed to predict the effect of mutations on protein stability; but most require features from experimental structure. Given the fast progress in protein structure prediction, this work explores the possibility to improve the mutation-induced stability change prediction using low-resolution structure modeling. Results: We developed a new method (STRUM) for predicting stability change caused by single-point mutations. Starting from wild-type sequences, 3D models are constructed by the iterative threading assembly refinement (I-TASSER) simulations, where physics- and knowledge-based energy functions are derived on the I-TASSER models and used to train STRUM models through gradient boosting regression. STRUM was assessed by 5-fold cross validation on 3421 experimentally determined mutations from 150 proteins. The Pearson correlation coefficient (PCC) between predicted and measured changes of Gibbs free-energy gap, ΔΔG, upon mutation reaches 0.79 with a root-mean-square error 1.2 kcal/mol in the mutation-based cross-validations. The PCC reduces if separating training and test mutations from non-homologous proteins, which reflects inherent correlations in the current mutation sample. Nevertheless, the results significantly outperform other state-of-the-art methods, including those built on experimental protein structures. Detailed analyses show that the most sensitive features in STRUM are the physics-based energy terms on I-TASSER models and the conservation scores from multiple-threading template alignments. However, the ΔΔG prediction accuracy has only a marginal dependence on the accuracy of protein structure models as long as the global fold is correct. These data demonstrate the feasibility to use low-resolution structure modeling for high-accuracy stability

Supersonic Wave Interference Affecting Stability

NASA Technical Reports Server (NTRS)

Love, Eugene S.

1958-01-01

Some of the significant interference fields that may affect stability of aircraft at supersonic speeds are briefly summarized. Illustrations and calculations are presented to indicate the importance of interference fields created by wings, bodies, wing-body combinations, jets, and nacelles.

Thermal precipitation fluorescence assay for protein stability screening.

PubMed

Fan, Junping; Huang, Bo; Wang, Xianping; Zhang, Xuejun C

2011-09-01

A simple and reliable method of protein stability assessment is desirable for high throughput expression screening of recombinant proteins. Here we described an assay termed thermal precipitation fluorescence (TPF) which can be used to compare thermal stabilities of recombinant protein samples directly from cell lysate supernatants. In this assay, target membrane proteins are expressed as recombinant fusions with a green fluorescence protein tag and solubilized with detergent, and the fluorescence signals are used to report the quantity of the fusion proteins in the soluble fraction of the cell lysate. After applying a heat shock, insoluble protein aggregates are removed by centrifugation. Subsequently, the amount of remaining protein in the supernatant is quantified by in-gel fluorescence analysis and compared to samples without a heat shock treatment. Over 60 recombinant membrane proteins from Escherichia coli were subject to this screening in the presence and absence of a few commonly used detergents, and the results were analyzed. Because no sophisticated protein purification is required, this TPF technique is suitable to high throughput expression screening of recombinant membrane proteins as well as soluble ones and can be used to prioritize target proteins based on their thermal stabilities for subsequent large scale expression and structural studies. Copyright © 2011 Elsevier Inc. All rights reserved.

Cold denaturation as a tool to measure protein stability

PubMed Central

Sanfelice, Domenico; Temussi, Piero Andrea

2016-01-01

Protein stability is an important issue for the interpretation of a wide variety of biological problems but its assessment is at times difficult. The most common parameter employed to describe protein stability is the temperature of melting, at which the populations of folded and unfolded species are identical. This parameter may yield ambiguous results. It would always be preferable to measure the whole stability curve. The calculation of this curve is greatly facilitated whenever it is possible to observe cold denaturation. Using Yfh1, one of the few proteins whose cold denaturation occurs at neutral pH and low ionic strength, we could measure the variation of its full stability curve under several environmental conditions. Here we show the advantages of gauging stability as a function of external variables using stability curves. PMID:26026885

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

PubMed Central

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

2017-01-01

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. PMID:28213515

Electrostatic contribution to the binding stability of protein-protein complexes.

PubMed

Dong, Feng; Zhou, Huan-Xiang

2006-10-01

To investigate roles of electrostatic interactions in protein binding stability, electrostatic calculations were carried out on a set of 64 mutations over six protein-protein complexes. These mutations alter polar interactions across the interface and were selected for putative dominance of electrostatic contributions to the binding stability. Three protocols of implementing the Poisson-Boltzmann model were tested. In vdW4 the dielectric boundary between the protein low dielectric and the solvent high dielectric is defined as the protein van der Waals surface and the protein dielectric constant is set to 4. In SE4 and SE20, the dielectric boundary is defined as the surface of the protein interior inaccessible to a 1.4-A solvent probe, and the protein dielectric constant is set to 4 and 20, respectively. In line with earlier studies on the barnase-barstar complex, the vdW4 results on the large set of mutations showed the closest agreement with experimental data. The agreement between vdW4 and experiment supports the contention of dominant electrostatic contributions for the mutations, but their differences also suggest van der Waals and hydrophobic contributions. The results presented here will serve as a guide for future refinement in electrostatic calculation and inclusion of nonelectrostatic effects. Proteins 2006. (c) 2006 Wiley-Liss, Inc.

Stability of the Influenza Virus Hemagglutinin Protein Correlates with Evolutionary Dynamics.

PubMed

Klein, Eili Y; Blumenkrantz, Deena; Serohijos, Adrian; Shakhnovich, Eugene; Choi, Jeong-Mo; Rodrigues, João V; Smith, Brendan D; Lane, Andrew P; Feldman, Andrew; Pekosz, Andrew

2018-01-01

Protein thermodynamics are an integral determinant of viral fitness and one of the major drivers of protein evolution. Mutations in the influenza A virus (IAV) hemagglutinin (HA) protein can eliminate neutralizing antibody binding to mediate escape from preexisting antiviral immunity. Prior research on the IAV nucleoprotein suggests that protein stability may constrain seasonal IAV evolution; however, the role of stability in shaping the evolutionary dynamics of the HA protein has not been explored. We used the full coding sequence of 9,797 H1N1pdm09 HA sequences and 16,716 human seasonal H3N2 HA sequences to computationally estimate relative changes in the thermal stability of the HA protein between 2009 and 2016. Phylogenetic methods were used to characterize how stability differences impacted the evolutionary dynamics of the virus. We found that pandemic H1N1 IAV strains split into two lineages that had different relative HA protein stabilities and that later variants were descended from the higher-stability lineage. Analysis of the mutations associated with the selective sweep of the higher-stability lineage found that they were characterized by the early appearance of highly stabilizing mutations, the earliest of which was not located in a known antigenic site. Experimental evidence further suggested that H1N1 HA stability may be correlated with in vitro virus production and infection. A similar analysis of H3N2 strains found that surviving lineages were also largely descended from viruses predicted to encode more-stable HA proteins. Our results suggest that HA protein stability likely plays a significant role in the persistence of different IAV lineages. IMPORTANCE One of the constraints on fast-evolving viruses, such as influenza virus, is protein stability, or how strongly the folded protein holds together. Despite the importance of this protein property, there has been limited investigation of the impact of the stability of the influenza virus

Deciphering the Dynamics of Non-Covalent Interactions Affecting Thermal Stability of a Protein: Molecular Dynamics Study on Point Mutant of Thermus thermophilus Isopropylmalate Dehydrogenase.

PubMed

Sharma, Reetu; Sastry, G Narahari

2015-01-01

Thermus thermophilius isopropylmalate dehydrogenase catalyzes oxidative decarboxylation and dehydrogenation of isopropylmalate. Substitution of leucine to alanine at position 172 enhances the thermal stability among the known point mutants. Exploring the dynamic properties of non-covalent interactions such as saltbridges, hydrogen bonds and hydrophobic interactions to explain thermal stability of a protein is interesting in its own right. In this study dynamic changes in the non-covalent interactions are studied to decipher the deterministic features of thermal stability of a protein considering a case study of a point mutant in Thermus thermophilus isopropylmalate dehydrogenase. A total of four molecular dynamic simulations of 0.2 μs were carried out on wild type and mutant's functional dimers at 300 K and 337 K. Higher thermal stability of the mutant as compared to wild type is revealed by root mean square deviation, root mean square fluctuations and Cα-Cα distance with an increase in temperature from 300 K to 337 K. Most of the regions of wild type fluctuate higher than the corresponding regions of mutant with an increase in temperature. Cα-Cα distance analysis suggests that long distance networks are significantly affected in wild type as compared to the mutant. Short lived contacts are higher in wild type, while long lived contacts are lost at 337 K. The mutant forms less hydrogen bonds with water as compared to wild type at 337 K. In contrast to wild type, the mutant shows significant increase in unique saltbridges, hydrogen bonds and hydrophobic contacts at 337 K. The current study indicates that there is a strong inter-dependence of thermal stability on the way in which non-covalent interactions reorganize, and it is rewarding to explore this connection in single mutant studies.

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.

Hsp90 shapes protein and RNA evolution to balance trade-offs between protein stability and aggregation.

PubMed

Geller, Ron; Pechmann, Sebastian; Acevedo, Ashley; Andino, Raul; Frydman, Judith

2018-05-03

Acquisition of mutations is central to evolution; however, the detrimental effects of most mutations on protein folding and stability limit protein evolvability. Molecular chaperones, which suppress aggregation and facilitate polypeptide folding, may alleviate the effects of destabilizing mutations thus promoting sequence diversification. To illuminate how chaperones can influence protein evolution, we examined the effect of reduced activity of the chaperone Hsp90 on poliovirus evolution. We find that Hsp90 offsets evolutionary trade-offs between protein stability and aggregation. Lower chaperone levels favor variants of reduced hydrophobicity and protein aggregation propensity but at a cost to protein stability. Notably, reducing Hsp90 activity also promotes clusters of codon-deoptimized synonymous mutations at inter-domain boundaries, likely to facilitate cotranslational domain folding. Our results reveal how a chaperone can shape the sequence landscape at both the protein and RNA levels to harmonize competing constraints posed by protein stability, aggregation propensity, and translation rate on successful protein biogenesis.

Automated selection of stabilizing mutations in designed and natural proteins.

PubMed

Borgo, Benjamin; Havranek, James J

2012-01-31

The ability to engineer novel protein folds, conformations, and enzymatic activities offers enormous potential for the development of new protein therapeutics and biocatalysts. However, many de novo and redesigned proteins exhibit poor hydrophobic packing in their predicted structures, leading to instability or insolubility. The general utility of rational, structure-based design would greatly benefit from an improved ability to generate well-packed conformations. Here we present an automated protocol within the RosettaDesign framework that can identify and improve poorly packed protein cores by selecting a series of stabilizing point mutations. We apply our method to previously characterized designed proteins that exhibited a decrease in stability after a full computational redesign. We further demonstrate the ability of our method to improve the thermostability of a well-behaved native protein. In each instance, biophysical characterization reveals that we were able to stabilize the original proteins against chemical and thermal denaturation. We believe our method will be a valuable tool for both improving upon designed proteins and conferring increased stability upon native proteins.

Automated selection of stabilizing mutations in designed and natural proteins

PubMed Central

Borgo, Benjamin; Havranek, James J.

2012-01-01

The ability to engineer novel protein folds, conformations, and enzymatic activities offers enormous potential for the development of new protein therapeutics and biocatalysts. However, many de novo and redesigned proteins exhibit poor hydrophobic packing in their predicted structures, leading to instability or insolubility. The general utility of rational, structure-based design would greatly benefit from an improved ability to generate well-packed conformations. Here we present an automated protocol within the RosettaDesign framework that can identify and improve poorly packed protein cores by selecting a series of stabilizing point mutations. We apply our method to previously characterized designed proteins that exhibited a decrease in stability after a full computational redesign. We further demonstrate the ability of our method to improve the thermostability of a well-behaved native protein. In each instance, biophysical characterization reveals that we were able to stabilize the original proteins against chemical and thermal denaturation. We believe our method will be a valuable tool for both improving upon designed proteins and conferring increased stability upon native proteins. PMID:22307603

STRUM: structure-based prediction of protein stability changes upon single-point mutation.

PubMed

Quan, Lijun; Lv, Qiang; Zhang, Yang

2016-10-01

Mutations in human genome are mainly through single nucleotide polymorphism, some of which can affect stability and function of proteins, causing human diseases. Several methods have been proposed to predict the effect of mutations on protein stability; but most require features from experimental structure. Given the fast progress in protein structure prediction, this work explores the possibility to improve the mutation-induced stability change prediction using low-resolution structure modeling. We developed a new method (STRUM) for predicting stability change caused by single-point mutations. Starting from wild-type sequences, 3D models are constructed by the iterative threading assembly refinement (I-TASSER) simulations, where physics- and knowledge-based energy functions are derived on the I-TASSER models and used to train STRUM models through gradient boosting regression. STRUM was assessed by 5-fold cross validation on 3421 experimentally determined mutations from 150 proteins. The Pearson correlation coefficient (PCC) between predicted and measured changes of Gibbs free-energy gap, ΔΔG, upon mutation reaches 0.79 with a root-mean-square error 1.2 kcal/mol in the mutation-based cross-validations. The PCC reduces if separating training and test mutations from non-homologous proteins, which reflects inherent correlations in the current mutation sample. Nevertheless, the results significantly outperform other state-of-the-art methods, including those built on experimental protein structures. Detailed analyses show that the most sensitive features in STRUM are the physics-based energy terms on I-TASSER models and the conservation scores from multiple-threading template alignments. However, the ΔΔG prediction accuracy has only a marginal dependence on the accuracy of protein structure models as long as the global fold is correct. These data demonstrate the feasibility to use low-resolution structure modeling for high-accuracy stability change prediction upon

Principles of Protein Stability and Their Application in Computational Design.

PubMed

Goldenzweig, Adi; Fleishman, Sarel

2018-01-26

Proteins are increasingly used in basic and applied biomedical research.Many proteins, however, are only marginally stable and can be expressed in limited amounts, thus hampering research and applications. Research has revealed the thermodynamic, cellular, and evolutionary principles and mechanisms that underlie marginal stability. With this growing understanding, computational stability design methods have advanced over the past two decades starting from methods that selectively addressed only some aspects of marginal stability. Current methods are more general and, by combining phylogenetic analysis with atomistic design, have shown drastic improvements in solubility, thermal stability, and aggregation resistance while maintaining the protein's primary molecular activity. Stability design is opening the way to rational engineering of improved enzymes, therapeutics, and vaccines and to the application of protein design methodology to large proteins and molecular activities that have proven challenging in the past. Expected final online publication date for the Annual Review of Biochemistry Volume 87 is June 20, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Screening of mutations affecting protein stability and dynamics of FGFR1-A simulation analysis.

PubMed

Doss, C George Priya; Rajith, B; Garwasis, Nimisha; Mathew, Pretty Raju; Raju, Anand Solomon; Apoorva, K; William, Denise; Sadhana, N R; Himani, Tanwar; Dike, I P

2012-12-01

Single amino acid substitutions in Fibroblast Growth Factor Receptor 1 ( FGFR1 ) destabilize protein and have been implicated in several genetic disorders like various forms of cancer, Kallamann syndrome, Pfeiffer syndrome, Jackson Weiss syndrome, etc. In order to gain functional insight into mutation caused by amino acid substitution to protein function and expression, special emphasis was laid on molecular dynamics simulation techniques in combination with in silico tools such as SIFT, PolyPhen 2.0, I-Mutant 3.0 and SNAP. It has been estimated that 68% nsSNPs were predicted to be deleterious by I-Mutant, slightly higher than SIFT (37%), PolyPhen 2.0 (61%) and SNAP (58%). From the observed results, P722S mutation was found to be most deleterious by comparing results of all in silico tools. By molecular dynamics approach, we have shown that P722S mutation leads to increase in flexibility, and deviated more from the native structure which was supported by the decrease in the number of hydrogen bonds. In addition, biophysical analysis revealed a clear insight of stability loss due to P722S mutation in FGFR1 protein. Majority of mutations predicted by these in silico tools were in good concordance with the experimental results.

Enzyme stabilization via computationally guided protein stapling.

PubMed

Moore, Eric J; Zorine, Dmitri; Hansen, William A; Khare, Sagar D; Fasan, Rudi

2017-11-21

Thermostabilization represents a critical and often obligatory step toward enhancing the robustness of enzymes for organic synthesis and other applications. While directed evolution methods have provided valuable tools for this purpose, these protocols are laborious and time-consuming and typically require the accumulation of several mutations, potentially at the expense of catalytic function. Here, we report a minimally invasive strategy for enzyme stabilization that relies on the installation of genetically encoded, nonreducible covalent staples in a target protein scaffold using computational design. This methodology enables the rapid development of myoglobin-based cyclopropanation biocatalysts featuring dramatically enhanced thermostability (Δ T m = +18.0 °C and Δ T 50 = +16.0 °C) as well as increased stability against chemical denaturation [Δ C m (GndHCl) = 0.53 M], without altering their catalytic efficiency and stereoselectivity properties. In addition, the stabilized variants offer superior performance and selectivity compared with the parent enzyme in the presence of a high concentration of organic cosolvents, enabling the more efficient cyclopropanation of a water-insoluble substrate. This work introduces and validates an approach for protein stabilization which should be applicable to a variety of other proteins and enzymes.

The βγ-crystallin domain of Lysinibacillus sphaericus phosphatidylinositol phospholipase C plays a central role in protein stability.

PubMed

Cerminati, Sebastián; Paoletti, Luciana; Peirú, Salvador; Menzella, Hugo G; Castelli, María Eugenia

2018-06-16

βγ-crystallin has emerged as a superfamily of structurally homologous proteins with representatives across all domains of life. A major portion of this superfamily is constituted by microbial members. This superfamily has also been recognized as a novel group of Ca 2+ -binding proteins with a large diversity and variable properties in Ca 2+ binding and stability. We have recently described a new phosphatidylinositol phospholipase C from Lysinibacillus sphaericus (LS-PIPLC) which was shown to efficiently remove phosphatidylinositol from crude vegetable oil. Here, the role of the C-terminal βγ-crystallin domain of LS-PIPLC was analyzed in the context of the whole protein. A truncated protein in which the C-terminal βγ-crystallin domain was deleted (LS-PIPLC ΔCRY ) is catalytically as efficient as the full-length protein (LS-PIPLC). However, the thermal and chemical stability of LS-PIPLC ΔCRY are highly affected, demonstrating a stabilizing role for this domain. It is also shown that the presence of Ca 2+ increases the thermal and chemical stability of the protein both in aqueous media and in oil, making LS-PIPLC an excellent candidate for use in industrial soybean oil degumming.

Analysis of protein stability and ligand interactions by thermal shift assay.

PubMed

Huynh, Kathy; Partch, Carrie L

2015-02-02

Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye. Copyright © 2015 John Wiley & Sons, Inc.

Effects of protein phosphorylation on color stability of ground meat.

PubMed

Li, Meng; Li, Xin; Xin, Jianzeng; Li, Zheng; Li, Guixia; Zhang, Yan; Du, Manting; Shen, Qingwu W; Zhang, Dequan

2017-03-15

The influence of protein phosphorylation on meat color stability was investigated in this study. Phosphatase and protein kinase inhibitors were added to minced ovine Longissimus thoracis et lumborum (LTL) muscle to manipulate the global phosphorylation of sarcoplasmic proteins. The data obtained show that the rate and extent of pH decline, along with lactate accumulation in postmortem muscle, were related to protein phosphorylation. Analysis of meat color and the relative content of myoglobin redox forms revealed that meat color stability was inversely related to the phosphorylation of sarcoplasmic proteins. Thus, this study suggests that protein phosphorylation may be involved in meat color development by regulating glycolysis and the redox stability of myoglobin. Copyright © 2016 Elsevier Ltd. All rights reserved.

Axin and GSK3- control Smad3 protein stability and modulate TGF- signaling.

PubMed

Guo, Xing; Ramirez, Alejandro; Waddell, David S; Li, Zhizhong; Liu, Xuedong; Wang, Xiao-Fan

2008-01-01

The broad range of biological responses elicited by transforming growth factor-beta (TGF-beta) in various types of tissues and cells is mainly determined by the expression level and activity of the effector proteins Smad2 and Smad3. It is not fully understood how the baseline properties of Smad3 are regulated, although this molecule is in complex with many other proteins at the steady state. Here we show that nonactivated Smad3, but not Smad2, undergoes proteasome-dependent degradation due to the concerted action of the scaffolding protein Axin and its associated kinase, glycogen synthase kinase 3-beta (GSK3-beta). Smad3 physically interacts with Axin and GSK3-beta only in the absence of TGF-beta. Reduction in the expression or activity of Axin/GSK3-beta leads to increased Smad3 stability and transcriptional activity without affecting TGF-beta receptors or Smad2, whereas overexpression of these proteins promotes Smad3 basal degradation and desensitizes cells to TGF-beta. Mechanistically, Axin facilitates GSK3-beta-mediated phosphorylation of Smad3 at Thr66, which triggers Smad3 ubiquitination and degradation. Thr66 mutants of Smad3 show altered protein stability and hence transcriptional activity. These results indicate that the steady-state stability of Smad3 is an important determinant of cellular sensitivity to TGF-beta, and suggest a new function of the Axin/GSK3-beta complex in modulating critical TGF-beta/Smad3-regulated processes during development and tumor progression.

Confinement in nanopores can destabilize α-helix folding proteins and stabilize the β structures

NASA Astrophysics Data System (ADS)

Javidpour, Leili; Sahimi, Muhammad

2011-09-01

Protein folding in confined media has attracted wide attention over the past decade due to its importance in both in vivo and in vitro applications. Currently, it is generally believed that protein stability increases by decreasing the size of the confining medium, if its interaction with the confining walls is repulsive, and that the maximum folding temperature in confinement occurs for a pore size only slightly larger than the smallest dimension of the folded state of a protein. Protein stability in pore sizes, very close to the size of the folded state, has not however received the attention that it deserves. Using detailed, 0.3-ms-long molecular dynamics simulations, we show that proteins with an α-helix native state can have an optimal folding temperature in pore sizes that do not affect the folded-state structure. In contradiction to the current theoretical explanations, we find that the maximum folding temperature occurs in larger pores for smaller α-helices. In highly confined pores the free energy surface becomes rough, and a new barrier for protein folding may appear close to the unfolded state. In addition, in small nanopores the protein states that contain the β structures are entropically stabilized, in contrast to the bulk. As a consequence, folding rates decrease notably and the free energy surface becomes rougher. The results shed light on many recent experimental observations that cannot be explained by the current theories, and demonstrate the importance of entropic effects on proteins' misfolded states in highly confined environments. They also support the concept of passive effect of chaperonin GroEL on protein folding by preventing it from aggregation in crowded environment of biological cells, and provide deeper clues to the α → β conformational transition, believed to contribute to Alzheimer's and Parkinson's diseases. The strategy of protein and enzyme stabilization in confined media may also have to be revisited in the case of tight

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.

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.

Prediction of protein mutant stability using classification and regression tool.

PubMed

Huang, Liang-Tsung; Saraboji, K; Ho, Shinn-Ying; Hwang, Shiow-Fen; Ponnuswamy, M N; Gromiha, M Michael

2007-02-01

Prediction of protein stability upon amino acid substitutions is an important problem in molecular biology and the solving of which would help for designing stable mutants. In this work, we have analyzed the stability of protein mutants using two different datasets of 1396 and 2204 mutants obtained from ProTherm database, respectively for free energy change due to thermal (DeltaDeltaG) and denaturant denaturations (DeltaDeltaG(H(2)O)). We have used a set of 48 physical, chemical energetic and conformational properties of amino acid residues and computed the difference of amino acid properties for each mutant in both sets of data. These differences in amino acid properties have been related to protein stability (DeltaDeltaG and DeltaDeltaG(H(2)O)) and are used to train with classification and regression tool for predicting the stability of protein mutants. Further, we have tested the method with 4 fold, 5 fold and 10 fold cross validation procedures. We found that the physical properties, shape and flexibility are important determinants of protein stability. The classification of mutants based on secondary structure (helix, strand, turn and coil) and solvent accessibility (buried, partially buried, partially exposed and exposed) distinguished the stabilizing/destabilizing mutants at an average accuracy of 81% and 80%, respectively for DeltaDeltaG and DeltaDeltaG(H(2)O). The correlation between the experimental and predicted stability change is 0.61 for DeltaDeltaG and 0.44 for DeltaDeltaG(H(2)O). Further, the free energy change due to the replacement of amino acid residue has been predicted within an average error of 1.08 kcal/mol and 1.37 kcal/mol for thermal and chemical denaturation, respectively. The relative importance of secondary structure and solvent accessibility, and the influence of the dataset on prediction of protein mutant stability have been discussed.

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.

Molecular basis of the osmolyte effect on protein stability: a lesson from the mechanical unfolding of lysozyme.

PubMed

Adamczak, Beata; Wieczór, Miłosz; Kogut, Mateusz; Stangret, Janusz; Czub, Jacek

2016-10-15

Osmolytes are a class of small organic molecules that shift the protein folding equilibrium. For this reason, they are accumulated by organisms under environmental stress and find applications in biotechnology where proteins need to be stabilized or dissolved. However, despite years of research, debate continues over the exact mechanisms underpinning the stabilizing and denaturing effect of osmolytes. Here, we simulated the mechanical denaturation of lysozyme in different solvent conditions to study the molecular mechanism by which two biologically relevant osmolytes, denaturing (urea) and stabilizing (betaine), affect the folding equilibrium. We found that urea interacts favorably with all types of residues via both hydrogen bonds and dispersion forces, and therefore accumulates in a diffuse solvation shell around the protein. This not only provides an enthalpic stabilization of the unfolded state, but also weakens the hydrophobic effect, as hydrophobic forces promote the association of urea with nonpolar residues, facilitating the unfolding. In contrast, we observed that betaine is excluded from the protein backbone and nonpolar side chains, but is accumulated near the basic residues, yielding a nonuniform distribution of betaine molecules at the protein surface. Spatially resolved solvent-protein interaction energies further suggested that betaine behaves in a ligand- rather than solvent-like manner and its exclusion from the protein surface arises mostly from the scarcity of favorable binding sites. Finally, we found that, in the presence of betaine, the reduced ability of water molecules to solvate the protein results in an additional enthalpic contribution to the betaine-induced stabilization. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

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

A Protein Aggregation Based Test for Screening of the Agents Affecting Thermostability of Proteins

PubMed Central

Eronina, Tatyana; Borzova, Vera; Maloletkina, Olga; Kleymenov, Sergey; Asryants, Regina; Markossian, Kira; Kurganov, Boris

2011-01-01

To search for agents affecting thermal stability of proteins, a test based on the registration of protein aggregation in the regime of heating with a constant rate was used. The initial parts of the dependences of the light scattering intensity (I) on temperature (T) were analyzed using the following empiric equation: I = K agg(T−T 0)2, where K agg is the parameter characterizing the initial rate of aggregation and T 0 is a temperature at which the initial increase in the light scattering intensity is registered. The aggregation data are interpreted in the frame of the model assuming the formation of the start aggregates at the initial stages of the aggregation process. Parameter T 0 corresponds to the moment of the origination of the start aggregates. The applicability of the proposed approach was demonstrated on the examples of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscles and bovine liver glutamate dehydrogenase studied in the presence of agents of different chemical nature. The elaborated approach to the study of protein aggregation may be used for rapid identification of small molecules that interact with protein targets. PMID:21760963

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.

Monodispersed silk fibroin microdroplets for protein stabilization

NASA Astrophysics Data System (ADS)

Liu, Qiang; Jiang, Nan; Liu, Dewen; Ying, Guoliang; Shi, Qiusheng; Yetisen, Ali K.; Liu, Haifeng; Fan, Yubo

2018-04-01

Low stability of globular protein droplets in emulsion significantly limits their applications in drug encapsulation, long-term storage, and controlled drug release. Here, a microfluidic flow-focusing device was utilized to synthesize horseradish peroxidase (HRP)-loaded silk fibroin microdroplets. The two immiscible streams of microfluidic flow-focusing were regenerated by silk fibroin solution and a mixture of 95 wt. % sunflower oil and 5 wt. % span 80 as the dispersed and continuous phases, respectively. In this study, the water-in-oil silk fibroin microdroplets were homogeneously produced by leveraging the discrete and periodic breakup of microdroplets and regulating the flow rates. Moreover, the result showed that the stability of encapsulated HRP in microdroplets was 25% higher than that of HRP after 6 weeks incubation. Thus, the microfluidic flow-focusing is a promising technique to form monodisperse microdroplets and maximize the stability of protein droplets.

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.

Screening of mutations affecting protein stability and dynamics of FGFR1—A simulation analysis

PubMed Central

Doss, C. George Priya; Rajith, B.; Garwasis, Nimisha; Mathew, Pretty Raju; Raju, Anand Solomon; Apoorva, K.; William, Denise; Sadhana, N.R.; Himani, Tanwar; Dike, IP.

2012-01-01

Single amino acid substitutions in Fibroblast Growth Factor Receptor 1 (FGFR1) destabilize protein and have been implicated in several genetic disorders like various forms of cancer, Kallamann syndrome, Pfeiffer syndrome, Jackson Weiss syndrome, etc. In order to gain functional insight into mutation caused by amino acid substitution to protein function and expression, special emphasis was laid on molecular dynamics simulation techniques in combination with in silico tools such as SIFT, PolyPhen 2.0, I-Mutant 3.0 and SNAP. It has been estimated that 68% nsSNPs were predicted to be deleterious by I-Mutant, slightly higher than SIFT (37%), PolyPhen 2.0 (61%) and SNAP (58%). From the observed results, P722S mutation was found to be most deleterious by comparing results of all in silico tools. By molecular dynamics approach, we have shown that P722S mutation leads to increase in flexibility, and deviated more from the native structure which was supported by the decrease in the number of hydrogen bonds. In addition, biophysical analysis revealed a clear insight of stability loss due to P722S mutation in FGFR1 protein. Majority of mutations predicted by these in silico tools were in good concordance with the experimental results. PMID:27896051

Long Non-coding RNA, PANDA, Contributes to the Stabilization of p53 Tumor Suppressor Protein.

PubMed

Kotake, Yojiro; Kitagawa, Kyoko; Ohhata, Tatsuya; Sakai, Satoshi; Uchida, Chiharu; Niida, Hiroyuki; Naemura, Madoka; Kitagawa, Masatoshi

2016-04-01

P21-associated noncoding RNA DNA damage-activated (PANDA) is induced in response to DNA damage and represses apoptosis by inhibiting the function of nuclear transcription factor Y subunit alpha (NF-YA) transcription factor. Herein, we report that PANDA affects regulation of p53 tumor-suppressor protein. U2OS cells were transfected with PANDA siRNAs. At 72 h post-transfection, cells were subjected to immunoblotting and quantitative reverse transcription-polymerase chain reaction. Depletion of PANDA was associated with decreased levels of p53 protein, but not p53 mRNA. The stability of p53 protein was markedly reduced by PANDA silencing. Degradation of p53 protein by silencing PANDA was prevented by treatment of MG132, a proteasome inhibitor. Moreover, depletion of PANDA prevented accumulation of p53 protein, as a result of DNA damage, induced by the genotoxic agent etoposide. These results suggest that PANDA stabilizes p53 protein in response to DNA damage, and provide new insight into the regulatory mechanisms of p53. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

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.

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

Thermodynamic effects of proline introduction on protein stability.

PubMed

Prajapati, Ravindra Singh; Das, Mili; Sreeramulu, Sridhar; Sirajuddin, Minhajuddin; Srinivasan, Sankaranarayanan; Krishnamurthy, Vaishnavi; Ranjani, Ranganathan; Ramakrishnan, C; Varadarajan, Raghavan

2007-02-01

The amino acid Pro is more rigid than other naturally occurring amino acids and, in proteins, lacks an amide hydrogen. To understand the structural and thermodynamic effects of Pro substitutions, it was introduced at 13 different positions in four different proteins, leucine-isoleucine-valine binding protein, maltose binding protein, ribose binding protein, and thioredoxin. Three of the maltose binding protein mutants were characterized by X-ray crystallography to confirm that no structural changes had occurred upon mutation. In the remaining cases, fluorescence and CD spectroscopy were used to show the absence of structural change. Stabilities of wild type and mutant proteins were characterized by chemical denaturation at neutral pH and by differential scanning calorimetry as a function of pH. The mutants did not show enhanced stability with respect to chemical denaturation at room temperature. However, 6 of the 13 single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, 2 mutants showed no change, and 5 were destabilized. In five of the six cases, the stabilization was because of reduced entropy of unfolding. However, the magnitude of the reduction in entropy of unfolding was typically several fold larger than the theoretical estimate of -4 cal K(-1) mol(-1) derived from the relative areas in the Ramachandran map accessible to Pro and Ala residues, respectively. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were nonadditive. Copyright 2006 Wiley-Liss, Inc.

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.

Role of naturally occurring osmolytes in protein folding and stability.

PubMed

Kumar, Raj

2009-11-01

Osmolytes are typically accumulated in the intracellular environment at relatively high concentrations when cells/tissues are subjected to stress conditions. Osmolytes are common in a variety of organisms, including microorganisms, plants, and animals. They enhance thermodynamic stability of proteins by providing natively folded conformations without perturbing other cellular processes. By burying the backbone into the core of folded proteins, osmolytes can provide significant stability to proteins. Two properties of osmolytes are particularly important: (i) their ability to impart increased thermodynamic stability to folded proteins; and (ii) their compatibility in the intracellular environment at high concentrations. Under physiological conditions, the cellular compositions of osmolytes may vary significantly. This may lead to different protein folding pathways utilized in cells depending upon the intracellular environment. Proper understanding of the role of osmolytes in cell regulation should allow predicting the action of osmolytes on macromolecular interactions in stressed and crowded environments typical of cellular conditions.

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. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rational Design of Protein Stability: Effect of (2S,4R)-4-Fluoroproline on the Stability and Folding Pathway of Ubiquitin

PubMed Central

Crespo, Maria D.; Rubini, Marina

2011-01-01

Background Many strategies have been employed to increase the conformational stability of proteins. The use of 4-substituted proline analogs capable to induce pre-organization in target proteins is an attractive tool to deliver an additional conformational stability without perturbing the overall protein structure. Both, peptides and proteins containing 4-fluorinated proline derivatives can be stabilized by forcing the pyrrolidine ring in its favored puckering conformation. The fluorinated pyrrolidine rings of proline can preferably stabilize either a Cγ-exo or a Cγ-endo ring pucker in dependence of proline chirality (4R/4S) in a complex protein structure. To examine whether this rational strategy can be generally used for protein stabilization, we have chosen human ubiquitin as a model protein which contains three proline residues displaying Cγ-exo puckering. Methodology/Principal Findings While (2S,4R)-4-fluoroproline ((4R)-FPro) containing ubiquitinin can be expressed in related auxotrophic Escherichia coli strain, all attempts to incorporate (2S,4S)-4-fluoroproline ((4S)-FPro) failed. Our results indicate that (4R)-FPro is favoring the Cγ-exo conformation present in the wild type structure and stabilizes the protein structure due to a pre-organization effect. This was confirmed by thermal and guanidinium chloride-induced denaturation profile analyses, where we observed an increase in stability of −4.71 kJ·mol−1 in the case of (4R)-FPro containing ubiquitin ((4R)-FPro-ub) compared to wild type ubiquitin (wt-ub). Expectedly, activity assays revealed that (4R)-FPro-ub retained the full biological activity compared to wt-ub. Conclusions/Significance The results fully confirm the general applicability of incorporating fluoroproline derivatives for improving protein stability. In general, a rational design strategy that enforces the natural occurring proline puckering conformation can be used to stabilize the desired target protein. PMID:21625626

Honey-Induced Protein Stabilization as Studied by Fluorescein Isothiocyanate Fluorescence

PubMed Central

Abdul Kadir, Habsah; Tayyab, Saad

2013-01-01

Protein stabilizing potential of honey was studied on a model protein, bovine serum albumin (BSA), using extrinsic fluorescence of fluorescein isothiocyanate (FITC) as the probe. BSA was labelled with FITC using chemical coupling, and urea and thermal denaturation studies were performed on FITC-labelled BSA (FITC-BSA) both in the absence and presence of 10% and 20% (w/v) honey using FITC fluorescence at 522 nm upon excitation at 495 nm. There was an increase in the FITC fluorescence intensity upon increasing urea concentration or temperature, suggesting protein denaturation. The results from urea and thermal denaturation studies showed increased stability of protein in the presence of honey as reflected from the shift in the transition curve along with the start point and the midpoint of the transition towards higher urea concentration/temperature. Furthermore, the increase in ΔG D H2O and ΔG D 25°C in presence of honey also suggested protein stabilization. PMID:24222758

Assessing the utility of gene co-expression stability in combination with correlation in the analysis of protein-protein interaction networks

PubMed Central

2011-01-01

Background Gene co-expression, in the form of a correlation coefficient, has been valuable in the analysis, classification and prediction of protein-protein interactions. However, it is susceptible to bias from a few samples having a large effect on the correlation coefficient. Gene co-expression stability is a means of quantifying this bias, with high stability indicating robust, unbiased co-expression correlation coefficients. We assess the utility of gene co-expression stability as an additional measure to support the co-expression correlation in the analysis of protein-protein interaction networks. Results We studied the patterns of co-expression correlation and stability in interacting proteins with respect to their interaction promiscuity, levels of intrinsic disorder, and essentiality or disease-relatedness. Co-expression stability, along with co-expression correlation, acts as a better classifier of hub proteins in interaction networks, than co-expression correlation alone, enabling the identification of a class of hubs that are functionally distinct from the widely accepted transient (date) and obligate (party) hubs. Proteins with high levels of intrinsic disorder have low co-expression correlation and high stability with their interaction partners suggesting their involvement in transient interactions, except for a small group that have high co-expression correlation and are typically subunits of stable complexes. Similar behavior was seen for disease-related and essential genes. Interacting proteins that are both disordered have higher co-expression stability than ordered protein pairs. Using co-expression correlation and stability, we found that transient interactions are more likely to occur between an ordered and a disordered protein while obligate interactions primarily occur between proteins that are either both ordered, or disordered. Conclusions We observe that co-expression stability shows distinct patterns in structurally and functionally

Mechanism of Stabilization of Labile Compounds by Silk Fibroin Proteins

DTIC Science & Technology

2017-04-05

AFRL-AFOSR-VA-TR-2017-0076 Mechanism of Stabilization of Labile Compounds by Silk Fibroin Proteins David Kaplan TRUSTEES OF TUFTS COLEGE INC 169... Proteins 5a.  CONTRACT NUMBER 5b.  GRANT NUMBER FA9550-14-1-0015 5c.  PROGRAM ELEMENT NUMBER 61102F 6. AUTHOR(S) David Kaplan 5d.  PROJECT NUMBER 5e...objective of this research was to elucidate the fundamental mechanisms by which labile compounds are entrapped and stabilized by silk fibroin protein . The

Stability of dilute solutions of tuberculin purified protein derivative.

PubMed

Landi, S; Held, H R

1978-06-01

The biological potency of 15 commercially available tuberculin solutions prepared from a master batch of tuberculin purified protein derivative (PPD) (PPD-CT68) and using a newly formulated diluent (Connaught diluent) containing 0.0005% Tween 80 as an anti-adsorption agent and 0.3% phenol as a preservative, was determined after storage for various intervals at 4, 24 and 37 degrees C. The 5 tuberculin units (TU) per 0.1 ml dose solutions were bioequivalent to a non-stabilized solution of PPD-S whereas the 1 TU and 250 TU per dose solutions were equivalent by calculation to a non-stabilized solution of PPD-S. It was found that the PPD solutions of all 3 strengths, 1, 5 and 250 TU per dose, were stable for at least 3 years at 4 degrees C and for 2 years at room temperature (24 degrees C). Even at 37 degrees C the solutions of all 3 strengths were stable for at least 1 year. The stability of Connaught tuberculin PPD solutions has not been affected by the changes in formulation. The stability data suggest that the expiry date of the newly formulated tuberculin products could be at least two years from the data of the last satisfactory potency test. Although these products are stable for at least 1 year even at 37 degrees C, we nonetheless agree with the Canadian and U.S. regulations that they be stored at 2 to 8 degrees C in their original containers.

Do Crisis Response Operations Affect Political and Economic Stability?

DTIC Science & Technology

2003-05-01

military presence itself actually affects overall levels of political and economic stability is still an open question. We look at the following two...relationship between military actions and political and economic stability . In this paper, we focus only on the crisis response piece of the overseas presence issue.

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry.

PubMed

Schön, Arne; Clarkson, Benjamin R; Jaime, Maria; Freire, Ernesto

2017-11-01

The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (∼100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day -1 . Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs. © 2017 Wiley Periodicals, Inc.

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry

PubMed Central

Schön, Arne; Clarkson, Benjamin R; Jaime, Maria; Freire, Ernesto

2017-01-01

The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (~100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day−1. Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs. PMID:28722205

Plant ecology. Anthropogenic environmental changes affect ecosystem stability via biodiversity.

PubMed

Hautier, Yann; Tilman, David; Isbell, Forest; Seabloom, Eric W; Borer, Elizabeth T; Reich, Peter B

2015-04-17

Human-driven environmental changes may simultaneously affect the biodiversity, productivity, and stability of Earth's ecosystems, but there is no consensus on the causal relationships linking these variables. Data from 12 multiyear experiments that manipulate important anthropogenic drivers, including plant diversity, nitrogen, carbon dioxide, fire, herbivory, and water, show that each driver influences ecosystem productivity. However, the stability of ecosystem productivity is only changed by those drivers that alter biodiversity, with a given decrease in plant species numbers leading to a quantitatively similar decrease in ecosystem stability regardless of which driver caused the biodiversity loss. These results suggest that changes in biodiversity caused by drivers of environmental change may be a major factor determining how global environmental changes affect ecosystem stability. Copyright © 2015, American Association for the Advancement of Science.

Functional Stability of HIV-1 Envelope Trimer Affects Accessibility to Broadly Neutralizing Antibodies at Its Apex.

PubMed

Gift, Syna Kuriakose; Leaman, Daniel P; Zhang, Lei; Kim, Arthur S; Zwick, Michael B

2017-12-15

The trimeric envelope glycoprotein spike (Env) of HIV-1 is the target of vaccine development to elicit broadly neutralizing antibodies (bnAbs). Env trimer instability and heterogeneity in principle make subunit interfaces inconsistent targets for the immune response. Here, we investigate how functional stability of Env relates to neutralization sensitivity to V2 bnAbs and V3 crown antibodies that engage subunit interfaces upon binding to unliganded Env. Env heterogeneity was inferred when antibodies neutralized a mutant Env with a plateau of less than 100% neutralization. A statistically significant correlation was found between the stability of mutant Envs and the MPN of V2 bnAb, PG9, as well as an inverse correlation between stability of Env and neutralization by V3 crown antibody, 447-52D. A number of Env-stabilizing mutations and V2 bnAb-enhancing mutations were identified in Env, but they did not always overlap, indicating distinct requirements of functional stabilization versus antibody recognition. Blocking complex glycosylation of Env affected V2 bnAb recognition, as previously described, but also notably increased functional stability of Env. This study shows how instability and heterogeneity affect antibody sensitivity of HIV-1 Env, which is relevant to vaccine design involving its dynamic apex. IMPORTANCE The Env trimer is the only viral protein on the surface of HIV-1 and is the target of neutralizing antibodies that reduce viral infectivity. Quaternary epitopes at the apex of the spike are recognized by some of the most potent and broadly neutralizing antibodies to date. Being that their glycan-protein hybrid epitopes are at subunit interfaces, the resulting heterogeneity can lead to partial neutralization. Here, we screened for mutations in Env that allowed for complete neutralization by the bnAbs. We found that when mutations outside V2 increased V2 bnAb recognition, they often also increased Env stability-of-function and decreased binding by

Functional Stability of HIV-1 Envelope Trimer Affects Accessibility to Broadly Neutralizing Antibodies at Its Apex

PubMed Central

Gift, Syna Kuriakose; Leaman, Daniel P.; Zhang, Lei; Kim, Arthur S.

2017-01-01

ABSTRACT The trimeric envelope glycoprotein spike (Env) of HIV-1 is the target of vaccine development to elicit broadly neutralizing antibodies (bnAbs). Env trimer instability and heterogeneity in principle make subunit interfaces inconsistent targets for the immune response. Here, we investigate how functional stability of Env relates to neutralization sensitivity to V2 bnAbs and V3 crown antibodies that engage subunit interfaces upon binding to unliganded Env. Env heterogeneity was inferred when antibodies neutralized a mutant Env with a plateau of less than 100% neutralization. A statistically significant correlation was found between the stability of mutant Envs and the MPN of V2 bnAb, PG9, as well as an inverse correlation between stability of Env and neutralization by V3 crown antibody, 447-52D. A number of Env-stabilizing mutations and V2 bnAb-enhancing mutations were identified in Env, but they did not always overlap, indicating distinct requirements of functional stabilization versus antibody recognition. Blocking complex glycosylation of Env affected V2 bnAb recognition, as previously described, but also notably increased functional stability of Env. This study shows how instability and heterogeneity affect antibody sensitivity of HIV-1 Env, which is relevant to vaccine design involving its dynamic apex. IMPORTANCE The Env trimer is the only viral protein on the surface of HIV-1 and is the target of neutralizing antibodies that reduce viral infectivity. Quaternary epitopes at the apex of the spike are recognized by some of the most potent and broadly neutralizing antibodies to date. Being that their glycan-protein hybrid epitopes are at subunit interfaces, the resulting heterogeneity can lead to partial neutralization. Here, we screened for mutations in Env that allowed for complete neutralization by the bnAbs. We found that when mutations outside V2 increased V2 bnAb recognition, they often also increased Env stability-of-function and decreased binding

Investigating the effect of mutation on the thermo stability of GB1 protein

NASA Astrophysics Data System (ADS)

Sawitri, K. N.; Sumaryada, T.; Ambarsari, L.; Wahyudi, S. T.

2018-04-01

The thermo stability of wild-type and mutants of the B1 domain of Protein G (GB1 protein) have been studied using molecular dynamics simulation and free energy perturbation simulation. This research is aimed to examine what residue or what interaction that has a major role in the thermo stability of GB1 protein thermo stability by using the point mutation method. Based on the analysis, the unfolding of wild-type protein occurred in 500 K simulation at 704 ps. The mutations were chosen based on the changes in some analysis parameters and the calculated net solvation free energy change. It was found that a simple replacement of a positively charged residue in the β-sheet (K4S) decreases the stability of GB1 protein (unfolding at 452 ps), while the replacement of a negatively charged residue in the α-helix (E27G) increases the stability (unfolding at 846 ps). It was also found that the K4A mutation will break the α-helix and all β-sheet into the coil and turn. All those results suggest that the non-bonded interaction has the major role in the thermo stability of GB1 protein with the β-sheets were identified as the most important structure in the thermo stability of GB1 protein..

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.

Tailoring in vitro evolution for protein affinity or stability

PubMed Central

Jermutus, Lutz; Honegger, Annemarie; Schwesinger, Falk; Hanes, Jozef; Plückthun, Andreas

2001-01-01

We describe a rapid and general technology working entirely in vitro to evolve either the affinity or the stability of ligand-binding proteins, depending on the chosen selection pressure. Tailored in vitro selection strategies based on ribosome display were combined with in vitro diversification by DNA shuffling to evolve either the off-rate or thermodynamic stability of single-chain Fv antibody fragments (scFvs). To demonstrate the potential of this method, we chose to optimize two proteins already possessing favorable properties. A scFv with an initial affinity of 1.1 nM (koff at 4°C of 10−4 s−1) was improved 30-fold by the use of off-rate selections over a period of several days. As a second example, a generic selection strategy for improved stability exploited the property of ribosome display that the conditions can be altered under which the folding of the displayed protein occurs. We used decreasing redox potentials in the selection step to select for molecules stable in the absence of disulfide bonds. They could be functionally expressed in the reducing cytoplasm, and, when allowed to form disulfides again, their stability had increased to 54 kJ/mol from an initial value of 24 kJ/mol. Sequencing revealed that the evolved mutant proteins had used different strategies of residue changes to adapt to the selection pressure. Therefore, by a combination of randomization and appropriate selection strategies, an in vitro evolution of protein properties in a predictable direction is possible. PMID:11134506

MSH3 polymorphisms and protein levels affect CAG repeat instability in Huntington's disease mice.

PubMed

Tomé, Stéphanie; Manley, Kevin; Simard, Jodie P; Clark, Greg W; Slean, Meghan M; Swami, Meera; Shelbourne, Peggy F; Tillier, Elisabeth R M; Monckton, Darren G; Messer, Anne; Pearson, Christopher E

2013-01-01

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)∼100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of

MSH3 Polymorphisms and Protein Levels Affect CAG Repeat Instability in Huntington's Disease Mice

PubMed Central

Simard, Jodie P.; Clark, Greg W.; Slean, Meghan M.; Swami, Meera; Shelbourne, Peggy F.; Tillier, Elisabeth R. M.; Monckton, Darren G.; Messer, Anne; Pearson, Christopher E.

2013-01-01

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)∼100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of

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.

Mechanical stability analysis of the protein L immunoglobulin-binding domain by full alanine screening using molecular dynamics simulations.

PubMed

Glyakina, Anna V; Likhachev, Ilya V; Balabaev, Nikolay K; Galzitskaya, Oxana V

2015-03-01

This article is the first to study the mechanical properties of the immunoglobulin-binding domain of protein L (referred to as protein L) and its mutants at the atomic level. In the structure of protein L, each amino acid residue (except for alanines and glycines) was replaced sequentially by alanine. Thus, 49 mutants of protein L were obtained. The proteins were stretched at their termini at constant velocity using molecular dynamics simulations in water, i.e. by forced unfolding. 19 out of 49 mutations resulted in a large decrease of mechanical protein stability. These amino acids were affecting either the secondary structure (11 mutations) or loop structures (8 mutations) of protein L. Analysis of mechanical unfolding of the generated protein that has the same topology as protein L but consists of only alanines and glycines allows us to suggest that the mechanical stability of proteins, and specifically protein L, is determined by interactions between certain amino acid residues, although the unfolding pathway depends on the protein topology. This insight can now be used to modulate the mechanical properties of proteins and their unfolding pathways in the desired direction for using them in various biochips, biosensors and biomaterials for medicine, industry, and household purposes. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Influence of protein fold stability on immunogenicity and its implications for vaccine design

PubMed Central

Scheiblhofer, Sandra; Laimer, Josef; Machado, Yoan; Weiss, Richard; Thalhamer, Josef

2017-01-01

ABSTRACT Introduction: 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. PMID:28290225

Mechanism of protein precipitation and stabilization by co-solvents

NASA Astrophysics Data System (ADS)

Timasheff, Serge N.; Arakawa, Tsutomu

1988-07-01

The interactions between proteins and a number of substances which, when present at high concentration, stabilize or precipitate proteins, have been analyzed in terms of the preferential interactions of these co-solvents with proteins. In all cases, stabilization or precipitation was accompanied by preferential exclusion of the co-solvent from the immediate domain of the protein, i.e., preferential hydration of the protein. This means that addition of the co-solvent to the aqueous protein solution increased the chemical potentials of both components. The thermodynamic interaction parameters derived from such data make it possible to calculate the salting out constant, Ks, as well as to construct a phase isotherm for any given solvent mixture which indicates the limiting protein solubility. The salting-out effect can be decomposed into contributions from non-specific preferential exclusion and specific binding of the ligand to the protein, the balance leading to solubilization or precipitation. In reactions, such as denaturation, the effect of co-solvent on the reaction depends on the difference in the preferential interactions of the two end states of the protein. Principal sources of preferential exclusion have been identified as steric exclusion, increase of the surface tension of water by the co-solvent, repulsion by charged loci on the protein and solvophobicity.

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rational modification of protein stability by targeting surface sites leads to complicated results

PubMed Central

Xiao, Shifeng; Patsalo, Vadim; Shan, Bing; Bi, Yuan; Green, David F.; Raleigh, Daniel P.

2013-01-01

The rational modification of protein stability is an important goal of protein design. Protein surface electrostatic interactions are not evolutionarily optimized for stability and are an attractive target for the rational redesign of proteins. We show that surface charge mutants can exert stabilizing effects in distinct and unanticipated ways, including ones that are not predicted by existing methods, even when only solvent-exposed sites are targeted. Individual mutation of three solvent-exposed lysines in the villin headpiece subdomain significantly stabilizes the protein, but the mechanism of stabilization is very different in each case. One mutation destabilizes native-state electrostatic interactions but has a larger destabilizing effect on the denatured state, a second removes the desolvation penalty paid by the charged residue, whereas the third introduces unanticipated native-state interactions but does not alter electrostatics. Our results show that even seemingly intuitive mutations can exert their effects through unforeseen and complex interactions. PMID:23798426

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

A high-throughput assay of membrane protein stability.

PubMed

Postis, Vincent L G; Deacon, Sarah E; Roach, Peter C J; Wright, Gareth S A; Xia, Xiaobing; Ingram, Jean C; Hadden, Jonathan M; Henderson, Peter J F; Phillips, Simon E V; McPherson, Michael J; Baldwin, Stephen A

2008-12-01

The preparation of purified, detergent-solubilized membrane proteins in a monodisperse and stable form is usually a prerequisite for investigation not only of their function but also for structural studies by X-ray crystallography and other approaches. Typically, it is necessary to explore a wide range of conditions, including detergent type, buffer pH, and the presence of additives such as glycerol, in order to identify those optimal for stability. Given the difficulty of expressing and purifying membrane proteins in large amounts, such explorations must ideally be performed on as small a scale as practicable. To achieve this objective in the UK Membrane Protein Structure Initiative, we have developed a rapid, economical, light-scattering assay of membrane protein aggregation that allows the testing of 48 buffer conditions in parallel on 6 protein targets, requiring less than 2 mg protein for each target. Testing of the assay on a number of unrelated membrane transporters has shown that it is of generic applicability. Proteins of sufficient purity for this plate-based assay are first rapidly prepared using simple affinity purification procedures performed in batch mode. Samples are then transferred by microdialysis into each of the conditions to be tested. Finally, attenuance at 340 nm is monitored in a 384-well plate using a plate reader. Optimal conditions for protein stability identified in the assay can then be exploited for the tailored purification of individual targets in as stable a form as possible.

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

Estimating conformation content of a protein using citrate-stabilized Au nanoparticles

NASA Astrophysics Data System (ADS)

Deka, Jashmini; Paul, Anumita; Chattopadhyay, Arun

2010-08-01

Herein we report the use of the optical properties of citrate-stabilized gold nanoparticles (Au NPs) for estimation of native or denatured conformation content in a mixture of a protein in solution. The UV-vis extinction spectrum of citrate-stabilized Au NPs is known to broaden differently in the presence of native and denatured states of α-amylase, bovine serum albumin (BSA) or amyloglucosidase (AMG). On the other hand, herein we show that when a mixture of native and denatured protein was present in the medium, the broadening of the spectrum differed for different fractional content of the conformations. Also, the total area under the extinction spectrum varied linearly with the change in the mole fraction content of a state and for a constant total protein concentration. Transmission electron microscopy (TEM) measurements revealed different levels of agglomeration for different fractional contents of the native or denatured state of a protein. In addition, time-dependent denaturation of a protein could be followed using the present method. The rate constants calculated for denaturation indicated a possible fast change in conformation of a protein before complete thermal denaturation. The observations have been explained based on the changes in extinction coefficient (thereby oscillator strength) upon interaction of citrate-stabilized NPs with proteins being in different states and levels of agglomeration.Herein we report the use of the optical properties of citrate-stabilized gold nanoparticles (Au NPs) for estimation of native or denatured conformation content in a mixture of a protein in solution. The UV-vis extinction spectrum of citrate-stabilized Au NPs is known to broaden differently in the presence of native and denatured states of α-amylase, bovine serum albumin (BSA) or amyloglucosidase (AMG). On the other hand, herein we show that when a mixture of native and denatured protein was present in the medium, the broadening of the spectrum differed for

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.

Stabilization of a protein conferred by an increase in folded state entropy.

PubMed

Dagan, Shlomi; Hagai, Tzachi; Gavrilov, Yulian; Kapon, Ruti; Levy, Yaakov; Reich, Ziv

2013-06-25

Entropic stabilization of native protein structures typically relies on strategies that serve to decrease the entropy of the unfolded state. Here we report, using a combination of experimental and computational approaches, on enhanced thermodynamic stability conferred by an increase in the configurational entropy of the folded state. The enhanced stability is observed upon modifications of a loop region in the enzyme acylphosphatase and is achieved despite significant enthalpy losses. The modifications that lead to increased stability, as well as those that result in destabilization, however, strongly compromise enzymatic activity, rationalizing the preservation of the native loop structure even though it does not provide the protein with maximal stability or kinetic foldability.

Effect of glycine on pH changes and protein stability during freeze-thawing in phosphate buffer systems.

PubMed

Pikal-Cleland, Katherine A; Cleland, Jeffrey L; Anchordoquy, Thomas J; Carpenter, John F

2002-09-01

Previous studies have established that the selective precipitation of a less soluble buffer component during freezing can induce a significant pH shift in the freeze concentrate. During freezing of sodium phosphate solutions, crystallization of the disodium salt can produce a pH decrease as great as 3 pH units which can dramatically affect protein stability. The objective of our study was to determine how the presence of glycine (0-500 mM), a commonly used bulking agent in pharmaceutical protein formulations, affects the pH changes normally observed during freezing in sodium phosphate buffer solutions and to determine whether these pH changes contribute to instability of model proteins in glycine/phosphate formulations. During freezing in sodium phosphate buffers, the presence of glycine significantly influenced the pH. Glycine at the lower concentrations (< or = 50 mM) suppressed the pH decrease normally observed during freezing in 10 and 100 mM sodium phosphate buffer, possibly by reducing the nucleation rate of salt and thereby decreasing the extent of buffer salt crystallization. The presence of glycine at higher concentration (> 100 mM) in the sodium phosphate buffer resulted in a more complete crystallization of the disodium salt as indicated by the frozen pH values closer to the equilibrium value (pH 3.6). Although high concentrations of glycine can facilitate more buffer salt crystallization and these pH shifts may prove to be potentially damaging to the protein, glycine, in its amorphous state, can also act to stabilize a protein via the preferential exclusion mechanism. Copyright 2002 Wiley-Liss Inc.

Conservative mutation Met8 --> Leu affects the folding process and structural stability of squash trypsin inhibitor CMTI-I.

PubMed Central

Zhukov, I.; Jaroszewski, L.; Bierzyński, A.

2000-01-01

Protein molecules can accommodate a large number of mutations without noticeable effects on their stability and folding kinetics. On the other hand, some mutations can have quite strong effects on protein conformational properties. Such mutations either destabilize secondary structures, e.g., alpha-helices, are incompatible with close packing of protein hydrophobic cores, or lead to disruption of some specific interactions such as disulfide cross links, salt bridges, hydrogen bonds, or aromatic-aromatic contacts. The Met8 --> Leu mutation in CMTI-I results in significant destabilization of the protein structure. This effect could hardly be expected since the mutation is highly conservative, and the side chain of residue 8 is situated on the protein surface. We show that the protein destabilization is caused by rearrangement of a hydrophobic cluster formed by side chains of residues 8, Ile6, and Leu17 that leads to partial breaking of a hydrogen bond formed by the amide group of Leu17 with water and to a reduction of a hydrophobic surface buried within the cluster. The mutation perturbs also the protein folding. In aerobic conditions the reduced wild-type protein folds effectively into its native structure, whereas more then 75% of the mutant molecules are trapped in various misfolded species. The main conclusion of this work is that conservative mutations of hydrophobic residues can destabilize a protein structure even if these residues are situated on the protein surface and partially accessible to water. Structural rearrangement of small hydrophobic clusters formed by such residues can lead to local changes in protein hydration, and consequently, can affect considerably protein stability and folding process. PMID:10716179

Small heat shock protein AgsA: an effective stabilizer of enzyme activities.

PubMed

Tomoyasu, Toshifumi; Tabata, Atsushi; Ishikawa, Yoko; Whiley, Robert A; Nagamune, Hideaki

2013-01-01

A small heat shock protein, AgsA, possesses chaperone activity that can reduce the amount of heat-aggregated protein in vivo, and suppress the aggregation of chemical- and heat-denatured proteins in vitro. Therefore, we examined the ability of AgsA to stabilize the activity of several enzymes by using this chaperone activity. We observed that AgsA can stabilize the enzymatic activities of Renilla (Renilla reniformis) luciferase, firefly (Photinus pyralis) luciferase, and β-galactosidase, and showed comparable or greater stabilization of these enzymes than bovine serum albumin (BSA), a well-known stabilizer of enzyme activities. In particular, AgsA revealed better stabilization of Renilla luciferase and β-galactosidase than BSA under disulfide bond-reducing conditions with dithiothreitol. In addition, AgsA also increased the enzymatic performance of β-galactosidase and various restriction enzymes to a comparable or greater extent than BSA. These data indicate that AgsA may be useful as a general stabilizer of enzyme activities. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Guar foaming albumin: a low molecular mass protein with high foaming activity and foam stability isolated from guar meal.

PubMed

Shimoyama, Ami; Kido, Shoko; Kinekawa, Yoh-ichi; Doi, Yukio

2008-10-08

The water extract of guar meal ( Cyamopsis tetragonolobus) was examined for its foamability. Compared with egg white, the extract showed an extraordinary foam stability: no drainage after 3 h of standing in contrast to 65% drainage for egg white at the same protein concentration. The acid-precipitated protein from the extract was responsible for the high foamability and designated guar foaming albumin (GFA). The foaming activity of GFA was 20 times higher than that of egg white. GFA consisted of two subunits with molecular masses of 6 and 11 kDa linked to each other through disulfide bonds. The cleavage of disulfide bonds in GFA affected the foamability only slightly. GFA remarkably decreased the surface tension of water at low protein concentrations. Immunoblotting analysis demonstrated that GFA did not react to the antisera from allergic patients against plant food. These results suggest that GFA serves as an effective food additive in developing protein-stabilized foam.

Impact of critical process and formulation parameters affecting in-process stability of lactate dehydrogenase during the secondary drying stage of lyophilization: a mini freeze dryer study.

PubMed

Luthra, Sumit; Obert, Jean-Philippe; Kalonia, Devendra S; Pikal, Michael J

2007-09-01

The stresses during the secondary-drying stage of lyophilization were investigated using a controlled humidity mini-freeze-dryer [Luthra S, Obert J-P, Kalonia DS, Pikal MJ. 2007. Investigation of drying stresses on proteins during lyophilization: Differentiation between primary and secondary-drying stresses on lactate dehydrogenase using a humidity controlled mini freeze-dryer. J Pharm Sci 96: 61-70.]. Lactate dehydrogenase (LDH), was formulated in: (1) Tween 80, (2) citrate buffer, and (3) both Tween 80 and citrate buffer. Protein activity recovery was measured as a function of relative humidity (RH), product temperature, and drying duration. Studies were also conducted with different concentrations of sucrose, sorbitol, and poly (vinyl pyrrolidone) (PVP). LDH stability was affected to a small extent by RH and significantly by drying temperature and duration. Complete stabilization of LDH was observed when lyophilized with sucrose and PVP but only a partial stabilization was observed with sorbitol. The mini-freeze-dryer enabled studying the process parameters independently, unlike a conventional study where these effects are generally convoluted. The results suggest that the stability of the protein is a function of the dynamics of the system during lyophilization. The origin of the stabilization effect of sucrose, which could, in principle, be attributed both to direct interaction with the protein or vitrification of the protein was elucidated using lyoprotectants that can either hydrogen bond well with the protein (sorbitol) or form a good glass (PVP). It appears both effects are required for complete stabilization of the protein. (c) 2007 Wiley-Liss, Inc. and the American Pharmacists Association.

Similar folds with different stabilization mechanisms: the cases of prion and doppel proteins

PubMed Central

Colacino, Stefano; Tiana, Guido; Colombo, Giorgio

2006-01-01

Background Protein misfolding is the main cause of a group of fatal neurodegenerative diseases in humans and animals. In particular, in Prion-related diseases the normal cellular form of the Prion Protein PrP (PrPC) is converted into the infectious PrPSc through a conformational process during which it acquires a high β-sheet content. Doppel is a protein that shares a similar native fold, but lacks the scrapie isoform. Understanding the molecular determinants of these different behaviours is important both for biomedical and biophysical research. Results In this paper, the dynamical and energetic properties of the two proteins in solution is comparatively analyzed by means of long time scale explicit solvent, all-atom molecular dynamics in different temperature conditions. The trajectories are analyzed by means of a recently introduced energy decomposition approach (Tiana et al, Prot. Sci. 2004) aimed at identifying the key residues for the stabilization and folding of the protein. Our analysis shows that Prion and Doppel have two different cores stabilizing the native state and that the relative contribution of the nucleus to the global stability of the protein for Doppel is sensitively higher than for PrP. Moreover, under misfolding conditions the Doppel core is conserved, while the energy stabilization network of PrP is disrupted. Conclusion These observations suggest that different sequences can share similar native topology with different stabilizing interactions and that the sequences of the Prion and Doppel proteins may have diverged under different evolutionary constraints resulting in different folding and stabilization mechanisms. PMID:16857062

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

Boosting protein stability with the computational design of β-sheet surfaces.

PubMed

Kim, Doo Nam; Jacobs, Timothy M; Kuhlman, Brian

2016-03-01

β-sheets often have one face packed against the core of the protein and the other facing solvent. Mutational studies have indicated that the solvent-facing residues can contribute significantly to protein stability, and that the preferred amino acid at each sequence position is dependent on the precise structure of the protein backbone and the identity of the neighboring amino acids. This suggests that the most advantageous methods for designing β-sheet surfaces will be approaches that take into account the multiple energetic factors at play including side chain rotamer preferences, van der Waals forces, electrostatics, and desolvation effects. Here, we show that the protein design software Rosetta, which models these energetic factors, can be used to dramatically increase protein stability by optimizing interactions on the surfaces of small β-sheet proteins. Two design variants of the β-sandwich protein from tenascin were made with 7 and 14 mutations respectively on its β-sheet surfaces. These changes raised the thermal midpoint for unfolding from 45°C to 64°C and 74°C. Additionally, we tested an empirical approach based on increasing the number of potential salt bridges on the surfaces of the β-sheets. This was not a robust strategy for increasing stability, as three of the four variants tested were unfolded. © 2016 The Protein Society.

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.

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

Stabilities and Dynamics of Protein Folding Nuclei by Molecular Dynamics Simulation

NASA Astrophysics Data System (ADS)

Song, Yong-Shun; Zhou, Xin; Zheng, Wei-Mou; Wang, Yan-Ting

2017-07-01

To understand how the stabilities of key nuclei fragments affect protein folding dynamics, we simulate by molecular dynamics (MD) simulation in aqueous solution four fragments cut out of a protein G, including one α-helix (seqB: KVFKQYAN), two β-turns (seqA: LNGKTLKG and seqC: YDDATKTF), and one β-strand (seqD: DGEWTYDD). The Markov State Model clustering method combined with the coarse-grained conformation letters method are employed to analyze the data sampled from 2-μs equilibrium MD simulation trajectories. We find that seqA and seqB have more stable structures than their native structures which become metastable when cut out of the protein structure. As expected, seqD alone is flexible and does not have a stable structure. Throughout our simulations, the native structure of seqC is stable but cannot be reached if starting from a structure other than the native one, implying a funnel-shape free energy landscape of seqC in aqueous solution. All the above results suggest that different nuclei have different formation dynamics during protein folding, which may have a major contribution to the hierarchy of protein folding dynamics. Supported by the National Basic Research Program of China under Grant No. 2013CB932804, the National Natural Science Foundation of China under Grant No. 11421063, and the CAS Biophysics Interdisciplinary Innovation Team Project

mRNA stability in mammalian cells.

PubMed Central

Ross, J

1995-01-01

This review concerns how cytoplasmic mRNA half-lives are regulated and how mRNA decay rates influence gene expression. mRNA stability influences gene expression in virtually all organisms, from bacteria to mammals, and the abundance of a particular mRNA can fluctuate manyfold following a change in the mRNA half-life, without any change in transcription. The processes that regulate mRNA half-lives can, in turn, affect how cells grow, differentiate, and respond to their environment. Three major questions are addressed. Which sequences in mRNAs determine their half-lives? Which enzymes degrade mRNAs? Which (trans-acting) factors regulate mRNA stability, and how do they function? The following specific topics are discussed: techniques for measuring eukaryotic mRNA stability and for calculating decay constants, mRNA decay pathways, mRNases, proteins that bind to sequences shared among many mRNAs [like poly(A)- and AU-rich-binding proteins] and proteins that bind to specific mRNAs (like the c-myc coding-region determinant-binding protein), how environmental factors like hormones and growth factors affect mRNA stability, and how translation and mRNA stability are linked. Some perspectives and predictions for future research directions are summarized at the end. PMID:7565413

Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins

PubMed Central

Song, Albert S.; Poor, Taylor A.; Abriata, Luciano A.; Jardetzky, Theodore S.; Dal Peraro, Matteo; Lamb, Robert A.

2016-01-01

Parainfluenza virus 5 (PIV5) is an enveloped, single-stranded, negative-sense RNA virus of the Paramyxoviridae family. PIV5 fusion and entry are mediated by the coordinated action of the receptor-binding protein, hemagglutinin–neuraminidase (HN), and the fusion protein (F). Upon triggering by HN, F undergoes an irreversible ATP- and pH-independent conformational change, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. Previous studies have highlighted key conformational changes in the F-protein refolding pathway, but a detailed understanding of prefusion F-protein metastability remains elusive. Here, using two previously described F-protein mutations (S443D or P22L), we examine the capacity to modulate PIV5 F stability and the mechanisms by which these point mutants act. The S443D mutation destabilizes prefusion F proteins by disrupting a hydrogen bond network at the base of the F-protein globular head. The introduction of a P22L mutation robustly rescues destabilized F proteins through a local hydrophobic interaction between the N-terminal helix and a hydrophobic pocket. Prefusion stabilization conferred by a P22L-homologous mutation is demonstrated in the F protein of Newcastle disease virus, a paramyxovirus of a different genus, suggesting a conserved stabilizing structural element within the paramyxovirus family. Taken together, the available data suggest that movement of the N-terminal helix is a necessary early step for paramyxovirus F-protein refolding and presents a novel target for structure-based drug design. PMID:27335462

Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins.

PubMed

Song, Albert S; Poor, Taylor A; Abriata, Luciano A; Jardetzky, Theodore S; Dal Peraro, Matteo; Lamb, Robert A

2016-07-05

Parainfluenza virus 5 (PIV5) is an enveloped, single-stranded, negative-sense RNA virus of the Paramyxoviridae family. PIV5 fusion and entry are mediated by the coordinated action of the receptor-binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F). Upon triggering by HN, F undergoes an irreversible ATP- and pH-independent conformational change, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. Previous studies have highlighted key conformational changes in the F-protein refolding pathway, but a detailed understanding of prefusion F-protein metastability remains elusive. Here, using two previously described F-protein mutations (S443D or P22L), we examine the capacity to modulate PIV5 F stability and the mechanisms by which these point mutants act. The S443D mutation destabilizes prefusion F proteins by disrupting a hydrogen bond network at the base of the F-protein globular head. The introduction of a P22L mutation robustly rescues destabilized F proteins through a local hydrophobic interaction between the N-terminal helix and a hydrophobic pocket. Prefusion stabilization conferred by a P22L-homologous mutation is demonstrated in the F protein of Newcastle disease virus, a paramyxovirus of a different genus, suggesting a conserved stabilizing structural element within the paramyxovirus family. Taken together, the available data suggest that movement of the N-terminal helix is a necessary early step for paramyxovirus F-protein refolding and presents a novel target for structure-based drug design.

In vivo protein stabilization based on fragment complementation and a split GFP system.

PubMed

Lindman, Stina; Hernandez-Garcia, Armando; Szczepankiewicz, Olga; Frohm, Birgitta; Linse, Sara

2010-11-16

Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12 °C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25 °C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state.

Conformational co-dependence between Plasmodium berghei LCCL proteins promotes complex formation and stability.

PubMed

Saeed, Sadia; Tremp, Annie Z; Dessens, Johannes T

2012-10-01

Malaria parasites express a conserved family of LCCL-lectin adhesive-like domain proteins (LAPs) that have essential functions in sporozoite transmission. In Plasmodium falciparum all six family members are expressed in gametocytes and form a multi-protein complex. Intriguingly, knockout of P. falciparum LCCL proteins adversely affects expression of other family members at protein, but not at mRNA level, a phenomenon termed co-dependent expression. Here, we investigate this in Plasmodium berghei by crossing a PbLAP1 null mutant parasite with a parasite line expressing GFP-tagged PbLAP3 that displays strong fluorescence in gametocytes. Selected and validated double mutants show normal synthesis and subcellular localization of PbLAP3::GFP. However, GFP-based fluorescence is dramatically reduced without PbLAP1 present, indicating that PbLAP1 and PbLAP3 interact. Moreover, absence of PbLAP1 markedly reduces the half-life of PbLAP3, consistent with a scenario of misfolding. These findings unveil a potential mechanism of conformational interdependence that facilitates assembly and stability of the functional LCCL protein complex. Copyright © 2012 Elsevier B.V. All rights reserved.

The Assembly-Activating Protein Promotes Stability and Interactions between AAV's Viral Proteins to Nucleate Capsid Assembly.

PubMed

Maurer, Anna C; Pacouret, Simon; Cepeda Diaz, Ana Karla; Blake, Jessica; Andres-Mateos, Eva; Vandenberghe, Luk H

2018-05-08

The adeno-associated virus (AAV) vector is a preferred delivery platform for in vivo gene therapy. Natural and engineered variations of the AAV capsid affect a plurality of phenotypes relevant to gene therapy, including vector production and host tropism. Fundamental to these aspects is the mechanism of AAV capsid assembly. Here, the role of the viral co-factor assembly-activating protein (AAP) was evaluated in 12 naturally occurring AAVs and 9 putative ancestral capsid intermediates. The results demonstrate increased capsid protein stability and VP-VP interactions in the presence of AAP. The capsid's dependence on AAP can be partly overcome by strengthening interactions between monomers within the assembly, as illustrated by the transfer of a minimal motif defined by a phenotype-to-phylogeny mapping method. These findings suggest that the emergence of AAP within the Dependovirus genus relaxes structural constraints on AAV assembly in favor of increasing the degrees of freedom for the capsid to evolve. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Stability of lutein encapsulated whey protein nano-emulsion during storage

PubMed Central

Guo, Mingruo

2018-01-01

Lutein is a hydrophobic carotenoid that has multiple health functions. However, the application of lutein is limited due to its poor solubility in water and instability under certain conditions during storage. Hereby we generated lutein loaded nano-emulsions using whey protein isolate (WPI) or polymerized whey protein isolate (PWP) with assistance of high intensity ultrasound and evaluate their stability during storage at different conditions. We measured the particle size, zeta-potential, physical stability and lutein content change. Results showed that the PWP based nano-emulsion system was not stable in the tested Oil/Water/Ethanol system indicated by the appearance of stratification within only one week. The WPI based nano-emulsion system showed stable physiochemical stability during the storage at 4°C. The lutein content of the system was reduced by only 4% after four weeks storage at 4°C. In conclusion, our whey protein based nano-emulsion system provides a promising strategy for encapsulation of lutein or other hydrophobic bioactive molecules to expand their applications. PMID:29415071

Contribution of Charged Groups to the Enthalpic Stabilization of the Folded States of Globular Proteins

PubMed Central

Dadarlat, Voichita M.; Post, Carol Beth

2016-01-01

In this paper we use the results from all atom MD simulations of proteins and peptides to assess individual contribution of charged atomic groups to the enthalpic stability of the native state of globular proteins and investigate how the distribution of charged atomic groups in terms of solvent accessibility relates to protein enthalpic stability. The contributions of charged groups is calculated using a comparison of nonbonded interaction energy terms from equilibrium simulations of charged amino acid dipeptides in water (the “unfolded state”) and charged amino acids in globular proteins (the “folded state”). Contrary to expectation, the analysis shows that many buried, charged atomic groups contribute favorably to protein enthalpic stability. The strongest enthalpic contributions favoring the folded state come from the carboxylate (COO−) groups of either Glu or Asp. The contributions from Arg guanidinium groups are generally somewhat stabilizing, while NH3+ groups from Lys contribute little toward stabilizing the folded state. The average enthalpic gain due to the transfer of a methyl group in an apolar amino acid from solution to the protein interior is described for comparison. Notably, charged groups that are less exposed to solvent contribute more favorably to protein native-state enthalpic stability than charged groups that are solvent exposed. While solvent reorganization/release has favorable contributions to folding for all charged atomic groups, the variation in folded state stability among proteins comes mainly from the change in the nonbonded interaction energy of charged groups between the unfolded and folded states. A key outcome is that the calculated enthalpic stabilization is found to be inversely proportional to the excess charge density on the surface, in support of an hypothesis proposed previously. PMID:18303881

Poly(zwitterionic)protein conjugates offer increased stability without sacrificing binding affinity or bioactivity

NASA Astrophysics Data System (ADS)

Keefe, Andrew J.; Jiang, Shaoyi

2012-01-01

Treatment with therapeutic proteins is an attractive approach to targeting a number of challenging diseases. Unfortunately, the native proteins themselves are often unstable in physiological conditions, reducing bioavailability and therefore increasing the dose that is required. Conjugation with poly(ethylene glycol) (PEG) is often used to increase stability, but this has a detrimental effect on bioactivity. Here, we introduce conjugation with zwitterionic polymers such as poly(carboxybetaine). We show that poly(carboxybetaine) conjugation improves stability in a manner similar to PEGylation, but that the new conjugates retain or even improve the binding affinity as a result of enhanced protein-substrate hydrophobic interactions. This chemistry opens a new avenue for the development of protein therapeutics by avoiding the need to compromise between stability and affinity.

TOPICAL REVIEW: Protein stability and enzyme activity at extreme biological temperatures

NASA Astrophysics Data System (ADS)

Feller, Georges

2010-08-01

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.

Protein Molecular Structures, Protein SubFractions, and Protein Availability Affected by Heat Processing: A Review

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

Yu,P.

2007-01-01

The utilization and availability of protein depended on the types of protein and their specific susceptibility to enzymatic hydrolysis (inhibitory activities) in the gastrointestine and was highly associated with protein molecular structures. Studying internal protein structure and protein subfraction profiles leaded to an understanding of the components that make up a whole protein. An understanding of the molecular structure of the whole protein was often vital to understanding its digestive behavior and nutritive value in animals. In this review, recently obtained information on protein molecular structural effects of heat processing was reviewed, in relation to protein characteristics affecting digestive behaviormore » and nutrient utilization and availability. The emphasis of this review was on (1) using the newly advanced synchrotron technology (S-FTIR) as a novel approach to reveal protein molecular chemistry affected by heat processing within intact plant tissues; (2) revealing the effects of heat processing on the profile changes of protein subfractions associated with digestive behaviors and kinetics manipulated by heat processing; (3) prediction of the changes of protein availability and supply after heat processing, using the advanced DVE/OEB and NRC-2001 models, and (4) obtaining information on optimal processing conditions of protein as intestinal protein source to achieve target values for potential high net absorbable protein in the small intestine. The information described in this article may give better insight in the mechanisms involved and the intrinsic protein molecular structural changes occurring upon processing.« less

Factors affecting the stability of reverse shoulder arthroplasty: a biomechanical study.

PubMed

Clouthier, Allison L; Hetzler, Markus A; Fedorak, Graham; Bryant, J Tim; Deluzio, Kevin J; Bicknell, Ryan T

2013-04-01

Despite the success of reverse shoulder arthroplasty (RSA) in treating patients with painful pseudoparalytic shoulders, instability is a common complication and currently the factors affecting stability are not well understood. The objective of this study was to investigate a number of factors as well as the interactions between factors to determine how they affect the stability of the prosthesis. These factors included: active arm posture (abduction and abduction plane angles), loading direction, glenosphere diameter and eccentricity, and humeral socket constraint. Force required to dislocate the joint, determined using a biomechanical shoulder simulator, was used as a measure of stability. A factorial design experiment was implemented to examine the factors and interactions. Actively increasing the abduction angle by 15° leads to a 30% increase in stability and use of an inferior-offset rather than a centered glenosphere improved stability by 17%. Use of a more constrained humeral socket also increased stability; but the effect was dependent on loading direction, with a 88% improvement for superior loading, 66% for posterior, 36% for anterior, and no change for inferior loading. Abduction plane angle and glenosphere diameter had no effect on stability. Increased glenohumeral abduction and the use of an inferior-offset glenosphere were found to increase the stability of RSA. Additionally, use of a more constrained humeral socket increased stability for anterior, posterior, and superior loading. These identified factor effects have the potential to decrease the risk of dislocation following RSA. Copyright © 2013 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Mosby, Inc. All rights reserved.

Semiautomated Sample Preparation for Protein Stability and Formulation Screening via Buffer Exchange.

PubMed

Ying, William; Levons, Jaquan K; Carney, Andrea; Gandhi, Rajesh; Vydra, Vicky; Rubin, A Erik

2016-06-01

A novel semiautomated buffer exchange process workflow was developed to enable efficient early protein formulation screening. An antibody fragment protein, BMSdab, was used to demonstrate the workflow. The process afforded 60% to 80% cycle time and scientist time savings and significant material efficiencies. These efficiencies ultimately facilitated execution of this stability work earlier in the drug development process, allowing this tool to inform the developability of potential candidates for development from a formulation perspective. To overcome the key technical challenges, the protein solution was buffer-exchanged by centrifuge filtration into formulations for stability screening in a 96-well plate with an ultrafiltration membrane, leveraging automated liquid handling and acoustic volume measurements to allow several cycles of exchanges. The formulations were transferred into a vacuum manifold and sterile filtered into a rack holding 96 glass vials. The vials were sealed with a capmat of individual caps and placed in stability stations. Stability of the samples prepared by this process and by the standard process was demonstrated to be comparable. This process enabled screening a number of formulations of a protein at an early pharmaceutical development stage with a short sample preparation time. © 2015 Society for Laboratory Automation and Screening.

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

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

PubMed

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

2011-10-11

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 K(p) for the accumulation or exclusion of urea in the water of hydration of these surfaces relative to bulk water are obtained. K(p) 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 K(p) values.

Chaperonin-based biolayer interferometry to assess the kinetic stability of metastable, aggregation-prone proteins

PubMed Central

Lea, Wendy A.; Naik, Subhashchandra; Chaudhri, Tapan; Machen, Alexandra J.; O’Neil, Pierce T.; McGinn-Straub, Wesley; Tischer, Alexander; Auton, Matthew T.; Burns, Joshua R.; Baldwin, Michael R.; Khar, Karen R.; Karanicolas, John; Fisher, Mark T.

2017-01-01

Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy to decrease disease pathologies brought on by protein folding defects or deleterious kinetic transitions. Current methods of examining ligand binding to these marginally stable native states are limited, because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, multi-domain) and metastable proteins (e.g. low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant-pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein immobilized on a BLI biosensor to increasing denaturant concentrations (urea or GnHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remain is detected by increased GroEL binding. Since this kinetic denaturant pulse is brief, the amplitude of the GroEL binding to the immobilized protein depends on the duration of exposure to denaturant, the concentration of denaturant, wash times, and the underlying protein unfolding/refolding kinetics; fixing all other parameters and plotting GroEL binding amplitude versus denaturant pulse concentration results in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein is manifested by a decreased GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be

Structural stability of proteins in aqueous and nonpolar environments

NASA Astrophysics Data System (ADS)

Yasuda, Satoshi; Oshima, Hiraku; Kinoshita, Masahiro

2012-10-01

A protein folds into its native structure with the α-helix and/or β-sheet in aqueous solution under the physiological condition. The relative content of these secondary structures largely varies from protein to protein. However, such structural variability is not exhibited in nonaqueous environment. For example, there is a strong trend that alcohol induces a protein to form α-helices, and many of the membrane proteins within the lipid bilayer consists of α-helices. Here we investigate the structural stability of proteins in aqueous and nonpolar environments using our recently developed free-energy function F = (Λ - TS)/(kBT0) = Λ/(kBT0) - S/kB (T0 = 298 K and the absolute temperature T is set at T0) which is based on statistical thermodynamics. Λ/(kBT0) and S/kB are the energetic and entropic components, respectively, and kB is Boltzmann's constant. A smaller value of the positive quantity, -S, represents higher efficiency of the backbone and side-chain packing promoted by the entropic effect arising from the translational displacement of solvent molecules or the CH2, CH3, and CH groups which constitute nonpolar chains of lipid molecules. As for Λ, in aqueous solution, a transition to a more compact structure of a protein accompanies the break of protein-solvent hydrogen bonds: As the number of donors and acceptors buried without protein intramolecular hydrogen bonding increases, Λ becomes higher. In nonpolar solvent, lower Λ simply implies more intramolecular hydrogen bonds formed. We find the following. The α-helix and β-sheet are advantageous with respect to -S as well as Λ and to be formed as much as possible. In aqueous solution, the solvent-entropy effect on the structural stability is so strong that the close packing of side chains is dominantly important, and the α-helix and β-sheet contents are judiciously adjusted to accomplish it. In nonpolar solvent, the solvent-entropy effect is substantially weaker than in aqueous solution. Λ is

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. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Unraveling protein stabilization mechanisms: vitrification and water replacement in a glass transition temperature controlled system.

PubMed

Grasmeijer, N; Stankovic, M; de Waard, H; Frijlink, H W; Hinrichs, W L J

2013-04-01

The aim of this study was to elucidate the role of the two main mechanisms used to explain the stabilization of proteins by sugar glasses during drying and subsequent storage: the vitrification and the water replacement theory. Although in literature protein stability is often attributed to either vitrification or water replacement, both mechanisms could play a role and they should be considered simultaneously. A model protein, alkaline phosphatase, was incorporated in either inulin or trehalose by spray drying. To study the storage stability at different glass transition temperatures, a buffer which acts as a plasticizer, ammediol, was incorporated in the sugar glasses. At low glass transition temperatures (<50°C), the enzymatic activity of the protein strongly decreased during storage at 60°C. Protein stability increased when the glass transition temperature was raised considerably above the storage temperature. This increased stability could be attributed to vitrification. A further increase of the glass transition temperature did not further improve stability. In conclusion, vitrification plays a dominant role in stabilization at glass transition temperatures up to 10 to 20°C above storage temperature, depending on whether trehalose or inulin is used. On the other hand, the water replacement mechanism predominantly determines stability at higher glass transition temperatures. Copyright © 2013 Elsevier B.V. All rights reserved.

Optimization of rotamers prior to template minimization improves stability predictions made by computational protein design.

PubMed

Davey, James A; Chica, Roberto A

2015-04-01

Computational protein design (CPD) predictions are highly dependent on the structure of the input template used. However, it is unclear how small differences in template geometry translate to large differences in stability prediction accuracy. Herein, we explored how structural changes to the input template affect the outcome of stability predictions by CPD. To do this, we prepared alternate templates by Rotamer Optimization followed by energy Minimization (ROM) and used them to recapitulate the stability of 84 protein G domain β1 mutant sequences. In the ROM process, side-chain rotamers for wild-type (WT) or mutant sequences are optimized on crystal or nuclear magnetic resonance (NMR) structures prior to template minimization, resulting in alternate structures termed ROM templates. We show that use of ROM templates prepared from sequences known to be stable results predominantly in improved prediction accuracy compared to using the minimized crystal or NMR structures. Conversely, ROM templates prepared from sequences that are less stable than the WT reduce prediction accuracy by increasing the number of false positives. These observed changes in prediction outcomes are attributed to differences in side-chain contacts made by rotamers in ROM templates. Finally, we show that ROM templates prepared from sequences that are unfolded or that adopt a nonnative fold result in the selective enrichment of sequences that are also unfolded or that adopt a nonnative fold, respectively. Our results demonstrate the existence of a rotamer bias caused by the input template that can be harnessed to skew predictions toward sequences displaying desired characteristics. © 2014 The Protein Society.

Effect of the geometry of confining media on the stability and folding rate of α -helix proteins

NASA Astrophysics Data System (ADS)

Wang, Congyue; Piroozan, Nariman; Javidpour, Leili; Sahimi, Muhammad

2018-05-01

Protein folding in confined media has attracted wide attention over the past 15 years due to its importance to both in vivo and in vitro applications. It is generally believed that protein stability increases by decreasing the size of the confining medium, if the medium's walls are repulsive, and that the maximum folding temperature in confinement is in a pore whose size D0 is only slightly larger than the smallest dimension of a protein's folded state. Until recently, the stability of proteins in pores with a size very close to that of the folded state has not received the attention it deserves. In a previous paper [L. Javidpour and M. Sahimi, J. Chem. Phys. 135, 125101 (2011)], we showed that, contrary to the current theoretical predictions, the maximum folding temperature occurs in larger pores for smaller α-helices. Moreover, in very tight pores, the free energy surface becomes rough, giving rise to a new barrier for protein folding close to the unfolded state. In contrast to unbounded domains, in small nanopores proteins with an α-helical native state that contain the β structures are entropically stabilized implying that folding rates decrease notably and that the free energy surface becomes rougher. In view of the potential significance of such results to interpretation of many sets of experimental data that could not be explained by the current theories, particularly the reported anomalously low rates of folding and the importance of entropic effects on proteins' misfolded states in highly confined environments, we address the following question in the present paper: To what extent the geometry of a confined medium affects the stability and folding rates of proteins? Using millisecond-long molecular dynamics simulations, we study the problem in three types of confining media, namely, cylindrical and slit pores and spherical cavities. Most importantly, we find that the prediction of the previous theories that the dependence of the maximum folding

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

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

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 humanmore » 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.« less

The impact of particle preparation methods and polymorphic stability of lipid excipients on protein distribution in microparticles.

PubMed

Liu, Jingying; Christophersen, Philip C; Yang, Mingshi; Nielsen, Hanne M; Mu, Huiling

2017-12-01

The present study aimed at elucidating the influence of polymorphic stability of lipid excipients on the physicochemical characters of different solid lipid microparticles (SLM), with the focus on the alteration of protein distribution in SLM. Labeled lysozyme was incorporated into SLM prepared with different excipients, i.e. trimyristin (TG14), glyceryl distearate (GDS), and glyceryl monostearate (GMS), by water-oil-water (w/o/w) or solid-oil-water (s/o/w) method. The distribution of lysozyme in SLM and the release of the protein from SLM were evaluated by confocal laser scanning microscopy. The storage stability of SLM was characterized by HPLC, differential scanning calorimetry, X-ray powder diffraction, and scanning electron microscopy. Lysozyme was displayed as small scattered domains inside GDS and GMS SLM, whereas it was incorporated in the core of TG14 SLM formulated by the w/o/w method or evenly distributed in TG14 SLM prepared by the s/o/w method. Stability study at 37 °C revealed that only TG14 SLM made by the w/o/w method was able to maintain the lysozyme amount both on the particle surface and released from the SLM. Elevated storage temperature induced polymorphic transition of lipids in GDS and GMS SLM, which was, however, not remarkable for the TG14 SLM. Lipid excipients and particle preparation methods were found to differently affect the lysozyme distribution in SLM, owning to varied storage stabilities of the lipids. The present study provides updated knowledge for rational development of lipid-based formulations for oral delivery of peptide or protein drugs.

The influence of disulfide bonds on the mechanical stability of proteins is context dependent.

PubMed

Manteca, Aitor; Alonso-Caballero, Álvaro; Fertin, Marie; Poly, Simon; De Sancho, David; Perez-Jimenez, Raul

2017-08-11

Disulfide bonds play a crucial role in proteins, modulating their stability and constraining their conformational dynamics. A particularly important case is that of proteins that need to withstand forces arising from their normal biological function and that are often disulfide bonded. However, the influence of disulfides on the overall mechanical stability of proteins is poorly understood. Here, we used single-molecule force spectroscopy (smFS) to study the role of disulfide bonds in different mechanical proteins in terms of their unfolding forces. For this purpose, we chose the pilus protein FimG from Gram-negative bacteria and a disulfide-bonded variant of the I91 human cardiac titin polyprotein. Our results show that disulfide bonds can alter the mechanical stability of proteins in different ways depending on the properties of the system. Specifically, disulfide-bonded FimG undergoes a 30% increase in its mechanical stability compared with its reduced counterpart, whereas the unfolding force of I91 domains experiences a decrease of 15% relative to the WT form. Using a coarse-grained simulation model, we rationalized that the increase in mechanical stability of FimG is due to a shift in the mechanical unfolding pathway. The simple topology-based explanation suggests a neutral effect in the case of titin. In summary, our results indicate that disulfide bonds in proteins act in a context-dependent manner rather than simply as mechanical lockers, underscoring the importance of considering disulfide bonds both computationally and experimentally when studying the mechanical properties of proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

Physicochemical properties and storage stability of soybean protein nanoemulsions prepared by ultra-high pressure homogenization.

PubMed

Xu, Jing; Mukherjee, Dipaloke; Chang, Sam K C

2018-02-01

This study investigated the effects of the ultrahigh pressure homogenization (pressure, protein concentration, oil phase fraction, pH, temperature, and ionic strength) and storage on the properties of nanoemulsions (100-500nm range), which were stabilized by laboratory-prepared soybean protein isolate (SPI), β-conglycinin (7S) and glycinin (11S). The nanoemulsions made with SPI, 7S and 11S proteins exhibited considerable stability over various ionic strengths (0-500mM NaCl), pH (<4 or >7), thermal treatments (30-60°C) and storage (0-45days). The far-UV spectra of SPI, 7S, 11S dispersions, and SPI-, 7S-, 11S protein-stabilized nanoemulsions were analyzed for the protein structural changes following lipid removal. The ultra-high pressure homogenization changed the secondary structure of SPI, 7S, 11S proteins in the nanoemulsions, and enhanced their stability. This study demonstrated that SPI, 7S, and 11S proteins can be used as effective emulsifiers in nanoemulsions prepared by ultra-high pressure homogenization. Copyright © 2017. Published by Elsevier Ltd.

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…

A Novel Acidic Matrix Protein, PfN44, Stabilizes Magnesium Calcite to Inhibit the Crystallization of Aragonite*

PubMed Central

Pan, Cong; Fang, Dong; Xu, Guangrui; Liang, Jian; Zhang, Guiyou; Wang, Hongzhong; Xie, Liping; Zhang, Rongqing

2014-01-01

Magnesium is widely used to control calcium carbonate deposition in the shell of pearl oysters. Matrix proteins in the shell are responsible for nucleation and growth of calcium carbonate crystals. However, there is no direct evidence supporting a connection between matrix proteins and magnesium. Here, we identified a novel acidic matrix protein named PfN44 that affected aragonite formation in the shell of the pearl oyster Pinctada fucata. Using immunogold labeling assays, we found PfN44 in both the nacreous and prismatic layers. In shell repair, PfN44 was repressed, whereas other matrix proteins were up-regulated. Disturbing the function of PfN44 by RNAi led to the deposition of porous nacreous tablets with overgrowth of crystals in the nacreous layer. By in vitro circular dichroism spectra and fluorescence quenching, we found that PfN44 bound to both calcium and magnesium with a stronger affinity for magnesium. During in vitro calcium carbonate crystallization and calcification of amorphous calcium carbonate, PfN44 regulated the magnesium content of crystalline carbonate polymorphs and stabilized magnesium calcite to inhibit aragonite deposition. Taken together, our results suggested that by stabilizing magnesium calcite to inhibit aragonite deposition, PfN44 participated in P. fucata shell formation. These observations extend our understanding of the connections between matrix proteins and magnesium. PMID:24302723

Stability of Magnetically-Suppressed Solutal Convection In Protein Crystal Growth

NASA Technical Reports Server (NTRS)

Leslie, F. W.; Ramachandran, N.

2005-01-01

The effect of convection during the crystallization of proteins is not very well understood. In a gravitational field, convection is caused by crystal sedimentation and by solutal buoyancy induced flow and these can lead to crystal imperfections. While crystallization in microgravity can approach diffusion limited growth conditions (no convection), terrestrially strong magnetic fields can be used to control fluid flow and sedimentation effects. In this work, a theory is presented on the stability of solutal convection of a magnetized fluid in the presence of a magnetic field. The requirements for stability are developed and compared to experiments performed within the bore of a superconducting magnet. The theoretical predictions are in good agreement with the experiments and show solutal convection can be stabilized if the surrounding fluid has larger magnetic susceptibility and the magnetic field has a specific structure. Discussion on the application of the technique to protein crystallization is also provided.

Tighter Ligand Binding Can Compensate for Impaired Stability of an RNA-Binding Protein.

PubMed

Wallis, Christopher P; Richman, Tara R; Filipovska, Aleksandra; Rackham, Oliver

2018-06-15

It has been widely shown that ligand-binding residues, by virtue of their orientation, charge, and solvent exposure, often have a net destabilizing effect on proteins that is offset by stability conferring residues elsewhere in the protein. This structure-function trade-off can constrain possible adaptive evolutionary changes of function and may hamper protein engineering efforts to design proteins with new functions. Here, we present evidence from a large randomized mutant library screen that, in the case of PUF RNA-binding proteins, this structural relationship may be inverted and that active-site mutations that increase protein activity are also able to compensate for impaired stability. We show that certain mutations in RNA-protein binding residues are not necessarily destabilizing and that increased ligand-binding can rescue an insoluble, unstable PUF protein. We hypothesize that these mutations restabilize the protein via thermodynamic coupling of protein folding and RNA binding.

Nitrous oxide emissions affected by biochar and nitrogen stabilizers

USDA-ARS?s Scientific Manuscript database

Both biochar and N fertilizer stabilizers (N transformation inhibitors) are potential strategies to reduce nitrous oxide (N2O) emissions from fertilization, but the mechanisms and/or N transformation processes affecting the N dynamics are not fully understood. This research investigated N2O emission...

Phosphorylation alters Bim-mediated Mcl-1 stabilization and priming.

PubMed

Conage-Pough, Jason E; Boise, Lawrence H

2018-05-18

Mcl-1 is a highly labile protein, subject to extensive post-translational regulation. This distinguishes Mcl-1 from other antiapoptotic proteins and necessitates further study to better understand how interactions with proapoptotic Bcl-2 proteins affect its regulation. One such protein, Bim, is known to stabilize Mcl-1, and Bim phosphorylation has been associated with increased Mcl-1 binding. Consequently, we investigated the potential impact of Bim phosphorylation on Mcl-1 stability. We found that Bim stabilizes and primes Mcl-1 in RPCI-WM1 cells and is constitutively phosphorylated. Additionally, introduction of several phospho-mimetic and unphosphosphorylateable Bim mutations resulted in altered Mcl-1 stability and distinct Bim binding to antiapoptotic proteins. These findings suggest Bim phosphorylation not only regulates Mcl-1 stability but also is a potential mechanism for enforcing Mcl-1 dependence. © 2018 Federation of European Biochemical Societies.

Novel microscale approaches for easy, rapid determination of protein stability in academic and commercial settings

PubMed Central

Alexander, Crispin G.; Wanner, Randy; Johnson, Christopher M.; Breitsprecher, Dennis; Winter, Gerhard; Duhr, Stefan; Baaske, Philipp; Ferguson, Neil

2014-01-01

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. PMID:25262836

Dynamic Stabilization of Expressed Proteins in Engineered Diatom Biosilica Matrices.

PubMed

Xiong, Yijia; Ford, Nicole R; Hecht, Karen A; Roesijadi, Guritno; Squier, Thomas C

2016-05-18

Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that will enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39-amino-acid targeting sequence (Sil3T8) that directs a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundance of >200 000 proteins per frustule. Using either a fluorescent ligand bound to the scFv or the intrinsic fluorescence of EGFP, we monitored protein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. Like proteins in solution, proteins within isolated frustules undergo isotropic rotational motion, but with 2-fold increases in rotational correlation times that are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibodies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). Together, these results argue that dramatic increases in protein conformational stability within the biosilica matrices arise through molecular crowding, acting to retain native protein folds and associated functionality with the potential to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

NASA Technical Reports Server (NTRS)

Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

2003-01-01

Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

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.

Can Supersaturation Affect Protein Crystal Quality?

NASA Technical Reports Server (NTRS)

Gorti, Sridhar

2013-01-01

In quiescent environments (microgravity, capillary tubes, gels) formation of a depletion zone is to be expected, due either to limited sedimentation, density driven convection or a combination of both. The formation of a depletion zone can: Modify solution supersaturation near crystal; Give rise to impurity partitioning. It is conjectured that both supersaturation and impurity partitioning affect protein crystal quality and size. Further detailed investigations on various proteins are needed to assess above hypothesis.

Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation.

PubMed

Schlimpert, Susan; Wasserstrom, Sebastian; Chandra, Govind; Bibb, Maureen J; Findlay, Kim C; Flärdh, Klas; Buttner, Mark J

2017-07-25

During sporulation, the filamentous bacteria Streptomyces undergo a massive cell division event in which the synthesis of ladders of sporulation septa convert multigenomic hyphae into chains of unigenomic spores. This process requires cytokinetic Z-rings formed by the bacterial tubulin homolog FtsZ, and the stabilization of the newly formed Z-rings is crucial for completion of septum synthesis. Here we show that two dynamin-like proteins, DynA and DynB, play critical roles in this process. Dynamins are a family of large, multidomain GTPases involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. Using a cell biological approach, we show that the two Streptomyces dynamins specifically localize to sporulation septa in an FtsZ-dependent manner. Moreover, dynamin mutants have a cell division defect due to the decreased stability of sporulation-specific Z-rings, as demonstrated by kymographs derived from time-lapse images of FtsZ ladder formation. This defect causes the premature disassembly of individual Z-rings, leading to the frequent abortion of septum synthesis, which in turn results in the production of long spore-like compartments with multiple chromosomes. Two-hybrid analysis revealed that the dynamins are part of the cell division machinery and that they mediate their effects on Z-ring stability during developmentally controlled cell division via a network of protein-protein interactions involving DynA, DynB, FtsZ, SepF, SepF2, and the FtsZ-positioning protein SsgB.

Controlled formation of emulsion gels stabilized by salted myofibrillar protein under malondialdehyde (MDA)-induced oxidative stress.

PubMed

Zhou, Feibai; Sun, Weizheng; Zhao, Mouming

2015-04-15

This study presented the cold-set gelation of emulsions stabilized by salted myofibrillar protein (MP) under oxidative stress originated from malondialdehyde (MDA). Gel properties were compared over a range of MDA/NaCl concentrations including gel viscoelastic properties, strength, water-holding capacity (WHC), amount of protein entrapped, and microstructure. The oxidative stability of emulsion gels as indicated by lipid hydroperoxide was further determined and compared. Results indicated that emulsion stabilized by MP at swollen state under certain ionic strengths (0.2-0.6 M) was the premise of gel formation under MDA. In the presence of intermediate MDA concentrations (2.5-10 mM), the emulsion gels showed an improved elasticity, strength, WHC, and oxidative stability. This improvement should be mainly attributed to the enhanced protein-protein cross-linkings via MDA, which were homogeneously formed among absorbed and/or unabsorbed proteins, entrapping a greater amount and fractions of protein within network. Therefore, the oil droplets were better adherent to the gel matrix. Nevertheless, addition of high MDA concentrations (25-50 mM) led to the formation of excessive covalent bonds, which might break protein-protein bonds and trigger the desorption of protein from the interface. This ultimately caused "oil leak" phenomena as well as the collapse of gel structure and, thus, overall decreased gel properties and oxidative stability.

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.

Distribution, transition and thermodynamic stability of protein conformations in the denaturant-induced unfolding of proteins.

PubMed

Bian, Liujiao; Ji, Xu

2014-01-01

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. 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. 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.

Stabilizing Protein Effects on the Pressure Sensitivity of Fluorescent Gold Nanoclusters

DTIC Science & Technology

2016-01-13

excess Au salt. The purified sample was lyophilized and resuspended at a concentration of 10 mg/mL in ultrapure water . BSA ( PDB :3v03) 100 % α...effect of scaffold protein secondary structure on the pressure response of protein-stabilized gold nanoclusters (P:NCs). These studies were...demonstrate that the pressure response of P:NCs is indeed dependent on the secondary structure of the protein. Proteins with high beta sheet content

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.

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

PubMed

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

2013-03-06

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.

Differential Effects of Hydrophobic Core Packing Residues for Thermodynamic and Mechanical Stability of a Hyperthermophilic Protein.

PubMed

Tych, Katarzyna M; Batchelor, Matthew; Hoffmann, Toni; Wilson, Michael C; Hughes, Megan L; Paci, Emanuele; Brockwell, David J; Dougan, Lorna

2016-07-26

Proteins from organisms that have adapted to environmental extremes provide attractive systems to explore and determine the origins of protein stability. Improved hydrophobic core packing and decreased loop-length flexibility can increase the thermodynamic stability of proteins from hyperthermophilic organisms. However, their impact on protein mechanical stability is not known. Here, we use protein engineering, biophysical characterization, single-molecule force spectroscopy (SMFS), and molecular dynamics (MD) simulations to measure the effect of altering hydrophobic core packing on the stability of the cold shock protein TmCSP from the hyperthermophilic bacterium Thermotoga maritima. We make two variants of TmCSP in which a mutation is made to reduce the size of aliphatic groups from buried hydrophobic side chains. In the first, a mutation is introduced in a long loop (TmCSP L40A); in the other, the mutation is introduced on the C-terminal β-strand (TmCSP V62A). We use MD simulations to confirm that the mutant TmCSP L40A shows the most significant increase in loop flexibility, and mutant TmCSP V62A shows greater disruption to the core packing. We measure the thermodynamic stability (ΔGD-N) of the mutated proteins and show that there is a more significant reduction for TmCSP L40A (ΔΔG = 63%) than TmCSP V62A (ΔΔG = 47%), as might be expected on the basis of the relative reduction in the size of the side chain. By contrast, SMFS measures the mechanical stability (ΔG*) and shows a greater reduction for TmCSP V62A (ΔΔG* = 8.4%) than TmCSP L40A (ΔΔG* = 2.5%). While the impact on the mechanical stability is subtle, the results demonstrate the power of tuning noncovalent interactions to modulate both the thermodynamic and mechanical stability of a protein. Such understanding and control provide the opportunity to design proteins with optimized thermodynamic and mechanical properties.

Neuronal ELAV proteins enhance mRNA stability by a PKCα-dependent pathway

PubMed Central

Pascale, Alessia; Amadio, Marialaura; Scapagnini, Giovanni; Lanni, Cristina; Racchi, Marco; Provenzani, Alessandro; Govoni, Stefano; Alkon, Daniel L.; Quattrone, Alessandro

2005-01-01

More than 1 in 20 human genes bear in the mRNA 3′ UTR a specific motif called the adenine- and uridine-rich element (ARE), which posttranscriptionally determines its expression in response to cell environmental signals. ELAV (embryonic lethal abnormal vision) proteins are the only known ARE-binding factors that are able to stabilize the bound mRNAs, thereby positively controlling gene expression. Here, we show that in human neuroblastoma SH-SY5Y cells, neuron-specific ELAV (nELAV) proteins (HuB, HuC, and HuD) are up-regulated and redistributed by 15 min of treatment with the activators of PKC phorbol esters and bryostatin-1. PKC stimulation also induces nELAV proteins to colocalize with the translocated PKCα isozyme preferentially on the cytoskeleton, with a concomitant increase of nELAV threonine phosphorylation. The same treatment promotes stabilization of growth-associated protein 43 (GAP-43) mRNA, a well known nELAV target, and induces an early increase in GAP-43 protein concentration, again only in the cytoskeletal cell fraction. Genetic or pharmacological inactivation of PKCα abolishes nELAV protein cytoskeletal up-regulation, GAP-43 mRNA stabilization, and GAP-43 protein increase, demonstrating the primary role of this specific PKC isozyme in the cascade of nELAV recruitment. Finally, in vivo PKC activation is associated with an up-regulation of nELAV proteins in the hippocampal rat brain. These findings suggest a model for gene expression regulation by nELAV proteins through a PKCα-dependent pathway that is relevant for the cellular programs in which ARE-mediated control plays a pivotal role. PMID:16099831

Protein Stability in Mixed Solvents: A Balance of Contact Interaction and Excluded Volume

PubMed Central

Schellman, John A.

2003-01-01

Changes in excluded volume and contact interaction with the surface of a protein have been suggested as mechanisms for the changes in stability induced by cosolvents. The aim of the present paper is to present an analysis that combines both effects in a quantitative manner. The result is that both processes are present in both stabilizing and destabilizing interactions and neither can be ignored. Excluded volume was estimated using accessible surface area calculations of the kind introduced by Lee and Richards. The change in excluded volume on unfolding, ΔX, is quite large. For example, ΔX for ribonuclease is 6.7 L in urea and ∼16 L in sucrose. The latter number is greater than the molar volume of the protein. Direct interaction with the protein is represented as the solvent exchange mechanism, which differs from ordinary association theory because of the weakness of the interaction and the high concentrations of cosolvents. The balance between the two effects and their contribution to overall stability are most simply presented as bar diagrams as in Fig. 3. Our finding for five proteins is that excluded volume contributes to the stabilization of the native structure and that contact interaction contributes to destabilization. This is true for five proteins and four cosolvents including both denaturants and osmolytes. Whether a substance stabilizes a protein or destabilizes it depends on the relative size of these two contributions. The constant for the cosolvent contact with the protein is remarkably uniform for four of the proteins, indicating a similarity of groups exposed during unfolding. One protein, staphylococcus nuclease, is anomalous in almost all respects. In general, the strength of the interaction with guanidinium is about twice that of urea, which is about twice that of trimethylamine-N-oxide and sucrose. Arguments are presented for the use of volume fractions in equilibrium equations and the ignoring of activity coefficients of the cosolvent. It

Cysteine residue is not essential for CPM protein thermal-stability assay.

PubMed

Wang, Zhaoshuai; Ye, Cui; Zhang, Xinyi; Wei, Yinan

2015-05-01

A popular thermal-stability assay developed especially for the study of membrane proteins uses a thiol-specific probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). The fluorescence emission of CPM surges when it forms a covalent bond with the side chain of a free Cys, which becomes more readily accessible upon protein thermal denaturation. Interestingly, the melting temperatures of membrane proteins determined using the CPM assay in literature are closely clustered in the temperature range 45-55 °C. A thorough understanding of the mechanism behind the observed signal change is critical for the accurate interpretation of the protein unfolding. Here we used two α-helical membrane proteins, AqpZ and AcrB, as model systems to investigate the nature of the fluorescence surge in the CPM assay. We found that the transition temperatures measured using circular-dichroism (CD) spectroscopy and the CPM assay were significantly different. To eliminate potential artifact that might arise from the presence of detergent, we monitored the unfolding of two soluble proteins. We found that, contrary to current understanding, the presence of a sulfhydryl group was not a prerequisite for the CPM thermal-stability assay. The observed fluorescence increase is probably caused by binding of the fluorophore to hydrophobic patches exposed upon protein unfolding.

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

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.

Elucidating Protein Involvement in the Stabilization of the Biogenic Silver Nanoparticles

NASA Astrophysics Data System (ADS)

Ballottin, Daniela; Fulaz, Stephanie; Souza, Michele L.; Corio, Paola; Rodrigues, Alexandre G.; Souza, Ana O.; Gaspari, Priscyla M.; Gomes, Alexandre F.; Gozzo, Fábio; Tasic, Ljubica

2016-06-01

Silver nanoparticles (AgNPs) have been broadly used as antibacterial and antiviral agents. Further, interests for green AgNP synthesis have increased in recent years and several results for AgNP biological synthesis have been reported using bacteria, fungi and plant extracts. The understanding of the role and nature of fungal proteins, their interaction with AgNPs and the subsequent stabilization of nanosilver is yet to be deeply investigated. Therefore, in an attempt to better understand biogenic AgNP stabilization with the extracellular fungal proteins and to describe these supramolecular interactions between proteins and silver nanoparticles, AgNPs, produced extracellularly by Aspergillus tubingensis—isolated as an endophytic fungus from Rizophora mangle—were characterized in order to study their physical characteristics, identify the involved proteins, and shed light into the interactions among protein-NPs by several techniques. AgNPs of around 35 nm in diameter as measured by TEM and a positive zeta potential of +8.48 mV were obtained. These AgNPs exhibited a surface plasmon resonance (SPR) band at 440 nm, indicating the nanoparticles formation, and another band at 280 nm, attributed to the electronic excitations in tryptophan, tyrosine, and/or phenylalanine residues in fungal proteins. Fungal proteins were covalently bounded to the AgNPs, mainly through S-Ag bonds due to cysteine residues (HS-) and with few N-Ag bonds from H2N- groups, as verified by Raman spectroscopy. Observed supramolecular interactions also occur by electrostatic and other protein-protein interactions. Furthermore, proteins that remain free on AgNP surface may perform hydrogen bonds with other proteins or water increasing thus the capping layer around the AgNPs and consequently expanding the hydrodynamic diameter of the particles (~264 nm, measured by DLS). FTIR results enabled us to state that proteins adsorbed to the AgNPs did not suffer relevant secondary structure alteration upon

Factors Affecting Impact Toughness in Stabilized Intermediate Purity 21Cr Ferritic Stainless Steels and Their Simulated Heat-Affected Zones

NASA Astrophysics Data System (ADS)

Anttila, Severi; Alatarvas, Tuomas; Porter, David A.

2017-12-01

The correlation between simulated weld heat-affected zone microstructures and toughness parameters has been investigated in four intermediate purity 21Cr ferritic stainless steels stabilized with titanium and niobium either separately or in combination. Extensive Charpy V impact toughness testing was carried out followed by metallography including particle analysis using electron microscopy. The results confirmed that the grain size and the number density of particle clusters rich in titanium nitride and carbide with an equivalent circular diameter of 2 µm or more are statistically the most critical factors influencing the ductile-to-brittle transition temperature. Other inclusions and particle clusters, as well as grain boundary precipitates, are shown to be relatively harmless. Stabilization with niobium avoids large titanium-rich inclusions and also suppresses excessive grain growth in the heat-affected zone when reasonable heat inputs are used. Thus, in order to maximize the limited heat-affected zone impact toughness of 21Cr ferritic stainless steels containing 380 to 450 mass ppm of interstitials, the stabilization should be either titanium free or the levels of titanium and nitrogen should be moderated.

Sublethal concentrations of silver nanoparticles affect the mechanical stability of biofilms.

PubMed

Grün, Alexandra Y; Meier, Jutta; Metreveli, George; Schaumann, Gabriele E; Manz, Werner

2016-12-01

Bacterial biofilms are most likely confronted with silver nanoparticles (Ag NPs) as a pollutant stressor in aquatic systems. In this study, biofilms of Aquabacterium citratiphilum were exposed for 20 h to 30 and 70 nm citrate stabilized Ag NPs in low-dose concentrations ranging from 600 to 2400 μg l -1 , and the Ag NP-mediated effects on descriptive, structural, and functional biofilm characteristics, including viability, protein content, architecture, and mechanical stability, were investigated. Viability, based on the bacterial cell membrane integrity of A. citratiphilum, as determined by epifluorescence microscopy, remained unaffected after Ag NP exposure. Moreover, in contrast to information in the current literature, protein contents of cells and extracellular polymeric substances (EPS) and biofilm architecture, including dry mass, thickness, and density, were not significantly impacted by exposure to Ag NPs. However, the biofilms themselves served as effective sinks for Ag NPs, exhibiting enrichment factors from 5 to 8. Biofilms showed a greater capacity to accumulate 30 nm sized Ag NPs than 70 nm Ag NPs. Furthermore, Ag NPs significantly threatened the mechanical stability of biofilms, as determined by a newly developed assay. For 30 nm Ag NPs, the mechanical stability of biofilms decreased as the Ag NP concentrations applied to them increased. In contrast, 70 nm Ag NPs produced a similar decrease in mechanical stability for each applied concentration. Overall, this finding demonstrates that exposure to Ag NPs triggers remarkable changes in biofilm adhesion and/or cohesiveness. Because of biofilm-mediated ecological services, this response raises environmental concerns regarding Ag NP release into freshwater systems, even in sublethal concentrations.

Direct measurements of protein-stabilized gold nanoparticle interactions.

PubMed

Eichmann, Shannon L; Bevan, Michael A

2010-09-21

We report integrated video and total internal reflection microscopy measurements of protein stabilized 110 nm Au nanoparticles confined in 280 nm gaps in physiological media. Measured potential energy profiles display quantitative agreement with Brownian dynamic simulations that include hydrodynamic interactions and camera exposure time and noise effects. Our results demonstrate agreement between measured nonspecific van der Waals and adsorbed protein interactions with theoretical potentials. Confined, lateral nanoparticle diffusivity measurements also display excellent agreement with predictions. These findings provide a basis to interrogate specific biomacromolecular interactions in similar experimental configurations and to design future improved measurement methods.

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

PubMed

Calhoun, Kara A; Swartz, James R

2006-05-17

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

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.

Deletion of internal structured repeats increases the stability of a leucine-rich repeat protein, YopM

PubMed Central

Barrick, Doug

2011-01-01

Mapping the stability distributions of proteins in their native folded states provides a critical link between structure, thermodynamics, and function. Linear repeat proteins have proven more amenable to this kind of mapping than globular proteins. C-terminal deletion studies of YopM, a large, linear leucine-rich repeat (LRR) protein, show that stability is distributed quite heterogeneously, yet a high level of cooperativity is maintained [1]. Key components of this distribution are three interfaces that strongly stabilize adjacent sequences, thereby maintaining structural integrity and promoting cooperativity. To better understand the distribution of interaction energy around these critical interfaces, we studied internal (rather than terminal) deletions of three LRRs in this region, including one of these stabilizing interfaces. Contrary to our expectation that deletion of structured repeats should be destabilizing, we find that internal deletion of folded repeats can actually stabilize the native state, suggesting that these repeats are destabilizing, although paradoxically, they are folded in the native state. We identified two residues within this destabilizing segment that deviate from the consensus sequence at a position that normally forms a stacked leucine ladder in the hydrophobic core. Replacement of these nonconsensus residues with leucine is stabilizing. This stability enhancement can be reproduced in the context of nonnative interfaces, but it requires an extended hydrophobic core. Our results demonstrate that different LRRs vary widely in their contribution to stability, and that this variation is context-dependent. These two factors are likely to determine the types of rearrangements that lead to folded, functional proteins, and in turn, are likely to restrict the pathways available for the evolution of linear repeat proteins. PMID:21764506

Probing the determinants of protein stability: comparison of class A beta-lactamases.

PubMed Central

Vanhove, M; Houba, S; b1motte-Brasseur, J; Frère, J M

1995-01-01

Five class A beta-lactamases produced by various mesophilic bacterial species have been compared. Although closely related in primary and overall structures, these enzymes exhibit very different stabilities. In order to investigate the factors responsible for these differences, several features deduced from the amino acid composition and three-dimensional structures were studied for the five proteins. This analysis revealed that higher stability appeared to correlate with increased numbers of intramolecular hydrogen bonds and of salt bridges. By contrast, the global hydrophobicity of the protein seemed to play a relatively minor role. A strongly unfavourable balance between charged residues and the presence of a cis-peptide bond preceding a non-proline residue might also contribute to the particularly low stability of two of the enzymes. PMID:8948443

Influence of pea protein aggregates on the structure and stability of pea protein/soybean polysaccharide complex emulsions.

PubMed

Yin, Baoru; Zhang, Rujing; Yao, Ping

2015-03-20

The applications of plant proteins in the food and beverage industry have been hampered by their precipitation in acidic solution. In this study, pea protein isolate (PPI) with poor dispersibility in acidic solution was used to form complexes with soybean soluble polysaccharide (SSPS), and the effects of PPI aggregates on the structure and stability of PPI/SSPS complex emulsions were investigated. Under acidic conditions, high pressure homogenization disrupts the PPI aggregates and the electrostatic attraction between PPI and SSPS facilitates the formation of dispersible PPI/SSPS complexes. The PPI/SSPS complex emulsions prepared from the PPI containing aggregates prove to possess similar droplet structure and similar stability compared with the PPI/SSPS emulsions produced from the PPI in which the aggregates have been previously removed by centrifugation. The oil droplets are protected by PPI/SSPS complex interfacial films and SSPS surfaces. The emulsions show long-term stability against pH and NaCl concentration changes. This study demonstrates that PPI aggregates can also be used to produce stable complex emulsions, which may promote the applications of plant proteins in the food and beverage industry.

Protein engineering of subtilisins to improve stability in detergent formulations.

PubMed

von der Osten, C; Branner, S; Hastrup, S; Hedegaard, L; Rasmussen, M D; Bisgård-Frantzen, H; Carlsen, S; Mikkelsen, J M

1993-03-01

Microbial proteases are used extensively in a large number of industrial processes and most importantly in detergent formulations facilitating the removal of proteinaceous stains. Site-directed mutagenesis has been employed in the construction of subtilisin variants with improved storage and oxidation stabilities. It is shown that in spite of significant structural homology between subtilisins subjected to protein engineering the effects of specific mutations can be quite different. Mutations that stabilize one subtilisin may destabilize another.

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

NASA Astrophysics Data System (ADS)

He, Yi-Ming; Ma, Bin-Guang

2016-05-01

Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions.

Stability of buffer-free freeze-dried formulations: A feasibility study of a monoclonal antibody at high protein concentrations.

PubMed

Garidel, Patrick; Pevestorf, Benjamin; Bahrenburg, Sven

2015-11-01

We studied the stability of freeze-dried therapeutic protein formulations over a range of initial concentrations (from 40 to 160 mg/mL) and employed a variety of formulation strategies (including buffer-free freeze dried formulations, or BF-FDF). Highly concentrated, buffer-free liquid formulations of therapeutic monoclonal antibodies (mAbs) have been shown to be a viable alternative to conventionally buffered preparations. We considered whether it is feasible to use the buffer-free strategy in freeze-dried formulations, as an answer to some of the known drawbacks of conventional buffers. We therefore conducted an accelerated stability study (24 weeks at 40 °C) to assess the feasibility of stabilizing freeze-dried formulations without "classical" buffer components. Factors monitored included pH stability, protein integrity, and protein aggregation. Because the protein solutions are inherently self-buffering, and the system's buffer capacity scales with protein concentration, we included highly concentrated buffer-free freeze-dried formulations in the study. The tested formulations ranged from "fully formulated" (containing both conventional buffer and disaccharide stabilizers) to "buffer-free" (including formulations with only disaccharide lyoprotectant stabilizers) to "excipient-free" (with neither added buffers nor stabilizers). We evaluated the impacts of varying concentrations, buffering schemes, pHs, and lyoprotectant additives. At the end of 24 weeks, no change in pH was observed in any of the buffer-free formulations. Unbuffered formulations were found to have shorter reconstitution times and lower opalescence than buffered formulations. Protein stability was assessed by visual inspection, sub-visible particle analysis, protein monomer content, charge variants analysis, and hydrophobic interaction chromatography. All of these measures found the stability of buffer-free formulations that included a disaccharide stabilizer comparable to buffer

Amaranth proteins foaming properties: Film rheology and foam stability - Part 2.

PubMed

Bolontrade, Agustín J; Scilingo, Adriana A; Añón, María C

2016-05-01

In this work the influence of pH and ionic strength on the stability of foams prepared with amaranth protein isolate was analyzed. The behaviour observed was related to the physico-chemical and structural changes undergone by amaranth protein as a result of those treatments. The results obtained show that foams prepared at acidic pH were more stable than the corresponding to alkaline pH. At pH 2.0 the foams presented higher times and more volumes of drainage. This behaviour is consistent with the characteristics of the interfacial film, which showed a higher viscoelasticity and a greater flexibility at acidic pH than alkaline pH value, which in turn increased by increasing the concentration of proteins in the foaming solution. It is also important to note that the presence of insoluble protein is not necessarily detrimental to the properties of the foam. Detected changes in the characteristics of the interfacial film as in the foam stability have been attributed to the increased unfolding, greater flexibility and net charge of amaranth proteins at acidic conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

Role of the Acidic Tail of High Mobility Group Protein B1 (HMGB1) in Protein Stability and DNA Bending

PubMed Central

Belgrano, Fabricio S.; de Abreu da Silva, Isabel C.; Bastos de Oliveira, Francisco M.; Fantappié, Marcelo R.; Mohana-Borges, Ronaldo

2013-01-01

High mobility group box (HMGB) proteins are abundant nonhistone proteins found in all eukaryotic nuclei and are capable of binding/bending DNA. The human HMGB1 is composed of two binding motifs, known as Boxes A and B, are L-shaped alpha-helix structures, followed by a random-coil acidic tail that consists of 30 Asp and Glu residues. This work aimed at evaluating the role of the acidic tail of human HMGB1 in protein stability and DNA interactions. For this purpose, we cloned, expressed and purified HMGB1 and its tailless form, HMGB1ΔC, in E. coli strain. Tryptophan fluorescence spectroscopy and circular dichroism (CD) experiments clearly showed an increase in protein stability promoted by the acidic tail under different conditions, such as the presence of the chemical denaturant guanidine hydrochloride (Gdn.HCl), high temperature and low pH. Folding intermediates found at low pH for both proteins were denatured only in the presence of chemical denaturant, thus showing a relatively high stability. The acidic tail did not alter the DNA-binding properties of the protein, although it enhanced the DNA bending capability from 76° (HMGB1ΔC) to 91° (HMGB1), as measured using the fluorescence resonance energy transfer technique. A model of DNA bending in vivo was proposed, which might help to explain the interaction of HMGB1 with DNA and other proteins, i.e., histones, and the role of that protein in chromatin remodeling. PMID:24255708

MEICPS: substitution mutations to engineer intracellular protein stability.

PubMed

Reddy, B V; Ramesh, P; Tiwari, S

1998-01-01

In MEICPS, results from earlier analyses are utilized to suggest possible substitution point mutations to engineer intracellular stability using a given sequence or structure of the protein. From bvbreddy@ccmb.ap.nic.in. This program needs data from other software, PSA and SSTRUC, available from sali@tamika.rockefeller.edu and tom@cryst.bioc.cam.ac.uk, respectively. bvbreddy@ccmb.ap.nic.in

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.

Complex Stability of Single Proteins Explored by Forced Unfolding Experiments

PubMed Central

Janovjak, Harald; Sapra, K. Tanuj; Müller, Daniel J.

2005-01-01

In the last decade atomic force microscopy has been used to measure the mechanical stability of single proteins. These force spectroscopy experiments have shown that many water-soluble and membrane proteins unfold via one or more intermediates. Recently, Li and co-workers found a linear correlation between the unfolding force of the native state and the intermediate in fibronectin, which they suggested indicated the presence of a molecular memory or multiple unfolding pathways (1). Here, we apply two independent methods in combination with Monte Carlo simulations to analyze the unfolding of α-helices E and D of bacteriorhodopsin (BR). We show that correlation analysis of unfolding forces is very sensitive to errors in force calibration of the instrument. In contrast, a comparison of relative forces provides a robust measure for the stability of unfolding intermediates. The proposed approach detects three energetically different states of α-helices E and D in trimeric BR. These states are not observed for monomeric BR and indicate that substantial information is hidden in forced unfolding experiments of single proteins. PMID:15792967

Complex stability of single proteins explored by forced unfolding experiments.

PubMed

Janovjak, Harald; Sapra, K Tanuj; Müller, Daniel J

2005-05-01

In the last decade atomic force microscopy has been used to measure the mechanical stability of single proteins. These force spectroscopy experiments have shown that many water-soluble and membrane proteins unfold via one or more intermediates. Recently, Li and co-workers found a linear correlation between the unfolding force of the native state and the intermediate in fibronectin, which they suggested indicated the presence of a molecular memory or multiple unfolding pathways (1). Here, we apply two independent methods in combination with Monte Carlo simulations to analyze the unfolding of alpha-helices E and D of bacteriorhodopsin (BR). We show that correlation analysis of unfolding forces is very sensitive to errors in force calibration of the instrument. In contrast, a comparison of relative forces provides a robust measure for the stability of unfolding intermediates. The proposed approach detects three energetically different states of alpha-helices E and D in trimeric BR. These states are not observed for monomeric BR and indicate that substantial information is hidden in forced unfolding experiments of single proteins.

TMV-Cg Coat Protein stabilizes DELLA proteins and in turn negatively modulates salicylic acid-mediated defense pathway during Arabidopsis thaliana viral infection.

PubMed

Rodriguez, Maria Cecilia; Conti, Gabriela; Zavallo, Diego; Manacorda, Carlos Augusto; Asurmendi, Sebastian

2014-08-03

Plant viral infections disturb defense regulatory networks during tissue invasion. Emerging evidence demonstrates that a significant proportion of these alterations are mediated by hormone imbalances. Although the DELLA proteins have been reported to be central players in hormone cross-talk, their role in the modulation of hormone signaling during virus infections remains unknown. This work revealed that TMV-Cg coat protein (CgCP) suppresses the salicylic acid (SA) signaling pathway without altering defense hormone SA or jasmonic acid (JA) levels in Arabidopsis thaliana. Furthermore, it was observed that the expression of CgCP reduces plant growth and delays the timing of floral transition. Quantitative RT-qPCR analysis of DELLA target genes showed that CgCP alters relative expression of several target genes, indicating that the DELLA proteins mediate transcriptional changes produced by CgCP expression. Analyses by fluorescence confocal microscopy showed that CgCP stabilizes DELLA proteins accumulation in the presence of gibberellic acid (GA) and that the DELLA proteins are also stabilized during TMV-Cg virus infections. Moreover, DELLA proteins negatively modulated defense transcript profiles during TMV-Cg infection. As a result, TMV-Cg accumulation was significantly reduced in the quadruple-DELLA mutant Arabidopsis plants compared to wild type plants. Taken together, these results demonstrate that CgCP negatively regulates the salicylic acid-mediated defense pathway by stabilizing the DELLA proteins during Arabidopsis thaliana viral infection, suggesting that CgCP alters the stability of DELLAs as a mechanism of negative modulation of antiviral defense responses.

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

The effect of denaturant on protein stability: a Monte Carlo lattice simulation

NASA Astrophysics Data System (ADS)

Choi, Ho Sup; Huh, June; Jo, Won Ho

2003-03-01

Denaturants are the reagents that decrease protein stability by interacting with both nonpolar and polar surfaces of protein when added to the aqueous solvent. However, the physical nature of these interactions has not been clearly understood. It is not easy to elucidate the nature of denaturant theoretically or experimentally. Even in computer simulation, the denaturant atoms are unable to be dealt explicitly due to computationally enormous costs. We have used a lattice model of protein and denaturant. By varying concentration of denaturant and interaction energy between protein and denaturant, we have measured the change of stability of the protein. This simple model reflects the experimental observation that the free energy of unfolding is a linear function of denaturant concentration in the transition range. We have also performed a simulation under isotropic perturbation. In this case, denaturant molecules are not included and a biasing potential is introduced in order to increase the radius of gyration of protein, which incorporates the effect of denaturant implicitly. The calculated free energy landscape and conformational ensembles sampled under this condition is very close to those of simulation using denaturant molecules interacting with protein. We have applied this simple approach for simulating the effect of denaturant to real proteins.

Structure and stability insights into tumour suppressor p53 evolutionary related proteins.

PubMed

Pagano, Bruno; Jama, Abdullah; Martinez, Pierre; Akanho, Ester; Bui, Tam T T; Drake, Alex F; Fraternali, Franca; Nikolova, Penka V

2013-01-01

The p53 family of genes and their protein products, namely, p53, p63 and p73, have over one billion years of evolutionary history. Advances in computational biology and genomics are enabling studies of the complexities of the molecular evolution of p53 protein family to decipher the underpinnings of key biological conditions spanning from cancer through to various metabolic and developmental disorders and facilitate the design of personalised medicines. However, a complete understanding of the inherent nature of the thermodynamic and structural stability of the p53 protein family is still lacking. This is due, to a degree, to the lack of comprehensive structural information for a large number of homologous proteins and to an incomplete knowledge of the intrinsic factors responsible for their stability and how these might influence function. Here we investigate the thermal stability, secondary structure and folding properties of the DNA-binding domains (DBDs) of a range of proteins from the p53 family using biophysical methods. While the N- and the C-terminal domains of the p53 family show sequence diversity and are normally targets for post-translational modifications and alternative splicing, the central DBD is highly conserved. Together with data obtained from Molecular Dynamics simulations in solution and with structure based homology modelling, our results provide further insights into the molecular properties of evolutionary related p53 proteins. We identify some marked structural differences within the p53 family, which could account for the divergence in biological functions as well as the subtleties manifested in the oligomerization properties of this family.

Characteristics of sugar surfactants in stabilizing proteins during freeze-thawing and freeze-drying.

PubMed

Imamura, Koreyoshi; Murai, Katsuyuki; Korehisa, Tamayo; Shimizu, Noriyuki; Yamahira, Ryo; Matsuura, Tsutashi; Tada, Hiroko; Imanaka, Hiroyuki; Ishida, Naoyuki; Nakanishi, Kazuhiro

2014-06-01

Sugar surfactants with different alkyl chain lengths and sugar head groups were compared for their protein-stabilizing effect during freeze-thawing and freeze-drying. Six enzymes, different in terms of tolerance against inactivation because of freeze-thawing and freeze-drying, were used as model proteins. The enzyme activities that remained after freeze-thawing and freeze-drying in the presence of a sugar surfactant were measured for different types and concentrations of sugar surfactants. Sugar surfactants stabilized all of the tested enzymes both during freeze-thawing and freeze-drying, and a one or two order higher amount of added sugar surfactant was required for achieving protein stabilization during freeze-drying than for the cryoprotection. The comprehensive comparison showed that the C10-C12 esters of sucrose or trehalose were the most effective through the freeze-drying process: the remaining enzyme activities after freeze-thawing and freeze-drying increased at the sugar ester concentrations of 1-10 and 10-100 μM, respectively, and increased to a greater extent than for the other surfactants at higher concentrations. Results also indicate that, when a decent amount of sugar was also added, the protein-stabilizing effect of a small amount of sugar ester through the freeze-drying process could be enhanced. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

[The absence of cyclin-dependent protein kinase Pho85 affects stability of mitochondrial DNA in yeast Saccharomyces cerevisiae].

PubMed

Fizikova, A Iu; Padkina, M V; Sambuk, E V

2009-06-01

The cyclin-dependent protein kinase Pho85 is involved in the regulation of phosphate metabolism in yeast Saccharomyces cerevisiae. Mutations in the PH085 gene lead to constitutive synthesis of Pho5 acidic phosphatase, a delay in cell growth on media containing nonfermentable carbon sources, and other pleiotropic effects. In this work, it was shown that the accumulation of respiratory incompetent cells occurs with high frequency in strains carrying pho85 mutations as early as during the first cell divisions, and the number of these cells at the early logarithmic growth phase of the culture promptly reaches virtually 100%. Cytological analysis revealed a high accumulation rate of [rho(0)] cells the background of gene pho85 that may be related to disturbances in the distribution of mitochondrial nucleoids rather than to changes in morphology of mitochondria and a delay in their transport into the bud. Genetic analysis revealed that the appearing secondary mutations pho4, pho81, pho84, and pho87 stabilize nucleoids and hamper the loss of mitochondrial DNA caused by pho85. These results provide evidence for the influence of intracellular phosphate concentration on the inheritance of mitochondrial nucleoids, but it is fully probable that the occurrence of mutation pho4 in the background of gene pho85 may change the expression level of other genes required for the stabilization of mitochondrial functions.

Protein stability and dynamics influenced by ligands in extremophilic complexes - a molecular dynamics investigation.

PubMed

Khan, Sara; Farooq, Umar; Kurnikova, Maria

2017-08-22

In this study, we explore the structural and dynamic adaptations of the Tryptophan synthase α-subunit in a ligand bound state in psychrophilic, mesophilic and hyperthermophilic organisms at different temperatures by MD simulations. We quantify the global and local fluctuations in the 40 ns time scale by analyzing the root mean square deviation/fluctuations. The distinct behavior of the active site and loop 6 is observed with the elevation of temperature. Protein stability relies more on electrostatic interactions, and these interactions might be responsible for the stability of varying temperature evolved proteins. The paper also focuses on the effect of temperature on protein dynamics and stability governed by the distinct behavior of the ligand associated with its retention, binding and dissociation over the course of time. The integration of principle component analysis and a free energy landscape was useful in identifying the conformational space accessible to ligand bound homologues and how the presence of the ligand alters the conformational and dynamic properties of the protein.

Lengths of Orthologous Prokaryotic Proteins Are Affected by Evolutionary Factors

PubMed Central

Tatarinova, Tatiana; Dien Bard, Jennifer; Cohen, Irit

2015-01-01

Proteins of the same functional family (for example, kinases) may have significantly different lengths. It is an open question whether such variation in length is random or it appears as a response to some unknown evolutionary driving factors. The main purpose of this paper is to demonstrate existence of factors affecting prokaryotic gene lengths. We believe that the ranking of genomes according to lengths of their genes, followed by the calculation of coefficients of association between genome rank and genome property, is a reasonable approach in revealing such evolutionary driving factors. As we demonstrated earlier, our chosen approach, Bubble-sort, combines stability, accuracy, and computational efficiency as compared to other ranking methods. Application of Bubble Sort to the set of 1390 prokaryotic genomes confirmed that genes of Archaeal species are generally shorter than Bacterial ones. We observed that gene lengths are affected by various factors: within each domain, different phyla have preferences for short or long genes; thermophiles tend to have shorter genes than the soil-dwellers; halophiles tend to have longer genes. We also found that species with overrepresentation of cytosines and guanines in the third position of the codon (GC3 content) tend to have longer genes than species with low GC3 content. PMID:26114113

Lengths of Orthologous Prokaryotic Proteins Are Affected by Evolutionary Factors.

PubMed

Tatarinova, Tatiana; Salih, Bilal; Dien Bard, Jennifer; Cohen, Irit; Bolshoy, Alexander

2015-01-01

Proteins of the same functional family (for example, kinases) may have significantly different lengths. It is an open question whether such variation in length is random or it appears as a response to some unknown evolutionary driving factors. The main purpose of this paper is to demonstrate existence of factors affecting prokaryotic gene lengths. We believe that the ranking of genomes according to lengths of their genes, followed by the calculation of coefficients of association between genome rank and genome property, is a reasonable approach in revealing such evolutionary driving factors. As we demonstrated earlier, our chosen approach, Bubble-sort, combines stability, accuracy, and computational efficiency as compared to other ranking methods. Application of Bubble Sort to the set of 1390 prokaryotic genomes confirmed that genes of Archaeal species are generally shorter than Bacterial ones. We observed that gene lengths are affected by various factors: within each domain, different phyla have preferences for short or long genes; thermophiles tend to have shorter genes than the soil-dwellers; halophiles tend to have longer genes. We also found that species with overrepresentation of cytosines and guanines in the third position of the codon (GC3 content) tend to have longer genes than species with low GC3 content.

Functional Properties of a High Protein Beverage Stabilized with Oat-β-Glucan.

PubMed

Vasquez-Orejarena, Eva; Simons, Christopher T; Litchfield, John H; Alvarez, Valente B

2018-05-01

This study evaluated the effect of oat flour and milk protein on the functional properties and sensory acceptability of shelf stable high protein dairy beverages containing at least 0.75 g of oat-β-glucan per serving size. Formulations adjusted to levels of 1.50% to 2.30% oat flour and 2.50% to 4.00% milk protein isolate (MPI) were thermal processed in a rotary retort. The finished product exhibited good suspension stability (>80%). The increase of oat and MPI contents lead to nectar-like beverages (51 to 100 mPas). However, oat flour was the component showing the highest effect on the viscosity coefficient values of the beverages. Sensory evaluation indicated that formulations with less than 1.9% oat flour and 2.5% MPI (thin liquid, <50 mPas) were the most accepted. Mouthfeel (perceived thickness), sweetness and aftertaste had the most influence on overall liking of the beverages. Overall, this study comprises the development of a functional food product. Supplementation of beverages with fiber from oats is an innovative approach to stabilize high protein beverages. Ready to drink protein beverage formulations use gums to stabilize the product and provide a desirable mouthfeel. The levels of oat-β-glucan used in the beverage increased the thickness and meet the requirement of the FDA approved health claim for reduction of the cardiovascular disease risk (21 CFR 101.81). © 2018 Institute of Food Technologists®.

In vivo architectonic stability of fully de novo designed protein-only nanoparticles.

PubMed

Céspedes, María Virtudes; Unzueta, Ugutz; Tatkiewicz, Witold; Sánchez-Chardi, Alejandro; Conchillo-Solé, Oscar; Álamo, Patricia; Xu, Zhikun; Casanova, Isolda; Corchero, José Luis; Pesarrodona, Mireia; Cedano, Juan; Daura, Xavier; Ratera, Imma; Veciana, Jaume; Ferrer-Miralles, Neus; Vazquez, Esther; Villaverde, Antonio; Mangues, Ramón

2014-05-27

The fully de novo design of protein building blocks for self-assembling as functional nanoparticles is a challenging task in emerging nanomedicines, which urgently demand novel, versatile, and biologically safe vehicles for imaging, drug delivery, and gene therapy. While the use of viruses and virus-like particles is limited by severe constraints, the generation of protein-only nanocarriers is progressively reachable by the engineering of protein-protein interactions, resulting in self-assembling functional building blocks. In particular, end-terminal cationic peptides drive the organization of structurally diverse protein species as regular nanosized oligomers, offering promise in the rational engineering of protein self-assembling. However, the in vivo stability of these constructs, being a critical issue for their medical applicability, needs to be assessed. We have explored here if the cross-molecular contacts between protein monomers, generated by end-terminal cationic peptides and oligohistidine tags, are stable enough for the resulting nanoparticles to overcome biological barriers in assembled form. The analyses of renal clearance and biodistribution of several tagged modular proteins reveal long-term architectonic stability, allowing systemic circulation and tissue targeting in form of nanoparticulate material. This observation fully supports the value of the engineered of protein building blocks addressed to the biofabrication of smart, robust, and multifunctional nanoparticles with medical applicability that mimic structure and functional capabilities of viral capsids.

β-lactoglobulin stabilized nanemulsions--Formulation and process factors affecting droplet size and nanoemulsion stability.

PubMed

Ali, Ali; Mekhloufi, Ghozlene; Huang, Nicolas; Agnely, Florence

2016-03-16

To avoid the toxicological concerns associated to synthetic surfactants, proteins might be an alternative for the stabilization of pharmaceutical nanoemulsions. The present study investigates the use of β-lactoglobulin (β-lg) to stabilize oil in water biocompatible nanoemulsions intended for a pharmaceutical use and prepared by high pressure homogenization (HPH). The effects of composition (nature and weight fraction of oil, β-lg concentration) and of process parameters (pressure and number of cycles) on the droplet size and on the stability of nanoemulsions were thoroughly assessed. The nanoemulsions prepared with β-lg at 1 wt% and with 5 wt% Miglyol 812 (the oil with the lowest viscosity) displayed a relatively small particle size (about 200 nm) and a low polydispersity when a homogenization pressure of 100 MPa was applied for 4 cycles. These nanoemulsions were the most stable formulations over 30 days at least. Emulsification efficiency of β-lg was reduced at higher homogenization pressures (200 MPa and 300 MPa). The effect of HPH process on the interfacial properties of β-lg was evaluated by drop shape analysis. This treatment had an effect neither on the interfacial tension nor on the interfacial dilatational rheology of β-lg at the Miglyol 812/water interface. Copyright © 2016 Elsevier B.V. All rights reserved.

An amphipathic polypeptide derived from poly-γ-glutamic acid for the stabilization of membrane proteins.

PubMed

Han, Seong-Gu; Na, Jung-Hyun; Lee, Won-Kyu; Park, Dongkook; Oh, Jihye; Yoon, Sung-Ho; Lee, Cheng-Kang; Sung, Moon-Hee; Shin, Yeon-Kyun; Yu, Yeon Gyu

2014-12-01

Difficulties in the extraction of membrane proteins from cell membrane and their solubilization in native conformations have hindered their structural and biochemical analysis. To overcome these difficulties, an amphipathic polypeptide was synthesized by the conjugation of octyl and glucosyl groups to the carboxyl groups of poly-γ-glutamic acid (PGA). This polymer, called amphipathic PGA (APG), self-assembles as mono-disperse oligomers consisted of 4-5 monomers. APG shows significantly low value of critical micelle concentration and stabilization activity toward membrane proteins. Most of the sodium dodecyl sulfate (SDS)-solubilized membrane proteins from Escherichia coli remain soluble state in the presence of APG even after the removal of SDS. In addition, APG stabilizes purified 7 transmembrane proteins such as bacteriorhodopsin and human endothelin receptor Type A (ETA ) in their active conformations. Furthermore, ETA in complex with APG is readily inserted into liposomes without disrupting the integrity of liposomes. These properties of APG can be applied to overcome the difficulties in the stabilization and reconstitution of membrane proteins. © 2014 The Protein Society.

Salt Potentiates Methylamine Counteraction System to Offset the Deleterious Effects of Urea on Protein Stability and Function

PubMed Central

Singh, Laishram R.; Warepam, Marina; Ahmad, Faizan; Dar, Tanveer Ali

2015-01-01

Cellular methylamines are osmolytes (low molecular weight organic compounds) believed to offset the urea’s harmful effects on the stability and function of proteins in mammalian kidney and marine invertebrates. Although urea and methylamines are found at 2:1 molar ratio in tissues, their opposing effects on protein structure and function have been questioned on several grounds including failure to counteraction or partial counteraction. Here we investigated the possible involvement of cellular salt, NaCl, in urea-methylamine counteraction on protein stability and function. We found that NaCl mediates methylamine counteracting system from no or partial counteraction to complete counteraction of urea’s effect on protein stability and function. These conclusions were drawn from the systematic thermodynamic stability and functional activity measurements of lysozyme and RNase-A. Our results revealed that salts might be involved in protein interaction with charged osmolytes and hence in the urea-methylamine counteraction. PMID:25793733

Salt potentiates methylamine counteraction system to offset the deleterious effects of urea on protein stability and function.

PubMed

Rahman, Safikur; Rehman, Md Tabish; Singh, Laishram R; Warepam, Marina; Ahmad, Faizan; Dar, Tanveer Ali

2015-01-01

Cellular methylamines are osmolytes (low molecular weight organic compounds) believed to offset the urea's harmful effects on the stability and function of proteins in mammalian kidney and marine invertebrates. Although urea and methylamines are found at 2:1 molar ratio in tissues, their opposing effects on protein structure and function have been questioned on several grounds including failure to counteraction or partial counteraction. Here we investigated the possible involvement of cellular salt, NaCl, in urea-methylamine counteraction on protein stability and function. We found that NaCl mediates methylamine counteracting system from no or partial counteraction to complete counteraction of urea's effect on protein stability and function. These conclusions were drawn from the systematic thermodynamic stability and functional activity measurements of lysozyme and RNase-A. Our results revealed that salts might be involved in protein interaction with charged osmolytes and hence in the urea-methylamine counteraction.

Ultra-High Pressure Homogenization enhances physicochemical properties of soy protein isolate-stabilized emulsions.

PubMed

Fernández-Ávila, C; Escriu, R; Trujillo, A J

2015-09-01

The effect of Ultra-High Pressure Homogenization (UHPH, 100-300MPa) on the physicochemical properties of oil-in-water emulsions prepared with 4.0% (w/v) of soy protein isolate (SPI) and soybean oil (10 and 20%, v/v) was studied and compared to emulsions treated by conventional homogenization (CH, 15MPa). CH emulsions were prepared with non-heated and heated (95°C for 15min) SPI dispersions. Emulsions were characterized by particle size determination with laser diffraction, rheological properties using a rotational rheometer by applying measurements of flow curve and by transmission electron microscopy. The variation on particle size and creaming was assessed by Turbiscan® analysis, and visual observation of the emulsions was also carried out. UHPH emulsions showed much smaller d 3.2 values and greater physical stability than CH emulsions. The thermal treatment of SPI prior CH process did not improve physical stability properties. In addition, emulsions containing 20% of oil exhibited greater physical stability compared to emulsions containing 10% of oil. Particularly, UHPH emulsions treated at 100 and 200MPa with 20% of oil were the most stable due to low particle size values (d 3.2 and Span), greater viscosity and partial protein denaturation. These results address the physical stability improvement of protein isolate-stabilized emulsions by using the emerging UHPH technology. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparative profiling of sarcoplasmic phosphoproteins in ovine muscle with different color stability.

PubMed

Li, Meng; Li, Zheng; Li, Xin; Xin, Jianzeng; Wang, Ying; Li, Guixia; Wu, Liguo; Shen, Qingwu W; Zhang, Dequan

2018-02-01

The phosphorylation of sarcoplasmic proteins in postmortem muscles was investigated in relationship to color stability in the present study. Although no difference was observed in the global phosphorylation level of sarcoplasmic proteins, difference was determined in the phosphorylation levels of individual protein bands from muscles with different color stability. Correlation analysis and liquid chromatography - tandem mass spectrometry (LC-MS/MS) identification of phosphoproteins showed that most of the color stability-related proteins were glycolytic enzymes. Interestingly, the phosphorylation level of myoglobin was inversely related to meat color stability. As the phosphorylation of myoglobin increased, color stability based on a ∗ value decreased and metMb content increased. In summary, the study revealed that protein phosphorylation might play a role in the regulation of meat color stability probably by regulating glycolysis and the redox stability of myoglobin, which might be affected by the phosphorylation of myoglobin. Copyright © 2017 Elsevier Ltd. All rights reserved.

Unfolding stabilities of two structurally similar proteins as probed by temperature-induced and force-induced molecular dynamics simulations.

PubMed

Gorai, Biswajit; Prabhavadhni, Arasu; Sivaraman, Thirunavukkarasu

2015-09-01

Unfolding stabilities of two homologous proteins, cardiotoxin III and short-neurotoxin (SNTX) belonging to three-finger toxin (TFT) superfamily, have been probed by means of molecular dynamics (MD) simulations. Combined analysis of data obtained from steered MD and all-atom MD simulations at various temperatures in near physiological conditions on the proteins suggested that overall structural stabilities of the two proteins were different from each other and the MD results are consistent with experimental data of the proteins reported in the literature. Rationalization for the differential structural stabilities of the structurally similar proteins has been chiefly attributed to the differences in the structural contacts between C- and N-termini regions in their three-dimensional structures, and the findings endorse the 'CN network' hypothesis proposed to qualitatively analyse the thermodynamic stabilities of proteins belonging to TFT superfamily of snake venoms. Moreover, the 'CN network' hypothesis has been revisited and the present study suggested that 'CN network' should be accounted in terms of 'structural contacts' and 'structural strengths' in order to precisely describe order of structural stabilities of TFTs.

High-throughput analysis of the protein sequence-stability landscape using a quantitative "yeast surface two-hybrid" system and fragment reconstitution

PubMed Central

Dutta, Sanjib; Koide, Akiko; Koide, Shohei

2008-01-01

Stability evaluation of many mutants can lead to a better understanding of the sequence determinants of a structural motif and of factors governing protein stability and protein evolution. The traditional biophysical analysis of protein stability is low throughput, limiting our ability to widely explore the sequence space in a quantitative manner. In this study, we have developed a high-throughput library screening method for quantifying stability changes, which is based on protein fragment reconstitution and yeast surface display. Our method exploits the thermodynamic linkage between protein stability and fragment reconstitution and the ability of the yeast surface display technique to quantitatively evaluate protein-protein interactions. The method was applied to a fibronectin type III (FN3) domain. Characterization of fragment reconstitution was facilitated by the co-expression of two FN3 fragments, thus establishing a "yeast surface two-hybrid" method. Importantly, our method does not rely on competition between clones and thus eliminates a common limitation of high-throughput selection methods in which the most stable variants are predominantly recovered. Thus, it allows for the isolation of sequences that exhibits a desired level of stability. We identified over one hundred unique sequences for a β-bulge motif, which was significantly more informative than natural sequences of the FN3 family in revealing the sequence determinants for the β-bulge. Our method provides a powerful means to rapidly assess stability of many variants, to systematically assess contribution of different factors to protein stability and to enhance protein stability. PMID:18674545

Analyzing bean extracts using time-dependent SDS trapping to quantify the kinetic stability of phaseolin proteins.

PubMed

Thibeault, Jane; Church, Jennifer; Ortiz-Perez, Brian; Addo, Samuel; Hill, Shakeema; Khalil, Areeg; Young, Malaney; Xia, Ke; Colón, Wilfredo

2017-09-30

In common beans and lima bean, the storage protein phaseolin is difficult to degrade and SDS-resistant, a sign of kinetic stability. Kinetically stable proteins (KSPs) are characterized by having a high-energy barrier between the native and denatured states that results in very slow unfolding. Such proteins are resistant to proteolytic degradation and detergents, such as SDS. Here the method SDS-Trapping of Proteins (S-TraP) is applied directly on bean extracts to quantify the kinetic stability of phaseolin in lima bean and several common beans, including black bean, navy bean, and small red bean. The bean extracts were incubated in SDS at various temperatures (60-75 °C) for different time periods, followed by SDS-PAGE analysis at room temperature, and subsequent band quantification to determine the kinetics of phaseolin unfolding. Eyring plot analysis showed that the phaseolin from each bean has high kinetic stability, with an SDS-trapping (i.e. unfolding) half-life ranging from about 20-100 years at 24 °C and 2-7 years at 37 °C. The remarkably high kinetic stability of these phaseolin proteins is consistent with the low digestibility of common beans and lima bean, as well as their relatively high germination temperatures. From a practical perspective, this work exemplifies that S-TraP is a useful and cost-effective method for quantifying the kinetic stability of proteins in biological extracts or lysates. Depending on the protein to be studied and its abundance, S-TraP may be performed directly on the extract without need for protein purification. Copyright © 2017 Elsevier Inc. All rights reserved.

Substrate uptake and protein stability relationship in mammalian histidine decarboxylase.

PubMed

Pino-Angeles, A; Morreale, A; Negri, A; Sánchez-Jiménez, F; Moya-García, A A

2010-01-01

There is some evidence linking the substrate entrance in the active site of mammalian histidine decarboxylase and an increased stability against proteolytic degradation. In this work, we study the basis of this relationship by means of protein structure network analysis and molecular dynamics simulations. We find that the substrate binding to the active site influences the conformation of a flexible region sensible to proteolytic degradation and observe how formation of the Michaelis-Menten complex increases stability in the conformation of this region. (c) 2009 Wiley-Liss, Inc.

Role of the disulfide bond in stabilizing and folding of the fimbrial protein DraE from uropathogenic Escherichia coli

PubMed Central

Pilipczuk, Justyna; Zalewska-Piątek, Beata; Bruździak, Piotr; Czub, Jacek; Wieczór, Miłosz; Olszewski, Marcin; Wanarska, Marta; Nowicki, Bogdan; Augustin-Nowacka, Danuta; Piątek, Rafał

2017-01-01

Dr fimbriae are homopolymeric adhesive organelles of uropathogenic Escherichia coli composed of DraE subunits, responsible for the attachment to host cells. These structures are characterized by enormously high stability resulting from the structural properties of an Ig-like fold of DraE. One feature of DraE and other fimbrial subunits that makes them peculiar among Ig-like domain-containing proteins is a conserved disulfide bond that joins their A and B strands. Here, we investigated how this disulfide bond affects the stability and folding/unfolding pathway of DraE. We found that the disulfide bond stabilizes self-complemented DraE (DraE-sc) by ∼50 kJ mol−1 in an exclusively thermodynamic manner, i.e. by lowering the free energy of the native state and with almost no effect on the free energy of the transition state. This finding was confirmed by experimentally determined folding and unfolding rate constants of DraE-sc and a disulfide bond-lacking DraE-sc variant. Although the folding of both proteins exhibited similar kinetics, the unfolding rate constant changed upon deletion of the disulfide bond by 10 orders of magnitude, from ∼10−17 s−1 to 10−7 s−1. Molecular simulations revealed that unfolding of the disulfide bond-lacking variant is initiated by strands A or G and that disulfide bond-mediated joining of strand A to the core strand B cooperatively stabilizes the whole protein. We also show that the disulfide bond in DraE is recognized by the DraB chaperone, indicating a mechanism that precludes the incorporation of less stable, non-oxidized DraE forms into the fimbriae. PMID:28739804

Food protein-stabilized nanoemulsions as potential delivery systems for poorly water-soluble drugs: preparation, in vitro characterization, and pharmacokinetics in rats

PubMed Central

He, Wei; Tan, Yanan; Tian, Zhiqiang; Chen, Lingyun; Hu, Fuqiang; Wu, Wei

2011-01-01

Nanoemulsions stabilized by traditional emulsifiers raise toxicological concerns for long-term treatment. The present work investigates the potential of food proteins as safer stabilizers for nanoemulsions to deliver hydrophobic drugs. Nanoemulsions stabilized by food proteins (soybean protein isolate, whey protein isolate, β-lactoglobulin) were prepared by high-pressure homogenization. The toxicity of the nanoemulsions was tested in Caco-2 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide viability assay. In vivo absorption in rats was also evaluated. Food protein-stabilized nanoemulsions, with small particle size and good size distribution, exhibited better stability and biocompatibility compared with nanoemulsions stabilized by traditional emulsifiers. Moreover, β-lactoglobulin had a better emulsifying capacity and biocompatibility than the other two food proteins. The pancreatic degradation of the proteins accelerated drug release. It is concluded that an oil/water nanoemulsion system with good biocompatibility can be prepared by using food proteins as emulsifiers, allowing better and more rapid absorption of lipophilic drugs. PMID:21468355

Food protein-stabilized nanoemulsions as potential delivery systems for poorly water-soluble drugs: preparation, in vitro characterization, and pharmacokinetics in rats.

PubMed

He, Wei; Tan, Yanan; Tian, Zhiqiang; Chen, Lingyun; Hu, Fuqiang; Wu, Wei

2011-01-01

Nanoemulsions stabilized by traditional emulsifiers raise toxicological concerns for long-term treatment. The present work investigates the potential of food proteins as safer stabilizers for nanoemulsions to deliver hydrophobic drugs. Nanoemulsions stabilized by food proteins (soybean protein isolate, whey protein isolate, β-lactoglobulin) were prepared by high-pressure homogenization. The toxicity of the nanoemulsions was tested in Caco-2 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide viability assay. In vivo absorption in rats was also evaluated. Food protein-stabilized nanoemulsions, with small particle size and good size distribution, exhibited better stability and biocompatibility compared with nanoemulsions stabilized by traditional emulsifiers. Moreover, β-lactoglobulin had a better emulsifying capacity and biocompatibility than the other two food proteins. The pancreatic degradation of the proteins accelerated drug release. It is concluded that an oil/water nanoemulsion system with good biocompatibility can be prepared by using food proteins as emulsifiers, allowing better and more rapid absorption of lipophilic drugs.

Spray dried microparticles of chia oil using emulsion stabilized by whey protein concentrate and pectin by electrostatic deposition.

PubMed

Noello, C; Carvalho, A G S; Silva, V M; Hubinger, M D

2016-11-01

Chia seed oil has a high content of α-linolenic acid (60%) and linoleic acid (20%). Use of this oil in different products is limited due to its liquid state, and the presence of insaturation is a trigger for oxidation. In this context, to facilitate the incorporation of chia oil in food products and increase its protection against oxidation, the aim of this work was to produce chia oil microparticles by spray drying using emulsions stabilized by whey protein concentrate (ζ-potential +13.4 at pH3.8) and pectin (ζ-potential -40.4 at pH3.8) through the electrostatic layer-by-layer deposition technique and emulsions prepared with only whey protein concentrate. Emulsions stabilized by whey protein concentrate and stabilized by whey protein concentrate-pectin were prepared using maltodextrin (10 DE) and modified starch (Hi-Cap® 100). They were characterized in relation to stability, droplet size, ζ-Potential and optical microscopy. The microparticles were characterized in relation to moisture content, water activity, particle size, microstructure and oxidative stability by the Rancimat method. Emulsions stabilized by whey protein concentrate-pectin with added maltodextrin 10 DE and emulsions stabilized by whey protein concentrate with added modified starch (Hi-Cap® 100) were stable after 24h. Emulsions stabilized by whey protein concentrate and by whey protein concentrate-pectin showed droplets with mean diameter ranging from 0.80 to 1.31μm, respectively and ζ-potential varying from -6.9 to -27.43mV, respectively. After spray drying, the microparticles showed an mean diameter ranging from 7.00 to 9.00μm. All samples presented high encapsulation efficiency values, above 99%. Microparticles produced with modified starch showed a smoother spherical surface than particles with maltodextrin 10 DE, which presented a wrinkled surface. All microparticles exhibited higher oxidative stability than chia oil in pure form. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mutations of Glucose-6-Phosphate Dehydrogenase Durham, Santa-Maria and A+ Variants Are Associated with Loss Functional and Structural Stability of the Protein

PubMed Central

Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Enríquez-Flores, Sergio; De la Mora-De la Mora, Ignacio; González-Valdez, Abigail; García-Torres, Itzhel; Martínez-Rosas, Víctor; Sierra-Palacios, Edgar; Lazcano-Pérez, Fernando; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

2015-01-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in the world. More than 160 mutations causing the disease have been identified, but only 10% of these variants have been studied at biochemical and biophysical levels. In this study we report on the functional and structural characterization of three naturally occurring variants corresponding to different classes of disease severity: Class I G6PD Durham, Class II G6PD Santa Maria, and Class III G6PD A+. The results showed that the G6PD Durham (severe deficiency), and the G6PD Santa Maria and A+ (less severe deficiency) (Class I, II and III, respectively) affect the catalytic efficiency of these enzymes, are more sensitive to temperature denaturing, and affect the stability of the overall protein when compared to the wild type WT-G6PD. In the variants, the exposure of more and buried hydrophobic pockets was induced and monitored with 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence, directly affecting the compaction of structure at different levels and probably reducing the stability of the protein. The degree of functional and structural perturbation by each variant correlates with the clinical severity reported in different patients. PMID:26633385

The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity.

PubMed

Ando, Maya; Fiesel, Fabienne C; Hudec, Roman; Caulfield, Thomas R; Ogaki, Kotaro; Górka-Skoczylas, Paulina; Koziorowski, Dariusz; Friedman, Andrzej; Chen, Li; Dawson, Valina L; Dawson, Ted M; Bu, Guojun; Ross, Owen A; Wszolek, Zbigniew K; Springer, Wolfdieter

2017-04-24

Mutations in PINK1 and PARKIN are the most common causes of recessive early-onset Parkinson's disease (EOPD). Together, the mitochondrial ubiquitin (Ub) kinase PINK1 and the cytosolic E3 Ub ligase PARKIN direct a complex regulated, sequential mitochondrial quality control. Thereby, damaged mitochondria are identified and targeted to degradation in order to prevent their accumulation and eventually cell death. Homozygous or compound heterozygous loss of either gene function disrupts this protective pathway, though at different steps and by distinct mechanisms. While structure and function of PARKIN variants have been well studied, PINK1 mutations remain poorly characterized, in particular under endogenous conditions. A better understanding of the exact molecular pathogenic mechanisms underlying the pathogenicity is crucial for rational drug design in the future. Here, we characterized the pathogenicity of the PINK1 p.I368N mutation on the clinical and genetic as well as on the structural and functional level in patients' fibroblasts and in cell-based, biochemical assays. Under endogenous conditions, PINK1 p.I368N is expressed, imported, and N-terminally processed in healthy mitochondria similar to PINK1 wild type (WT). Upon mitochondrial damage, however, full-length PINK1 p.I368N is not sufficiently stabilized on the outer mitochondrial membrane (OMM) resulting in loss of mitochondrial quality control. We found that binding of PINK1 p.I368N to the co-chaperone complex HSP90/CDC37 is reduced and stress-induced interaction with TOM40 of the mitochondrial protein import machinery is abolished. Analysis of a structural PINK1 p.I368N model additionally suggested impairments of Ub kinase activity as the ATP-binding pocket was found deformed and the substrate Ub was slightly misaligned within the active site of the kinase. Functional assays confirmed the lack of Ub kinase activity. Here we demonstrated that mutant PINK1 p.I368N can not be stabilized on the OMM upon

Independent of Their Localization in Protein the Hydrophobic Amino Acid Residues Have No Effect on the Molten Globule State of Apomyoglobin and the Disulfide Bond on the Surface of Apomyoglobin Stabilizes This Intermediate State

PubMed Central

Melnik, Tatiana N.; Majorina, Maria A.; Larina, Daria S.; Kashparov, Ivan A.; Samatova, Ekaterina N.; Glukhov, Anatoly S.; Melnik, Bogdan S.

2014-01-01

At present it is unclear which interactions in proteins reveal the presence of intermediate states, their stability and formation rate. In this study, we have investigated the effect of substitutions of hydrophobic amino acid residues in the hydrophobic core of protein and on its surface on a molten globule type intermediate state of apomyoglobin. It has been found that independent of their localization in protein, substitutions of hydrophobic amino acid residues do not affect the stability of the molten globule state of apomyoglobin. It has been shown also that introduction of a disulfide bond on the protein surface can stabilize the molten globule state. However in the case of apomyoglobin, stabilization of the intermediate state leads to relative destabilization of the native state of apomyoglobin. The result obtained allows us not only to conclude which mutations can have an effect on the intermediate state of the molten globule type, but also explains why the introduction of a disulfide bond (which seems to “strengthen” the protein) can result in destabilization of the protein native state of apomyoglobin. PMID:24892675

Independent of their localization in protein the hydrophobic amino acid residues have no effect on the molten globule state of apomyoglobin and the disulfide bond on the surface of apomyoglobin stabilizes this intermediate state.

PubMed

Melnik, Tatiana N; Majorina, Maria A; Larina, Daria S; Kashparov, Ivan A; Samatova, Ekaterina N; Glukhov, Anatoly S; Melnik, Bogdan S

2014-01-01

At present it is unclear which interactions in proteins reveal the presence of intermediate states, their stability and formation rate. In this study, we have investigated the effect of substitutions of hydrophobic amino acid residues in the hydrophobic core of protein and on its surface on a molten globule type intermediate state of apomyoglobin. It has been found that independent of their localization in protein, substitutions of hydrophobic amino acid residues do not affect the stability of the molten globule state of apomyoglobin. It has been shown also that introduction of a disulfide bond on the protein surface can stabilize the molten globule state. However in the case of apomyoglobin, stabilization of the intermediate state leads to relative destabilization of the native state of apomyoglobin. The result obtained allows us not only to conclude which mutations can have an effect on the intermediate state of the molten globule type, but also explains why the introduction of a disulfide bond (which seems to "strengthen" the protein) can result in destabilization of the protein native state of apomyoglobin.

Effect of hydrogen peroxide on improving the heat stability of whey protein isolate solutions.

PubMed

Sutariya, Suresh; Patel, Hasmukh

2017-05-15

Whey protein isolate (WPI) solutions (12.8%w/w protein) were treated with varying concentrations of H 2 O 2 in the range of 0-0.144 H 2 O 2 to protein ratios (HTPR) by the addition of the required quantity of H 2 O 2 and deionized water. The samples were analyzed for heat stability, rheological properties, denaturation level of β-lactoglobulin (β-LG) and α-lactalbumin (α-LA). The samples treated with H 2 O 2 concentration >0.072 (HTPR) showed significant improvement in the heat stability, and decreased whey protein denaturation and aggregation. The WPI solution treated with H 2 O 2 (>0.072 HTPR) remained in the liquid state after heat treatment at 120°C, whereas the control samples formed gel upon heat treatment. Detailed analysis of these samples suggested that the improvement in the heat stability of H 2 O 2 treated WPI solution was attributed to the significant reduction in the sulfhydryl-disulfide interchange reaction during denaturation of β-LG and α-LA. Copyright © 2016 Elsevier Ltd. All rights reserved.

The amino terminal end determines the stability and assembling capacity of eukaryotic ribosomal stalk proteins P1 and P2.

PubMed

Camargo, Hendricka; Nusspaumer, Gretel; Abia, David; Briceño, Verónica; Remacha, Miguel; Ballesta, Juan P G

2011-05-01

The eukaryotic ribosomal proteins P1 and P2 bind to protein P0 through their N-terminal domain to form the essential ribosomal stalk. A mutational analysis points to amino acids at positions 2 and 3 as determinants for the drastic difference of Saccharomyces cerevisiae P1 and P2 half-life, and suggest different degradation mechanisms for each protein type. Moreover, the capacity to form P1/P2 heterodimers is drastically affected by mutations in the P2β four initial amino acids, while these mutations have no effect on P1β. Binding of P2β and, to a lesser extent, P1β to the ribosome is also seriously affected showing the high relevance of the amino acids in the first turn of the NTD α-helix 1 for the stalk assembly. The negative effect of some mutations on ribosome binding can be reversed by the presence of the second P1/P2 couple in the ribosome, indicating a stabilizing structural influence between the two heterodimers. Unexpectedly, some mutations totally abolish heterodimer formation but allow significant ribosome binding and, therefore, a previous P1 and P2 association seems not to be an absolute requirement for stalk assembly. Homology modeling of the protein complexes suggests that the mutated residues can affect the overall protein conformation. © The Author(s) 2011. Published by Oxford University Press.

Nitrous Oxide Emissions Affected by Biochar and Nitrogen Stabilizers

NASA Astrophysics Data System (ADS)

Gao, S.; Cai, Z.; Xu, M.

2016-12-01

Both biochar and N fertilizer stabilizers (N transformation inhibitors) are potential strategies to reduce nitrous oxide (N2O) emissions from fertilization, but the mechanisms and/or N transformation processes affecting the N dynamics are not fully understood. This research investigated N2O emissions and N transformations in soil amended with biochar and N transformation inhibitors. The soil was a sandy loam soil and adjusted to 10% soil water content and incubated at 25oC. Biochar amendment at 1% (w/w), Agrotain® Ultra (urease inhibitor), Agrotain® Plus (urease and nitrification inhibitor), and N-Serve® 24 (nitrification inhibitor) as well as another potential nitrification inhibitor, potassium thiosulfate (KTS), at 0.25-1:1 K2O/N ratios (w/w) were tested. Emissions of N2O, soil mineral N species change, and soil pH were determined for 35 days after fertilizers were applied. Biochar, Agrotain® Ultra or Plus, or N-Serve® 24 all effectively reduced N2O emissions by more than 60% as compared to no amendment control. The KTS, however, was only effective in reducing N2O emissions at a high ratio (1:1 K2O/N, w/w). There was a strong correlation between N2O emission and the concentration of nitrite (NO2-) in soil but not other mineral species. All the amendments showed that their effects on N transformation and N2O emissions were completed within a few weeks after application. Laboratory analysis indicated that biochar affected the N dynamics most likely via adsorption of ammonium (NH4+) and the inhibitors by affecting N transformation rate. This research has gained further understanding on how biochar and N stabilizers affect N2O emissions and the knowledge can assist in developing mitigation strategies.

Stage structure alters how complexity affects stability of ecological networks

USGS Publications Warehouse

Rudolf, V.H.W.; Lafferty, Kevin D.

2011-01-01

Resolving how complexity affects stability of natural communities is of key importance for predicting the consequences of biodiversity loss. Central to previous stability analysis has been the assumption that the resources of a consumer are substitutable. However, during their development, most species change diets; for instance, adults often use different resources than larvae or juveniles. Here, we show that such ontogenetic niche shifts are common in real ecological networks and that consideration of these shifts can alter which species are predicted to be at risk of extinction. Furthermore, niche shifts reduce and can even reverse the otherwise stabilizing effect of complexity. This pattern arises because species with several specialized life stages appear to be generalists at the species level but act as sequential specialists that are hypersensitive to resource loss. These results suggest that natural communities are more vulnerable to biodiversity loss than indicated by previous analyses.

An amphipathic polypeptide derived from poly-γ-glutamic acid for the stabilization of membrane proteins

PubMed Central

Han, Seong-Gu; Na, Jung-Hyun; Lee, Won-Kyu; Park, Dongkook; Oh, Jihye; Yoon, Sung-Ho; Lee, Cheng-Kang; Sung, Moon-Hee; Shin, Yeon-Kyun; Yu, Yeon Gyu

2014-01-01

Difficulties in the extraction of membrane proteins from cell membrane and their solubilization in native conformations have hindered their structural and biochemical analysis. To overcome these difficulties, an amphipathic polypeptide was synthesized by the conjugation of octyl and glucosyl groups to the carboxyl groups of poly-γ-glutamic acid (PGA). This polymer, called amphipathic PGA (APG), self-assembles as mono-disperse oligomers consisted of 4–5 monomers. APG shows significantly low value of critical micelle concentration and stabilization activity toward membrane proteins. Most of the sodium dodecyl sulfate (SDS)-solubilized membrane proteins from Escherichia coli remain soluble state in the presence of APG even after the removal of SDS. In addition, APG stabilizes purified 7 transmembrane proteins such as bacteriorhodopsin and human endothelin receptor Type A (ETA) in their active conformations. Furthermore, ETA in complex with APG is readily inserted into liposomes without disrupting the integrity of liposomes. These properties of APG can be applied to overcome the difficulties in the stabilization and reconstitution of membrane proteins. PMID:25283538

The role of surface electrostatics on the stability, function and regulation of human cystathionine β-synthase, a complex multidomain and oligomeric protein.

PubMed

Pey, Angel L; Majtan, Tomas; Kraus, Jan P

2014-09-01

Human cystathionine β-synthase (hCBS) is a key enzyme of sulfur amino acid metabolism, controlling the commitment of homocysteine to the transsulfuration pathway and antioxidant defense. Mutations in hCBS cause inherited homocystinuria (HCU), a rare inborn error of metabolism characterized by accumulation of toxic homocysteine in blood and urine. hCBS is a complex multidomain and oligomeric protein whose activity and stability are independently regulated by the binding of S-adenosyl-methionine (SAM) to two different types of sites at its C-terminal regulatory domain. Here we study the role of surface electrostatics on the complex regulation and stability of hCBS using biophysical and biochemical procedures. We show that the kinetic stability of the catalytic and regulatory domains is significantly affected by the modulation of surface electrostatics through noticeable structural and energetic changes along their denaturation pathways. We also show that surface electrostatics strongly affect SAM binding properties to those sites responsible for either enzyme activation or kinetic stabilization. Our results provide new insight into the regulation of hCBS activity and stability in vivo with implications for understanding HCU as a conformational disease. We also lend experimental support to the role of electrostatic interactions in the recently proposed binding modes of SAM leading to hCBS activation and kinetic stabilization. Copyright © 2014 Elsevier B.V. All rights reserved.

Universal distribution of mutational effects on protein stability, uncoupling of protein robustness from sequence evolution and distinct evolutionary modes of prokaryotic and eukaryotic proteins

NASA Astrophysics Data System (ADS)

Faure, Guilhem; Koonin, Eugene V.

2015-05-01

Robustness to destabilizing effects of mutations is thought of as a key factor of protein evolution. The connections between two measures of robustness, the relative core size and the computationally estimated effect of mutations on protein stability (ΔΔG), protein abundance and the selection pressure on protein-coding genes (dN/dS) were analyzed for the organisms with a large number of available protein structures including four eukaryotes, two bacteria and one archaeon. The distribution of the effects of mutations in the core on protein stability is universal and indistinguishable in eukaryotes and bacteria, centered at slightly destabilizing amino acid replacements, and with a heavy tail of more strongly destabilizing replacements. The distribution of mutational effects in the hyperthermophilic archaeon Thermococcus gammatolerans is significantly shifted toward strongly destabilizing replacements which is indicative of stronger constraints that are imposed on proteins in hyperthermophiles. The median effect of mutations is strongly, positively correlated with the relative core size, in evidence of the congruence between the two measures of protein robustness. However, both measures show only limited correlations to the expression level and selection pressure on protein-coding genes. Thus, the degree of robustness reflected in the universal distribution of mutational effects appears to be a fundamental, ancient feature of globular protein folds whereas the observed variations are largely neutral and uncoupled from short term protein evolution. A weak anticorrelation between protein core size and selection pressure is observed only for surface residues in prokaryotes but a stronger anticorrelation is observed for all residues in eukaryotic proteins. This substantial difference between proteins of prokaryotes and eukaryotes is likely to stem from the demonstrable higher compactness of prokaryotic proteins.

Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin

PubMed Central

Oulhen, Nathalie; Wessel, Gary M.

2016-01-01

Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3′UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability. PMID:27424271

Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin.

PubMed

Oulhen, Nathalie; Wessel, Gary M

2016-10-01

Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3'UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability. Copyright © 2016 Elsevier Inc. All rights reserved.

27-Hydroxycholesterol increases Myc protein stability via suppressing PP2A, SCP1 and FBW7 transcription in MCF-7 breast cancer cells

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

Ma, Li-Ming; Liang, Zi-Rui; Zhou, Ke-Ren

27-hydroxycholesterol (27-HC), the most abundant metabolite of cholesterol, is a risk factor for breast cancer. It can increase the proliferation of breast cancer cells and promote the metastasis of breast tumours in mouse models. Myc is a critical oncoprotein overexpressed in breast cancer. However, whether 27-HC affects Myc expression has not been reported. In the current study, we aimed to investigate the effects of 27-HC on Myc and the underlying mechanisms in MCF-7 breast cancer cells. Our data demonstrated that 27-HC activated Myc via increasing its protein stability. Three key negative modulators of Myc protein stability, PP2A, SCP1 and FBW7,more » were suppressed by 27-HC at the transcriptional level. We performed a data-mining analysis of the chromatin immunoprecipitation with next-generation DNA sequencing (ChIP-Seq) data in the ChIPBase, and discovered that a number of putative transcription factors (TFs), including Myc itself, were involved in the transcriptional regulation of PP2A, SCP1 and FBW7. Our results provide a novel mechanistic insight into the activation of Myc by 27-HC via transcriptional repression of PP2A, SCP1 and FBW7 to increase Myc protein stability in breast cancer cells. - Highlights: • 27-Hydroxycholesterol (27-HC) activates Myc via increasing its protein stability. • 27-HC inhibits PP2A and SCP1 transcription to block pS62-Myc dephosphorylation. • 27-HC suppresses FBW7 transcription to prevent pT58-Myc degradation.« less

The Linear Interaction Energy Method for the Prediction of Protein Stability Changes Upon Mutation

PubMed Central

Wickstrom, Lauren; Gallicchio, Emilio; Levy, Ronald M.

2011-01-01

The coupling of protein energetics and sequence changes is a critical aspect of computational protein design, as well as for the understanding of protein evolution, human disease, and drug resistance. In order to study the molecular basis for this coupling, computational tools must be sufficiently accurate and computationally inexpensive enough to handle large amounts of sequence data. We have developed a computational approach based on the linear interaction energy (LIE) approximation to predict the changes in the free energy of the native state induced by a single mutation. This approach was applied to a set of 822 mutations in 10 proteins which resulted in an average unsigned error of 0.82 kcal/mol and a correlation coefficient of 0.72 between the calculated and experimental ΔΔG values. The method is able to accurately identify destabilizing hot spot mutations however it has difficulty in distinguishing between stabilizing and destabilizing mutations due to the distribution of stability changes for the set of mutations used to parameterize the model. In addition, the model also performs quite well in initial tests on a small set of double mutations. Based on these promising results, we can begin to examine the relationship between protein stability and fitness, correlated mutations, and drug resistance. PMID:22038697

Does soy protein affect circulating levels of unbound IGF-1?

PubMed

Messina, Mark; Magee, Pamela

2018-03-01

Despite the enormous amount of research that has been conducted on the role of soyfoods in the prevention and treatment of chronic disease, the mechanisms by which soy exerts its physiological effects are not fully understood. The clinical data show that neither soyfoods nor soy protein nor isoflavones affect circulating levels of reproductive hormones in men or women. However, some research suggests that soy protein, but not isoflavones, affects insulin-like growth factor I (IGF-1). Since IGF-1 may have wide-ranging physiological effects, we sought to determine the effect of soy protein on IGF-1 and its major binding protein insulin-like growth factor-binding protein (IGFBP-3). Six clinical studies were identified that compared soy protein with a control protein, albeit only two studies measured IGFBP-3 in addition to IGF-1. Although the data are difficult to interpret because of the different experimental designs employed, there is some evidence that large amounts of soy protein (>25 g/day) modestly increase IGF-1 levels above levels observed with the control protein. The clinical data suggest that a decision to incorporate soy into the diet should not be based on its possible effects on IGF-1.

Molecular Dynamics Driven Design of pH-Stabilized Mutants of MNEI, a Sweet Protein

PubMed Central

Picone, Delia

2016-01-01

MNEI is a single chain derivative of monellin, a plant protein that can interact with the human sweet taste receptor, being therefore perceived as sweet. This unusual physiological activity makes MNEI a potential template for the design of new sugar replacers for the food and beverage industry. Unfortunately, applications of MNEI have been so far limited by its intrinsic sensitivity to some pH and temperature conditions, which could occur in industrial processes. Changes in physical parameters can, in fact, lead to irreversible protein denaturation, as well as aggregation and precipitation. It has been previously shown that the correlation between pH and stability in MNEI derives from the presence of a single glutamic residue in a hydrophobic pocket of the protein. We have used molecular dynamics to study the consequences, at the atomic level, of the protonation state of such residue and have identified the network of intramolecular interactions responsible for MNEI stability at acidic pH. Based on this information, we have designed a pH-independent, stabilized mutant of MNEI and confirmed its increased stability by both molecular modeling and experimental techniques. PMID:27340829

Molecular Dynamics Driven Design of pH-Stabilized Mutants of MNEI, a Sweet Protein.

PubMed

Leone, Serena; Picone, Delia

2016-01-01

MNEI is a single chain derivative of monellin, a plant protein that can interact with the human sweet taste receptor, being therefore perceived as sweet. This unusual physiological activity makes MNEI a potential template for the design of new sugar replacers for the food and beverage industry. Unfortunately, applications of MNEI have been so far limited by its intrinsic sensitivity to some pH and temperature conditions, which could occur in industrial processes. Changes in physical parameters can, in fact, lead to irreversible protein denaturation, as well as aggregation and precipitation. It has been previously shown that the correlation between pH and stability in MNEI derives from the presence of a single glutamic residue in a hydrophobic pocket of the protein. We have used molecular dynamics to study the consequences, at the atomic level, of the protonation state of such residue and have identified the network of intramolecular interactions responsible for MNEI stability at acidic pH. Based on this information, we have designed a pH-independent, stabilized mutant of MNEI and confirmed its increased stability by both molecular modeling and experimental techniques.

Analysis of SAT Type Foot-And-Mouth Disease Virus Capsid Proteins and the Identification of Putative Amino Acid Residues Affecting Virus Stability

PubMed Central

Maree, Francois F.; Blignaut, Belinda; de Beer, Tjaart A. P.; Rieder, Elizabeth

2013-01-01

Foot-and-mouth disease virus (FMDV) initiates infection by adhering to integrin receptors on target cells, followed by cell entry and disassembly of the virion through acidification within endosomes. Mild heating of the virions also leads to irreversible dissociation into pentamers, a characteristic linked to reduced vaccine efficacy. In this study, the structural stability of intra- and inter-serotype chimeric SAT2 and SAT3 virus particles to various conditions including low pH, mild temperatures or high ionic strength, was compared. Our results demonstrated that while both the SAT2 and SAT3 infectious capsids displayed different sensitivities in a series of low pH buffers, their stability profiles were comparable at high temperatures or high ionic strength conditions. Recombinant vSAT2 and intra-serotype chimeric viruses were used to map the amino acid differences in the capsid proteins of viruses with disparate low pH stabilities. Four His residues at the inter-pentamer interface were identified that change protonation states at pH 6.0. Of these, the H145 of VP3 appears to be involved in interactions with A141 in VP3 and K63 in VP2, and may be involved in orientating H142 of VP3 for interaction at the inter-pentamer interfaces. PMID:23717387

PPAR-γ agonist stabilizes KLF4 protein via activating Akt signaling and reducing KLF4 ubiquitination

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

Sun, Yan; Zheng, Bin; Zhang, Xin-hua

2014-01-10

Highlights: •PPAR-γ increases KLF4 protein level but does not influence KLF4 gene transcription. •The increase of KLF4 protein levels induced by pioglitazone is PPAR-γ-dependent. •Pioglitazone stabilizes KLF4 protein via activating Akt signaling and reducing KLF4 ubiquitination. -- Abstract: Peroxisome proliferator activated receptor γ (PPAR-γ) plays important roles in cell cycle regulation, differentiation and apoptosis. Krüppel-like factor 4 (KLF4) modulates vascular smooth muscle cell (VSMC) phenotype. Both KLF4 and PPAR-γ are involved in VSMC proliferation and differentiation. However, the actual relationship between KLF4 and PPAR-γ in VSMCs is not clear. In this study, we found that PPAR-γ agonist pioglitazone increases KLF4more » protein levels but does not influence KLF4 gene transcription. PPAR-γ overexpression increases, while PPAR-γ knockdown reduces KLF4 expression, suggesting that the increase in KLF4 protein levels induced by pioglitazone is PPAR-γ-dependent. Further study showed that pioglitazone enhances KLF4 protein stability through reducing KLF4 ubiquitination. Furthermore, we demonstrated that stabilization of KLF4 by pioglitazone was related to the activation of Akt signaling pathway. Taken together, we revealed that PPAR-γ agonist pioglitazone stabilizes KLF4 protein via activating Akt signaling and reducing KLF4 ubiquitination, providing further insights into PPAR-γ and KLF4 in regulating each other’s expression in VSMCs.« less

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.

[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.

Correlation between mechanical behavior of protein films at the air/water interface and intrinsic stability of protein molecules.

PubMed

Martin, Anneke H; Cohen Stuart, Martien A; Bos, Martin A; van Vliet, Ton

2005-04-26

The relation between mechanical film properties of various adsorbed protein layers at the air/water interface and intrinsic stability of the corresponding proteins is discussed. Mechanical film properties were determined by surface deformation in shear and dilation. In shear, fracture stress, sigma(f), and fracture strain, gamma(f), were determined, as well as the relaxation behavior after macroscopic fracture. The dilatational measurements were performed in a Langmuir trough equipped with an infra-red reflection absorption spectroscopy (IRRAS) accessory. During compression and relaxation of the surface, the surface pressure, Pi, and adsorbed amount, Gamma (determined from the IRRAS spectra), were determined simultaneously. In addition, IRRAS spectra revealed information on conformational changes in terms of secondary structure. Possible correlations between macroscopic film properties and intrinsic stability of the proteins were determined and discussed in terms of molecular dimensions of single proteins and interfacial protein films. Molecular properties involved the area per protein molecule at Pi approximately 0 mN/m (A(0)), A(0)/M (M = molecular weight) and the maximum slope of the Pi-Gamma curves (dPi/dGamma). The differences observed in mechanical properties and relaxation behavior indicate that the behavior of a protein film subjected to large deformation may vary widely from predominantly viscous (yielding) to more elastic (fracture). This transition is also observed in gradual changes in A(0)/M. It appeared that in general protein layers with high A(0)/M have a high gamma(f) and behave more fluidlike, whereas solidlike behavior is characterized by low A(0)/M and low gamma(f). Additionally, proteins with a low A(0)/M value have a low adaptability in changing their conformation upon adsorption at the air/water interface. Both results support the conclusion that the hardness (internal cohesion) of protein molecules determines predominantly the mechanical

Physicochemical Properties of Whey-Protein-Stabilized Astaxanthin Nanodispersion and Its Transport via a Caco-2 Monolayer.

PubMed

Shen, Xue; Zhao, Changhui; Lu, Jing; Guo, Mingruo

2018-02-14

Astaxanthin nanodispersion was prepared using whey protein isolate (WPI) and polymerized whey protein (PWP) through an emulsification-evaporation technique. The physicochemical properties of the astaxanthin nanodispersion were evaluated, and the transport of astaxanthin was assessed using a Caco-2 cell monolayer model. The astaxanthin nanodispersions stabilized by WPI and PWP (2.5%, w/w) had a small particle size (121 ± 4.9 and 80.4 ± 5.9 nm, respectively), negative ζ potential (-19.3 ± 1.5 and -35.0 ± 2.2 mV, respectively), and high encapsulation efficiency (92.1 ± 2.9 and 93.5 ± 2.4%, respectively). Differential scanning calorimetry curves indicated that amorphous astaxanthin existed in both astaxanthin nanodispersions. Whey-protein-stabilized astaxanthin nanodispersion showed resistance to pepsin digestion but readily released astaxanthin after trypsin digestion. The nanodispersions showed no cytotoxicity to Caco-2 cells at a protein concentration below 10 mg/mL. WPI- and PWP-stabilized nanodispersions improved the apparent permeability coefficient (P app ) of Caco-2 cells to astaxanthin by 10.3- and 16.1-fold, respectively. The results indicated that whey-protein-stabilized nanodispersion is a good vehicle to deliver lipophilic bioactive compounds, such as astaxanthin, and to improve their bioavailability.

Activation of Akt, not connexin 43 protein ubiquitination, regulates gap junction stability.

PubMed

Dunn, Clarence A; Su, Vivian; Lau, Alan F; Lampe, Paul D

2012-01-20

The pore-forming gap junctional protein connexin 43 (Cx43) has a short (1-3 h) half-life in cells in tissue culture and in whole tissues. Although critical for cellular function in all tissues, the process of gap junction turnover is not well understood because treatment of cells with a proteasomal inhibitor results in larger gap junctions but little change in total Cx43 protein whereas lysosomal inhibitors increase total, mostly nonjunctional Cx43. To better understand turnover and identify potential sites of Cx43 ubiquitination, we prepared constructs of Cx43 with different lysines converted to arginines. However, when transfected into cells, a mutant version of Cx43 with all lysines converted to arginines behaved similarly to wild type in the presence of proteasomal and lysosomal inhibitors, indicating that ubiquitination of Cx43 did not appear to be playing a role in gap junction stability. Through the use of inhibitors and dominant negative constructs, we found that Akt (protein kinase B) activity controlled gap junction stability and was necessary to form larger stable gap junctions. Akt activation was increased upon proteasomal inhibition and resulted in phosphorylation of Cx43 at Akt phosphorylation consensus sites. Thus, we conclude that Cx43 ubiquitination is not necessary for the regulation of Cx43 turnover; rather, Akt activity, probably through direct phosphorylation of Cx43, controls gap junction stability. This linkage of a kinase involved in controlling cell survival and growth to gap junction stability may mechanistically explain how gap junctions and Akt play similar regulatory roles.

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.

Whey protein/polysaccharide-stabilized emulsions: Effect of polymer type and pH on release and topical delivery of salicylic acid.

PubMed

Combrinck, Johann; Otto, Anja; du Plessis, Jeanetta

2014-06-01

Emulsions are widely used as topical formulations in the pharmaceutical and cosmetic industries. They are thermodynamically unstable and require emulsifiers for stabilization. Studies have indicated that emulsifiers could affect topical delivery of actives, and this study was therefore designed to investigate the effects of different polymers, applied as emulsifiers, as well as the effects of pH on the release and topical delivery of the active. O/w emulsions were prepared by the layer-by-layer technique, with whey protein forming the first layer around the oil droplets, while either chitosan or carrageenan was subsequently adsorbed to the protein at the interface. Additionally, the emulsions were prepared at three different pH values to introduce different charges to the polymers. The active ingredient, salicylic acid, was incorporated into the oil phase of the emulsions. Physical characterization of the resulting formulations, i.e., droplet size, zeta potential, stability, and turbidity in the water phase, was performed. Release studies were conducted, after which skin absorption studies were performed on the five most stable emulsions, by using Franz type diffusion cells and utilizing human, abdominal skin membranes. It was found that an increase in emulsion droplet charge could negatively affect the release of salicylic acid from these formulations. Contrary, positively charged emulsion droplets were found to enhance dermal and transdermal delivery of salicylic acid from emulsions. It was hypothesized that electrostatic complex formation between the emulsifier and salicylic acid could affect its release, whereas electrostatic interaction between the emulsion droplets and skin could influence dermal/transdermal delivery of the active.

Interaction of arginine, lysine, and guanidine with surface residues of lysozyme: implication to protein stability.

PubMed

Shah, Dhawal; Shaikh, Abdul Rajjak

2016-01-01

Additives are widely used to suppress aggregation of therapeutic proteins. However, the molecular mechanisms of effect of additives to stabilize proteins are still unclear. To understand this, we herein perform molecular dynamics simulations of lysozyme in the presence of three commonly used additives: arginine, lysine, and guanidine. These additives have different effects on stability of proteins and have different structures with some similarities; arginine and lysine have aliphatic side chain, while arginine has a guanidinium group. We analyze atomic contact frequencies to study the interactions of the additives with individual residues of lysozyme. Contact coefficient, quantified from contact frequencies, is helpful in analyzing the interactions with the guanidine groups as well as aliphatic side chains of arginine and lysine. Strong preference for contacts to the additives (over water) is seen for the acidic followed by polar and the aromatic residues. Further analysis suggests that the hydration layer around the protein surface is depleted more in the presence of arginine, followed by lysine and guanidine. Molecular dynamics simulations also reveal that the internal dynamics of protein, as indicated by the lifetimes of the hydrogen bonds within the protein, changes depending on the additives. Particularly, we note that the side-chain hydrogen-bonding patterns within the protein differ with the additives, with several side-chain hydrogen bonds missing in the presence of guanidine. These results collectively indicate that the aliphatic chain of arginine and lysine plays a critical role in the stabilization of the protein.

In silico prediction of a disease-associated STIL mutant and its affect on the recruitment of centromere protein J (CENPJ).

PubMed

Kumar, Ambuj; Rajendran, Vidya; Sethumadhavan, Rao; Purohit, Rituraj

2012-01-01

Human STIL (SCL/TAL1 interrupting locus) protein maintains centriole stability and spindle pole localisation. It helps in recruitment of CENPJ (Centromere protein J)/CPAP (centrosomal P4.1-associated protein) and other centrosomal proteins. Mutations in STIL protein are reported in several disorders, especially in deregulation of cell cycle cascades. In this work, we examined the non-synonymous single nucleotide polymorphisms (nsSNPs) reported in STIL protein for their disease association. Different SNP prediction tools were used to predict disease-associated nsSNPs. Our evaluation technique predicted rs147744459 (R242C) as a highly deleterious disease-associated nsSNP and its interaction behaviour with CENPJ protein. Molecular modelling, docking and molecular dynamics simulation were conducted to examine the structural consequences of the predicted disease-associated mutation. By molecular dynamic simulation we observed structural consequences of R242C mutation which affects interaction of STIL and CENPJ functional domains. The result obtained in this study will provide a biophysical insight into future investigations of pathological nsSNPs using a computational platform.

Nanoporous microbead supported bilayers: stability, physical characterization, and incorporation of functional transmembrane proteins.

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

Davis, Ryan W.; Brozik, James A.; Brozik, Susan Marie

2007-03-01

The introduction of functional transmembrane proteins into supported bilayer-based biomimetic systems presents a significant challenge for biophysics. Among the various methods for producing supported bilayers, liposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined. Supported bilayers on porous silica microspheres show a significant increasemore » in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature. Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin (bR) and the human type 3 serotonin receptor (5HT3R) are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency. In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are significantly enhanced through detergent-mediated protein reconstitution. The results of these experiments provide a basis for bulk ionic and fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform.« less

Chemistry and stability of thiol based polyethylene glycol surface coatings on colloidal gold and their relationship to protein adsorption and clearance in vivo

NASA Astrophysics Data System (ADS)

Carpinone, Paul

Nanomaterials have presented a wide range of novel biomedical applications, with particular emphasis placed on advances in imaging and treatment delivery. Of the many particulate nanomaterials researched for biomedical applications, gold is one of the most widely used. Colloidal gold has been of great interest due to its chemical inertness and its ability to perform multiple functions, such as drug delivery, localized heating of tissues (hyperthermia), and imaging (as a contrast agent). It is also readily functionalized through the use of thiols, which spontaneously form sulfur to gold bonds with the surface. Polyethylene glycol (PEG) is the most widely used coating material for these particles as it provides both steric stability to the suspension and protein resistance. These properties extend the circulation time of the particles in blood, and consequently the efficacy of the treatment. Despite widespread use of PEG coated gold particles, the coating chemistry and stability of these particles are largely unknown. The goal of this work was to identify the mechanisms leading to degradation and stability of thiol based polyethylene glycol coatings on gold particles and to relate this behavior to protein adsorption and clearance in vivo. The results indicate that the protective PEG coating is susceptible to sources of oxidation (including dissolved oxygen) and competing adsorbates, among other factors. The quality of commercially available thiolated PEG reagents was also found to play a key role in the quality and protein resistance of the final PEG coating. Analysis of the stability of these coatings indicated that they rapidly degrade under physiological conditions, leading to the onset of protein adsorption when exposed to plasma or blood. Paralleling the protein adsorption behavior and onset of coating degradation observed in vitro, blood clearance of parenterally administered PEG coated particles in mice began after approximately 2h of circulation time. Taken

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...

Kinetic Stability of Proteins in Beans and Peas: Implications for Protein Digestibility, Seed Germination, and Plant Adaptation.

PubMed

Xia, Ke; Pittelli, Sandy; Church, Jennifer; Colón, Wilfredo

2016-10-12

Kinetically stable proteins (KSPs) are resistant to the denaturing detergent sodium dodecyl sulfate (SDS). Such resilience makes KSPs resistant to proteolytic degradation and may have arisen in nature as a mechanism for organismal adaptation and survival against harsh conditions. Legumes are well-known for possessing degradation-resistant proteins that often diminish their nutritional value. Here we applied diagonal two-dimensional (D2D) SDS-polyacrylamide gel electrophoresis (PAGE), a method that allows for the proteomics-level identification of KSPs, to a group of 12 legumes (mostly beans and peas) of agricultural and nutritional importance. Our proteomics results show beans that are more difficult to digest, such as soybean, lima beans, and various common beans, have high contents of KSPs. In contrast, mung bean, red lentil, and various peas that are highly digestible contain low amounts of KSPs. Identified proteins with high kinetic stability are associated with warm-season beans, which germinate at higher temperatures. In contrast, peas and red lentil, which are cool-season legumes, contain low levels of KSPs. Thus, our results show protein kinetic stability is an important factor in the digestibility of legume proteins and may relate to nutrition efficiency, timing of seed germination, and legume resistance to biotic stressors. Furthermore, we show D2D SDS-PAGE is a powerful method that could be applied for determining the abundance and identity of KSPs in engineered and wild legumes and for advancing basic research and associated applications.

Increased helix and protein stability through the introduction of a new tertiary hydrogen bond.

PubMed

Peterson, R W; Nicholson, E M; Thapar, R; Klevit, R E; Scholtz, J M

1999-03-12

In an effort to quantify the importance of hydrogen bonding and alpha-helix formation to protein stability, a capping box motif was introduced into the small phosphocarrier protein HPr. Previous studies had confirmed that Ser46, at the N-cap position of the short helix-B in HPr, serves as an N-cap in solution. Thus, only a single-site mutation was required to produce a canonical S-X-X-E capping box: Lys49 at the N3 position was substituted with a glutamic acid residue. Thermal and chemical denaturation studies on the resulting K49E HPr show that the designed variant is approximately 2 kcal mol-1 more stable than the wild-type protein. However, NMR studies indicate that the side-chain of Glu49 does not participate in the expected capping H-bond interaction, but instead forms a new tertiary H-bond that links helix-B to the four-stranded beta-sheet of HPr. Here, we demonstrate that a strategy in which new non-native H-bonds are introduced can generate proteins with increased stability. We discuss why the original capping box design failed, and compare the energetic consequences of the new tertiary side-chain to main-chain H-bond with a local (helix-capping) side-chain to main-chain H-bond on the protein's global stability. Copyright 1999 Academic Press.

Protein adsorption at the electrified air-water interface: implications on foam stability.

PubMed

Engelhardt, Kathrin; Rumpel, Armin; Walter, Johannes; Dombrowski, Jannika; Kulozik, Ulrich; Braunschweig, Björn; Peukert, Wolfgang

2012-05-22

The surface chemistry of ions, water molecules, and proteins as well as their ability to form stable networks in foams can influence and control macroscopic properties such as taste and texture of dairy products considerably. Despite the significant relevance of protein adsorption at liquid interfaces, a molecular level understanding on the arrangement of proteins at interfaces and their interactions has been elusive. Therefore, we have addressed the adsorption of the model protein bovine serum albumin (BSA) at the air-water interface with vibrational sum-frequency generation (SFG) and ellipsometry. SFG provides specific information on the composition and average orientation of molecules at interfaces, while complementary information on the thickness of the adsorbed layer can be obtained with ellipsometry. Adsorption of charged BSA proteins at the water surface leads to an electrified interface, pH dependent charging, and electric field-induced polar ordering of interfacial H(2)O and BSA. Varying the bulk pH of protein solutions changes the intensities of the protein related vibrational bands substantially, while dramatic changes in vibrational bands of interfacial H(2)O are simultaneously observed. These observations have allowed us to determine the isoelectric point of BSA directly at the electrolyte-air interface for the first time. BSA covered air-water interfaces with a pH near the isoelectric point form an amorphous network of possibly agglomerated BSA proteins. Finally, we provide a direct correlation of the molecular structure of BSA interfaces with foam stability and new information on the link between microscopic properties of BSA at water surfaces and macroscopic properties such as the stability of protein foams.

Weak Links: Stabilizers of Complex Systems from Proteins to Social Networks

NASA Astrophysics Data System (ADS)

Csermely, Peter

Why do women stabilize our societies? Why can we enjoy and understand Shakespeare? Why are fruitflies uniform? Why do omnivorous eating habits aid our survival? Why is Mona Lisa's smile beautiful? -- Is there any answer to these questions? This book shows that the statement: "weak links stabilize complex systems" holds the answers to all of the surprising questions above. The author (recipientof several distinguished science communication prizes) uses weak (low affinity, low probability) interactions as a thread to introduce a vast varietyof networks from proteins to ecosystems.

Positive affect and cognitive control: approach-motivation intensity influences the balance between cognitive flexibility and stability.

PubMed

Liu, Ya; Wang, Zhenhong

2014-05-01

In most prior research, positive affect has been consistently found to promote cognitive flexibility. However, the motivational dimensional model of affect assumes that the influence of positive affect on cognitive processes is modulated by approach-motivation intensity. In the present study, we extended the motivational dimensional model to the domain of cognitive control by examining the effect of low- versus high-approach-motivated positive affect on the balance between cognitive flexibility and stability in an attentional-set-shifting paradigm. Results showed that low-approach-motivated positive affect promoted cognitive flexibility but also caused higher distractibility, whereas high-approach-motivated positive affect enhanced perseverance but simultaneously reduced distractibility. These results suggest that the balance between cognitive flexibility and stability is modulated by the approach-motivation intensity of positive affective states. Therefore, it is essential to incorporate motivational intensity into studies on the influence of affect on cognitive control.

StAR Protein Stability in Y1 and Kin-8 Mouse Adrenocortical Cells.

PubMed

Clark, Barbara J; Hudson, Elizabeth A

2015-03-04

The steroidogenic acute regulatory protein (STAR) protein expression is required for cholesterol transport into mitochondria to initiate steroidogenesis in the adrenal and gonads. STAR is synthesized as a 37 kDa precursor protein which is targeted to the mitochondria and imported and processed to an intra-mitochondrial 30 kDa protein. Tropic hormone stimulation of the cAMP-dependent protein kinase A (PKA) signaling pathway is the major contributor to the transcriptional and post-transcriptional regulation of STAR synthesis. Many studies have focused on the mechanisms of cAMP-PKA mediated control of STAR synthesis while there are few reports on STAR degradation pathways. The objective of this study was to determine the effect of cAMP-PKA-dependent signaling on STAR protein stability. We have used the cAMP-PKA responsive Y1 mouse adrenocortical cells and the PKA-deficient Kin-8 cells to measure STAR phosphorylation and protein half-life. Western blot analysis and standard radiolabeled pulse-chase experiments were used to determine STAR phosphorylation status and protein half-life, respectively. Our data demonstrate that PKA-dependent STAR phosphorylation does not contribute to 30 kDa STAR protein stability in the mitochondria. We further show that inhibition of the 26S proteasome does not block precursor STAR phosphorylation or steroid production in Y1 cells. These data suggest STAR can maintain function and promote steroidogenesis under conditions of proteasome inhibition.

StAR Protein Stability in Y1 and Kin-8 Mouse Adrenocortical Cells

PubMed Central

Clark, Barbara J.; Hudson, Elizabeth A.

2015-01-01

The steroidogenic acute regulatory protein (STAR) protein expression is required for cholesterol transport into mitochondria to initiate steroidogenesis in the adrenal and gonads. STAR is synthesized as a 37 kDa precursor protein which is targeted to the mitochondria and imported and processed to an intra-mitochondrial 30 kDa protein. Tropic hormone stimulation of the cAMP-dependent protein kinase A (PKA) signaling pathway is the major contributor to the transcriptional and post-transcriptional regulation of STAR synthesis. Many studies have focused on the mechanisms of cAMP-PKA mediated control of STAR synthesis while there are few reports on STAR degradation pathways. The objective of this study was to determine the effect of cAMP-PKA-dependent signaling on STAR protein stability. We have used the cAMP-PKA responsive Y1 mouse adrenocortical cells and the PKA-deficient Kin-8 cells to measure STAR phosphorylation and protein half-life. Western blot analysis and standard radiolabeled pulse-chase experiments were used to determine STAR phosphorylation status and protein half-life, respectively. Our data demonstrate that PKA-dependent STAR phosphorylation does not contribute to 30 kDa STAR protein stability in the mitochondria. We further show that inhibition of the 26S proteasome does not block precursor STAR phosphorylation or steroid production in Y1 cells. These data suggest STAR can maintain function and promote steroidogenesis under conditions of proteasome inhibition. PMID:25749137

Stability of halophilic proteins: from dipeptide attributes to discrimination classifier.

PubMed

Zhang, Guangya; Huihua, Ge; Yi, Lin

2013-02-01

To investigate the molecular features responsible for protein halophilicity is of great significance for understanding the structure basis of protein halo-stability and would help to develop a practical strategy for designing halophilic proteins. In this work, we have systematically analyzed the dipeptide composition of the halophilic and non-halophilic protein sequences. We observed the halophilic proteins contained more DA, RA, AD, RR, AP, DD, PD, EA, VG and DV at the expense of LK, IL, II, IA, KK, IS, KA, GK, RK and AI. We identified some macromolecular signatures of halo-adaptation, and thought the dipeptide composition might contain more information than amino acid composition. Based on the dipeptide composition, we have developed a machine learning method for classifying halophilic and non-halophilic proteins for the first time. The accuracy of our method for the training dataset was 100.0%, and for the 10-fold cross-validation was 93.1%. We also discussed the influence of some specific dipeptides on prediction accuracy. Copyright © 2012 Elsevier B.V. All rights reserved.

Stability of Proteins in Dried Blood Spot Biobanks*

PubMed Central

Björkesten, Johan; Enroth, Stefan; Shen, Qiujin; Wik, Lotta; Hougaard, David M.; Cohen, Arieh S.; Sörensen, Lene; Giedraitis, Vilmantas; Ingelsson, Martin; Larsson, Anders; Kamali-Moghaddam, Masood; Landegren, Ulf

2017-01-01

An important motivation for the construction of biobanks is to discover biomarkers that identify diseases at early, potentially curable stages. This will require biobanks from large numbers of individuals, preferably sampled repeatedly, where the samples are collected and stored under conditions that preserve potential biomarkers. Dried blood samples are attractive for biobanking because of the ease and low cost of collection and storage. Here we have investigated their suitability for protein measurements. Ninety-two proteins with relevance for oncology were analyzed using multiplex proximity extension assays (PEA) in dried blood spots collected on paper and stored for up to 30 years at either +4 °C or −24 °C. Our main findings were that (1) the act of drying only slightly influenced detection of blood proteins (average correlation of 0.970), and in a reproducible manner (correlation of 0.999), (2) detection of some proteins was not significantly affected by storage over the full range of three decades (34 and 76% of the analyzed proteins at +4 °C and −24 °C, respectively), whereas levels of others decreased slowly during storage with half-lives in the range of 10 to 50 years, and (3) detectability of proteins was less affected in dried samples stored at −24 °C compared with at +4 °C, as the median protein abundance had decreased to 80 and 93% of starting levels after 10 years of storage at +4 °C or −24 °C, respectively. The results of our study are encouraging as they suggest an inexpensive means to collect large numbers of blood samples, even by the donors themselves, and to transport, and store biobanked samples as spots of whole blood dried on paper. Combined with emerging means to measure hundreds or thousands of protein, such biobanks could prove of great medical value by greatly enhancing discovery as well as routine analysis of blood biomarkers. PMID:28501802

Purification of barley dimeric α-amylase inhibitor-1 (BDAI-1) and avenin-like protein-a (ALP) from beer and their impact on beer foam stability.

PubMed

Iimure, Takashi; Kihara, Makoto; Sato, Kazuhiro; Ogushi, Kensuke

2015-04-01

Foam stability is a key factor of beer quality for consumers and brewers. Recent beer proteome analyses have suggested that barley dimeric α-amylase inhibitor-1 (BDAI-1) and avenin-like protein-a (ALP) derived from barley are important for beer foam stability. In this study, BDAI-1 and ALP were purified from a Japanese commercial beer sample using salt precipitation and column chromatography. The purification level was verified using two-dimensional gel electrophoresis, mass spectrometry, and database searches. Purified BDAI-1 and ALP were added to a beer sample to compare the foam stability to that of a control beer sample. As a result, beer foam stability was significantly improved by BDAI-1 but not by ALP, thereby suggesting that BDAI-1 affects beer foam stability whereas ALP does not. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mechanistic insights into osmolyte action in protein stabilization under harsh conditions: N-methylacetamide in glycine betaine-urea mixture

NASA Astrophysics Data System (ADS)

Kumar, Narendra; Kishore, Nand

2014-10-01

Glycine betaine (GB), a small naturally occurring osmolyte, stabilizes proteins and counteracts harsh denaturing conditions such as extremes of temperature, cellular dehydration, and presence of high concentration of urea. In spite of several studies on understanding mechanism of protein stabilization and counteraction of these harsh conditions by osmolytes, studies centred on GB, one of the most important osmolyte, are scarce, hence, there is need for more investigations. To explore mechanism of protein stabilization and counteraction of denaturing property of urea by GB, molecular dynamics studies of N-methylacetamide (NMA), a model peptide representing denatured state of a protein, in the presence of GB, urea, and GB-urea mixture were carried out. The results show that GB and urea work such that the strength of GB as a protecting osmolyte is increased and the denaturing ability of urea is decreased in the GB-urea mixture. It can be inferred that GB counteracts urea by decreasing its hydrophobic interactions with proteins. The mutual interactions between GB and urea also play an important role in protein stabilization. This study provides insights on osmolyte induced counteraction of denaturing property of urea.

Biophysics of protein evolution and evolutionary protein biophysics

PubMed Central

Sikosek, Tobias; Chan, Hue Sun

2014-01-01

The study of molecular evolution at the level of protein-coding genes often entails comparing large datasets of sequences to infer their evolutionary relationships. Despite the importance of a protein's structure and conformational dynamics to its function and thus its fitness, common phylogenetic methods embody minimal biophysical knowledge of proteins. To underscore the biophysical constraints on natural selection, we survey effects of protein mutations, highlighting the physical basis for marginal stability of natural globular proteins and how requirement for kinetic stability and avoidance of misfolding and misinteractions might have affected protein evolution. The biophysical underpinnings of these effects have been addressed by models with an explicit coarse-grained spatial representation of the polypeptide chain. Sequence–structure mappings based on such models are powerful conceptual tools that rationalize mutational robustness, evolvability, epistasis, promiscuous function performed by ‘hidden’ conformational states, resolution of adaptive conflicts and conformational switches in the evolution from one protein fold to another. Recently, protein biophysics has been applied to derive more accurate evolutionary accounts of sequence data. Methods have also been developed to exploit sequence-based evolutionary information to predict biophysical behaviours of proteins. The success of these approaches demonstrates a deep synergy between the fields of protein biophysics and protein evolution. PMID:25165599

Factors Affecting Lateral Stability and Controllability

NASA Technical Reports Server (NTRS)

Campbell, John P; Toll, Thomas A

1948-01-01

The effects on dynamic lateral stability and controllability of some of the important aerodynamic and mass characteristics are discussed and methods are presented for estimating the various stability parameters to be used in the calculation of the dynamic lateral stability of airplanes with swept and low-aspect-ratio wings.

Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy

PubMed Central

Greco, Todd M.; Guise, Amanda J.; Cristea, Ileana M.

2016-01-01

In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions. In this chapter, we describe an approach that uses quantitative mass spectrometry (MS) to assess the specificity and the relative stability of protein interactions. Isolation of protein complexes from mammalian cells is performed by rapid immunoaffinity purification, and followed by in-solution digestion and high-resolution mass spectrometry analysis. We employ complementary quantitative MS workflows to assess the specificity of protein interactions using label-free MS and statistical analysis, and the relative stability of the interactions using a metabolic labeling technique. For each candidate protein interaction, scores from the two workflows can be correlated to minimize nonspecific background and profile protein complex composition and relative stability. PMID:26867737

The impact of the concentration of casein micelles and whey protein-stabilized fat globules on the rennet-induced gelation of milk.

PubMed

Gaygadzhiev, Zafir; Corredig, Milena; Alexander, Marcela

2009-02-01

The rennet-induced aggregation of skim milk recombined with whey protein-stabilized emulsion droplets was studied using diffusing wave spectroscopy (DSW) and small deformation rheology. The effect of different volume fractions of casein micelles and fat globules was investigated by observing changes in turbidity (1/l*), apparent radius, elastic modulus and mean square displacement (MSD), in addition to confocal imaging of the gels. Skim milk containing different concentration of casein micelles showed comparable light-scattering profiles; a higher volume fraction of caseins led to the development of more elastic gels. By following the development of 1/l* in recombined milks, it was possible to describe the behaviour of the fat globules during the initial stages of rennet coagulation. Increasing the volume fraction of fat globules showed a significant increase in gel elasticity, caused by flocculation of the oil droplets. The presence of flocculated oil globules within the gel structure was confirmed by confocal microscopy observations. Moreover, a lower degree of kappa-casein hydrolysis was needed to initiate casein micelles aggregation in milk containing whey protein-stabilized oil droplets compared to skim milk. This study for the first time clearly describes the impact of a mixture of casein micelles and whey protein-stabilized fat globules on the pre-gelation stages of rennet coagulation, and further highlights the importance of the flocculation state of the emulsion droplets in affecting the structure formation of the gel.

Light assisted drying (LAD) for protein stabilization: optimization of laser processing parameters

NASA Astrophysics Data System (ADS)

Young, Madison A.; Antczak, Andrew T.; Elliott, Gloria D.; Trammell, Susan R.

2017-02-01

In this study, a novel light-based processing method to create an amorphous trehalose matrix for the stabilization of proteins is discussed. Near-IR radiation is used to remove water from samples, leaving behind an amorphous solid with embedded protein. This method has potential applications in the stabilization of protein-based therapeutics and diagnostics that are becoming widely used in the treatment and diagnosis of a variety of diseases. Freeze-drying or freezing are currently the standard for the preservation of proteins, but these methods are expensive and can be challenging in some environments due to a lack of available infrastructure. Light-assisted drying offers a relatively inexpensive method for drying samples. Proteins suspended in a trehalose solution are dehydrated using near-infrared laser light. The laser radiation speeds drying and as water is removed the sugar forms a protective matrix. The goal of this study is to determine processing parameters that result in fast processing times and low end moisture contents (EMC), while maintaining the functionality of embedded proteins. We compare the effect of changing processing wavelength, power and resulting sample temperature, and substrate material on the EMC for two NIR laser sources (1064 nm and 1850 nm). The 1850 nm laser resulted in the lowest EMC (0.1836+/-0.09 gH2O/gDryWeight) after 10 minutes of processing on borosilicate glass microfiber paper. This suggests a storage temperature of 3°C.

Fetal bovine serum influences the stability and bioactivity of resveratrol analogues: A polyphenol-protein interaction approach.

PubMed

Tang, Fen; Xie, Yixi; Cao, Hui; Yang, Hua; Chen, Xiaoqing; Xiao, Jianbo

2017-03-15

Fetal bovine serum (FBS) is a universal growth supplement of cell and tissue culture media. Herein, the influences of FBS on the stability and antioxidant activity of 21 resveratrol analogues were investigated using a polyphenol-protein interaction approach. The structure-stability relationships of resveratrol analogues in FBS showed a clear decrease in the stability of hydroxylated resveratrol analogues in the order: resorcinol-type>pyrogallol-type>catechol-type. The glycosylation and methoxylation of resveratrol analogues enhanced their stability. A linear relationship between the stability of resveratrol analogues in FBS and the affinity of resveratrol analogues-FBS interaction was found. The oxidation process is not the only factor governing the stability of resveratrol analogues in FBS. These results facilitated the insightful investigation of the role of polyphenol-protein interactions in serum, thereby providing some fundamental clues for future clinical research and pharmacological studies on natural small molecules. Copyright © 2016 Elsevier Ltd. All rights reserved.

Stability of spray-dried beetroot extract using oligosaccharides and whey proteins.

PubMed

Carmo, Eloá Lourenço do; Teodoro, Rhana Amanda Ribeiro; Félix, Pedro Henrique Campelo; Fernandes, Regiane Victória de Barros; Oliveira, Érica Resende de; Veiga, Taís Regina Lima Abreu; Borges, Soraia Vilela; Botrel, Diego Alvarenga

2018-05-30

The properties and stability of spray-dried beetroot extract using maltodextrin (MD), inulin (IN), and whey protein isolate (WPI) as carrier agents were evaluated. The values of moisture, betalains content, and retention were 3.33-4.24%, 348.79-385.47 mg/100 g (dry-basis), and 88.45-95.69%, respectively. Higher values of antioxidant activity were observed for the treatments using WPI. The treatment with inulin alone presented higher hygroscopicity in the moisture adsorption isotherms at 25 °C and lower thermal stability when evaluating the thermogravimetric curves. When stored at 60 °C, the use of WPI alone conferred lower stability to the beetroot extract powder. In general, the simultaneous use of IN and WPI as carrier agents resulted in good stability of the beetroot extract powder, representing an opportunity for innovation in food products. Copyright © 2017 Elsevier Ltd. All rights reserved.

Factors affecting fixation of heavy metals in solidified/stabilized matrix: a review.

PubMed

Malviya, Rachana; Chaudhary, Rubina

2010-07-01

In this paper, an effort has been made to understand the factors, which affect fixation of heavy metals in solidified/stabilized matrix. Various aspects related to the solidification/stabilization of different heavy metals (Ar, Ba, Cu, Cr, Pb, Zn, Hg) are reviewed. A comparative study of different binders for the fixation of each metal has also been carried out to suggest the most suitable binder, pretreatment required for the metal. Valence, speciation, pH and other factors are also considered while reviewing metal retention capacity of different matrix.

Protein accumulation and rumen stability of wheat γ-gliadin fusion proteins in tobacco and alfalfa.

PubMed

Sun, Xiaodong; Chi-Ham, Cecilia L; Cohen-Davidyan, Tamar; DeBen, Christopher; Getachew, Girma; DePeters, Edward; Putnam, Daniel; Bennett, Alan

2015-09-01

The nutritional value of various crops can be improved by engineering plants to produce high levels of proteins. For example, because methionine deficiency limits the protein quality of Medicago Sativa (alfalfa) forage, producing alfalfa plants that accumulate high levels of a methionine-rich protein could increase the nutritional value of that crop. We used three strategies in designing methionine-rich recombinant proteins that could accumulate to high levels in plants and thereby serve as candidates for improving the protein quality of alfalfa forage. In tobacco, two fusion proteins, γ-gliadin-δ-zein and γ-δ-zein, as well as δ-zein co-expressed with β-zein, all formed protein bodies. However, the γ-gliadin-δ-zein fusion protein accumulated to the highest level, representing up to 1.5% of total soluble protein (TSP) in one transformant. In alfalfa, γ-gliadin-δ-zein accumulated to 0.2% of TSP, and in an in vitro rumen digestion assay, γ-gliadin-δ-zein was more resistant to microbial degradation than Rubisco. Additionally, although it did not form protein bodies, a γ-gliadin-GFP fusion protein accumulated to much higher levels, 7% of TSP, than a recombinant protein comprised of an ER localization signal fused to GFP in tobacco. Based on our results, we conclude that γ-gliadin-δ-zein is a potential candidate protein to use for enhancing methionine levels in plants and for improving rumen stability of forage protein. γ-gliadin fusion proteins may provide a general platform for increasing the accumulation of recombinant proteins in transgenic plants. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Yap4 PKA- and GSK3-dependent phosphorylation affects its stability but not its nuclear localization.

PubMed

Pereira, Jorge; Pimentel, Catarina; Amaral, Catarina; Menezes, Regina A; Rodrigues-Pousada, Claudina

2009-12-01

Yap4 is a nuclear-resident transcription factor induced in Saccharomyces cerevisiae when exposed to several stress conditions, which include mild hyperosmotic and oxidative stress, temperature shift or metal exposure. This protein is also phosphorylated. Here we report that this modification is driven by PKA and GSK3. In order to ascertain whether Yap4 is directly or indirectly phosphorylated by PKA, we searched for stress and PKA-related kinases that could phosphorylate Yap4. We show that phosphorylation is independent of the kinases Rim15, Yak1, Sch9, Slt2, Ste20 and Ptk2. In addition, we showed that Yap4 phosphorylation is also abrogated in the triple GSK3 mutant mck1 rim11 yol128c. Furthermore, our data reveal that Yap4 nuclear localization is independent of its phosphorylation state. This protein has several putative phosphorylation sites, but only the mutation of residues T192 and S196 impairs its phosphorylation under different stress conditions. The ability of the non-phosphorylated forms of Yap4 to partially rescue the hog1 severe sensitivity phenotype is not affected, suggesting that Yap4 activity is maintained in the absence of phosphorylation. However, this modification seems to be required for stability of the protein, as the non-phosphorylated form has a shorter half-life than the phosphorylated one.

ZMYND10 stabilizes intermediate chain proteins in the cytoplasmic pre-assembly of dynein arms.

PubMed

Cho, Kyeong Jee; Noh, Shin Hye; Han, Soo Min; Choi, Won-Il; Kim, Hye-Youn; Yu, Seyoung; Lee, Joon Suk; Rim, John Hoon; Lee, Min Goo; Hildebrandt, Friedhelm; Gee, Heon Yung

2018-03-01

Zinc finger MYND-type-containing 10 (ZMYND10), a cytoplasmic protein expressed in ciliated cells, causes primary ciliary dyskinesia (PCD) when mutated; however, its function is poorly understood. Therefore, in this study, we examined the roles of ZMYND10 using Zmynd10-/-mice exhibiting typical PCD phenotypes, including hydrocephalus and laterality defects. In these mutants, morphology, the number of motile cilia, and the 9+2 axoneme structure were normal; however, inner and outer dynein arms (IDA and ODA, respectively) were absent. ZMYND10 interacted with ODA components and proteins, including LRRC6, DYX1C1, and C21ORF59, implicated in the cytoplasmic pre-assembly of DAs, whose levels were significantly reduced in Zmynd10-/-mice. LRRC6 and DNAI1 were more stable when co-expressed with ZYMND10 than when expressed alone. DNAI2, which did not interact with ZMYND10, was not stabilized by co-expression with ZMYND10 alone, but was stabilized by co-expression with DNAI1 and ZMYND10, suggesting that ZMYND10 stabilized DNAI1, which subsequently stabilized DNAI2. Together, these results demonstrated that ZMYND10 regulated the early stage of DA cytoplasmic pre-assembly by stabilizing DNAI1.

Stabilizing and destabilizing protein surfactant-based foams in the presence of a chemical surfactant: Effect of adsorption kinetics.

PubMed

Li, Huazhen; Le Brun, Anton P; Agyei, Dominic; Shen, Wei; Middelberg, Anton P J; He, Lizhong

2016-01-15

Stimuli-responsive protein surfactants promise alternative foaming materials that can be made from renewable sources. However, the cost of protein surfactants is still higher than their chemical counterparts. In order to reduce the required amount of protein surfactant for foaming, we investigated the foaming and adsorption properties of the protein surfactant, DAMP4, with addition of low concentrations of the chemical surfactant sodium dodecylsulfate (SDS). The results show that the small addition of SDS can enhance foaming functions of DAMP4 at a lowered protein concentration. Dynamic surface tension measurements suggest that there is a synergy between DAMP4 and SDS which enhances adsorption kinetics of DAMP4 at the initial stage of adsorption (first 60s), which in turn stabilizes protein foams. Further interfacial properties were revealed by X-ray reflectometry measurements, showing that there is a re-arrangement of adsorbed protein-surfactant layer over a long period of 1h. Importantly, the foaming switchability of DAMP4 by metal ions is not affected by the presence of SDS, and foams can be switched off by the addition of zinc ions at permissive pH. This work provides fundamental knowledge to guide formulation using a mixture of protein and chemical surfactants towards a high performance of foaming at a low cost. Copyright © 2015 Elsevier Inc. All rights reserved.

Plant protein and animal proteins: do they differentially affect cardiovascular disease risk?

PubMed

Richter, Chesney K; Skulas-Ray, Ann C; Champagne, Catherine M; Kris-Etherton, Penny M

2015-11-01

Proteins from plant-based compared with animal-based food sources may have different effects on cardiovascular disease (CVD) risk factors. Numerous epidemiologic and intervention studies have evaluated their respective health benefits; however, it is difficult to isolate the role of plant or animal protein on CVD risk. This review evaluates the current evidence from observational and intervention studies, focusing on the specific protein-providing foods and populations studied. Dietary protein is derived from many food sources, and each provides a different composite of nonprotein compounds that can also affect CVD risk factors. Increasing the consumption of protein-rich foods also typically results in lower intakes of other nutrients, which may simultaneously influence outcomes. Given these complexities, blanket statements about plant or animal protein may be too general, and greater consideration of the specific protein food sources and the background diet is required. The potential mechanisms responsible for any specific effects of plant and animal protein are similarly multifaceted and include the amino acid content of particular foods, contributions from other nonprotein compounds provided concomitantly by the whole food, and interactions with the gut microbiome. Evidence to date is inconclusive, and additional studies are needed to further advance our understanding of the complexity of plant protein vs. animal protein comparisons. Nonetheless, current evidence supports the idea that CVD risk can be reduced by a dietary pattern that provides more plant sources of protein compared with the typical American diet and also includes animal-based protein foods that are unprocessed and low in saturated fat. © 2015 American Society for Nutrition.

Force spectroscopy studies on protein-ligand interactions: a single protein mechanics perspective.

PubMed

Hu, Xiaotang; Li, Hongbin

2014-10-01

Protein-ligand interactions are ubiquitous and play important roles in almost every biological process. The direct elucidation of the thermodynamic, structural and functional consequences of protein-ligand interactions is thus of critical importance to decipher the mechanism underlying these biological processes. A toolbox containing a variety of powerful techniques has been developed to quantitatively study protein-ligand interactions in vitro as well as in living systems. The development of atomic force microscopy-based single molecule force spectroscopy techniques has expanded this toolbox and made it possible to directly probe the mechanical consequence of ligand binding on proteins. Many recent experiments have revealed how ligand binding affects the mechanical stability and mechanical unfolding dynamics of proteins, and provided mechanistic understanding on these effects. The enhancement effect of mechanical stability by ligand binding has been used to help tune the mechanical stability of proteins in a rational manner and develop novel functional binding assays for protein-ligand interactions. Single molecule force spectroscopy studies have started to shed new lights on the structural and functional consequence of ligand binding on proteins that bear force under their biological settings. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

In-frame seven amino-acid duplication in AIP arose over the last 3000 years, disrupts protein interaction and stability and is associated with gigantism.

PubMed

Salvatori, Roberto; Radian, Serban; Diekmann, Yoan; Iacovazzo, Donato; David, Alessia; Gabrovska, Plamena; Grassi, Giorgia; Bussell, Anna-Marie; Stals, Karen; Weber, Astrid; Quinton, Richard; Crowne, Elizabeth C; Corazzini, Valentina; Metherell, Lou; Kearney, Tara; Du Plessis, Daniel; Sinha, Ajay Kumar; Baborie, Atik; Lecoq, Anne-Lise; Chanson, Philippe; Ansorge, Olaf; Ellard, Sian; Trainer, Peter J; Balding, David; Thomas, Mark G; Korbonits, Márta

2017-09-01

Mutations in the aryl hydrocarbon receptor-interacting protein ( AIP ) gene are associated with pituitary adenoma, acromegaly and gigantism. Identical alleles in unrelated pedigrees could be inherited from a common ancestor or result from recurrent mutation events. Observational, inferential and experimental study, including: AIP mutation testing; reconstruction of 14 AIP -region (8.3 Mbp) haplotypes; coalescent-based approximate Bayesian estimation of the time to most recent common ancestor (tMRCA) of the derived allele; forward population simulations to estimate current number of allele carriers; proposal of mutation mechanism; protein structure predictions; co-immunoprecipitation and cycloheximide chase experiments. Nine European-origin, unrelated c.805_825dup-positive pedigrees (four familial, five sporadic from the UK, USA and France) included 16 affected (nine gigantism/four acromegaly/two non-functioning pituitary adenoma patients and one prospectively diagnosed acromegaly patient) and nine unaffected carriers. All pedigrees shared a 2.79 Mbp haploblock around AIP with additional haploblocks privately shared between subsets of the pedigrees, indicating the existence of an evolutionarily recent common ancestor, the 'English founder', with an estimated median tMRCA of 47 generations (corresponding to 1175 years) with a confidence interval (9-113 generations, equivalent to 225-2825 years). The mutation occurred in a small tandem repeat region predisposed to slipped strand mispairing. The resulting seven amino-acid duplication disrupts interaction with HSP90 and leads to a marked reduction in protein stability. The c.805_825dup allele, originating from a common ancestor, associates with a severe clinical phenotype and a high frequency of gigantism. The mutation is likely to be the result of slipped strand mispairing and affects protein-protein interactions and AIP protein stability. © 2017 The authors.

The HCM-linked W792R mutation in cardiac myosin-binding protein C reduces C6 FnIII domain stability.

PubMed

Smelter, Dan F; de Lange, Willem J; Cai, Wenxuan; Ge, Ying; Ralphe, J Carter

2018-06-01

Cardiac myosin-binding protein C (cMyBP-C) is a functional sarcomeric protein that regulates contractility in response to contractile demand, and many mutations in cMyBP-C lead to hypertrophic cardiomyopathy (HCM). To gain insight into the effects of disease-causing cMyBP-C missense mutations on contractile function, we expressed the pathogenic W792R mutation (substitution of a highly conserved tryptophan residue by an arginine residue at position 792) in mouse cardiomyocytes lacking endogenous cMyBP-C and studied the functional effects using three-dimensional engineered cardiac tissue constructs (mECTs). Based on complete conservation of tryptophan at this location in fibronectin type II (FnIII) domains, we hypothesized that the W792R mutation affects folding of the C6 FnIII domain, destabilizing the mutant protein. Adenoviral transduction of wild-type (WT) and W792R cDNA achieved equivalent mRNA transcript abundance, but not equivalent protein levels, with W792R compared with WT controls. mECTs expressing W792R demonstrated abnormal contractile kinetics compared with WT mECTs that were nearly identical to cMyBP-C-deficient mECTs. We studied whether common pathways of protein degradation were responsible for the rapid degradation of W792R cMyBP-C. Inhibition of both ubiquitin-proteasome and lysosomal degradation pathways failed to increase full-length mutant protein abundance to WT equivalence, suggesting rapid cytosolic degradation. Bacterial expression of WT and W792R protein fragments demonstrated decreased mutant stability with altered thermal denaturation and increased susceptibility to trypsin digestion. These data suggest that the W792R mutation destabilizes the C6 FnIII domain of cMyBP-C, resulting in decreased full-length protein expression. This study highlights the vulnerability of FnIII-like domains to mutations that alter domain stability and further indicates that missense mutations in cMyBP-C can cause disease through a mechanism of

Thermodynamics of coupled protein adsorption and stability using hybrid Monte Carlo simulations.

PubMed

Zhong, Ellen D; Shirts, Michael R

2014-05-06

A better understanding of changes in protein stability upon adsorption can improve the design of protein separation processes. In this study, we examine the coupling of the folding and the adsorption of a model protein, the B1 domain of streptococcal protein G, as a function of surface attraction using a hybrid Monte Carlo (HMC) approach with temperature replica exchange and umbrella sampling. In our HMC implementation, we are able to use a molecular dynamics (MD) time step that is an order of magnitude larger than in a traditional MD simulation protocol and observe a factor of 2 enhancement in the folding and unfolding rate. To demonstrate the convergence of our systems, we measure the travel of our order parameter the fraction of native contacts between folded and unfolded states throughout the length of our simulations. Thermodynamic quantities are extracted with minimum statistical variance using multistate reweighting between simulations at different temperatures and harmonic distance restraints from the surface. The resultant free energies, enthalpies, and entropies of the coupled unfolding and absorption processes are in qualitative agreement with previous experimental and computational observations, including entropic stabilization of the adsorbed, folded state relative to the bulk on surfaces with low attraction.

Effect of chitosan on the heat stability of whey protein solution as a function of pH.

PubMed

Zhao, Zhengtao; Xiao, Qian

2017-03-01

Chitosan was reported to interact with proteins through electrostatic interactions. Their interaction was influenced by pH, which was not fully characterized. Further research on the interactions between protein and chitosan at different pH and their influence on the thermal denaturation of proteins is necessary. In this research, the effect of chitosan on the heat stability of whey protein solution at pH 4.0-6.0 was studied. At pH 4.0, a small amount chitosan was able to prevent the heat-induced denaturation and aggregation of whey protein molecules. At higher pH values (5.5 and 6.0), whey proteins complexed with chitosan through electrostatic attraction. The formation of chitosan-whey protein complexes at pH 5.5 improved the heat stability of dispersions and no precipitation could be detected up to 20 days. The dispersion with a medium amount of chitosan (chitosan:whey protein 1:5) produced the most stable particles, which had an average radius of 135 ± 14 nm and a zeta potential value of 36 ± 1 mV. In contrast, at pH 6.0 only the dispersion with a high amount of chitosan (chitosan:whey protein 1:2) showed good shelf stability up to 20 days. It was possible to produce heat-stable whey protein beverages by regulating the interaction between chitosan and whey protein molecules. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

NeEMO: a method using residue interaction networks to improve prediction of protein stability upon mutation.

PubMed

Giollo, Manuel; Martin, Alberto J M; Walsh, Ian; Ferrari, Carlo; Tosatto, Silvio C E

2014-01-01

The rapid growth of un-annotated missense variants poses challenges requiring novel strategies for their interpretation. From the thermodynamic point of view, amino acid changes can lead to a change in the internal energy of a protein and induce structural rearrangements. This is of great relevance for the study of diseases and protein design, justifying the development of prediction methods for variant-induced stability changes. Here we propose NeEMO, a tool for the evaluation of stability changes using an effective representation of proteins based on residue interaction networks (RINs). RINs are used to extract useful features describing interactions of the mutant amino acid with its structural environment. Benchmarking shows NeEMO to be very effective, allowing reliable predictions in different parts of the protein such as β-strands and buried residues. Validation on a previously published independent dataset shows that NeEMO has a Pearson correlation coefficient of 0.77 and a standard error of 1 Kcal/mol, outperforming nine recent methods. The NeEMO web server can be freely accessed from URL: http://protein.bio.unipd.it/neemo/. NeEMO offers an innovative and reliable tool for the annotation of amino acid changes. A key contribution are RINs, which can be used for modeling proteins and their interactions effectively. Interestingly, the approach is very general, and can motivate the development of a new family of RIN-based protein structure analyzers. NeEMO may suggest innovative strategies for bioinformatics tools beyond protein stability prediction.

Interfacial composition and stability of emulsions made with mixtures of commercial sodium caseinate and whey protein concentrate.

PubMed

Ye, Aiqian

2008-10-15

The interfacial composition and the stability of oil-in-water emulsion droplets (30% soya oil, pH 7.0) made with mixtures of sodium caseinate and whey protein concentrate (WPC) (1:1 by protein weight) at various total protein concentrations were examined. The average volume-surface diameter (d32) and the total surface protein concentration of emulsion droplets were similar to those of emulsions made with both sodium caseinate alone and WPC alone. Whey proteins were adsorbed in preference to caseins at low protein concentrations (<3%), whereas caseins were adsorbed in preference to whey proteins at high protein concentrations. The creaming stability of the emulsions decreased markedly as the total protein concentration of the system was increased above 2% (sodium caseinate >1%). This was attributed to depletion flocculation caused by the sodium caseinate in these emulsions. Whey proteins did not retard this instability in the emulsions made with mixtures of sodium caseinate and WPC. Copyright © 2008 Elsevier Ltd. All rights reserved.

Non-Arrhenius protein aggregation.

PubMed

Wang, Wei; Roberts, Christopher J

2013-07-01

Protein aggregation presents one of the key challenges in the development of protein biotherapeutics. It affects not only product quality but also potentially impacts safety, as protein aggregates have been shown to be linked with cytotoxicity and patient immunogenicity. Therefore, investigations of protein aggregation remain a major focus in pharmaceutical companies and academic institutions. Due to the complexity of the aggregation process and temperature-dependent conformational stability, temperature-induced protein aggregation is often non-Arrhenius over even relatively small temperature windows relevant for product development, and this makes low-temperature extrapolation difficult based simply on accelerated stability studies at high temperatures. This review discusses the non-Arrhenius nature of the temperature dependence of protein aggregation, explores possible causes, and considers inherent hurdles for accurately extrapolating aggregation rates from conventional industrial approaches for selecting accelerated conditions and from conventional or more advanced methods of analyzing the resulting rate data.

Temperature stability of proteins essential for the intracellular survival of Mycobacterium tuberculosis.

PubMed

Lack, Nathan A; Kawamura, Akane; Fullam, Elizabeth; Laurieri, Nicola; Beard, Stacey; Russell, Angela J; Evangelopoulos, Dimitrios; Westwood, Isaac; Sim, Edith

2009-03-01

In Mycobacterium tuberculosis, the genes hsaD (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase) and nat (arylamine N-acetyltransferase) are essential for survival inside of host macrophages. These genes act as an operon and have been suggested to be involved in cholesterol metabolism. However, the role of NAT in this catabolic pathway has not been determined. In an effort to better understand the function of these proteins, we have expressed, purified and characterized TBNAT (NAT from M. tuberculosis) and HsaD (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase) from M. tuberculosis. Both proteins demonstrated remarkable heat stability with TBNAT and HsaD retaining >95% of their activity after incubation at 60 degrees C for 30 min. The first and second domains of TBNAT were demonstrated to be very important to the heat stability of the protein, as the transfer of these domains caused a dramatic reduction in the heat stability. The specific activity of TBNAT was tested against a broad range of acyl-CoA cofactors using hydralazine as a substrate. TBNAT was found to be able to utilize not just acetyl-CoA, but also n-propionyl-CoA and acetoacetyl-CoA, although at a lower rate. As propionyl-CoA is a product of cholesterol catabolism, we propose that NAT could have a role in the utilization of this important cofactor.

Free energy calculations on the stability of the 14-3-3ζ protein.

PubMed

Jandova, Zuzana; Trosanova, Zuzana; Weisova, Veronika; Oostenbrink, Chris; Hritz, Jozef

2018-03-01

Mutations of cysteine are often introduced to e.g. avoid formation of non-physiological inter-molecular disulfide bridges in in-vitro experiments, or to maintain specificity in labeling experiments. Alanine or serine is typically preferred, which usually do not alter the overall protein stability, when the original cysteine was surface exposed. However, selecting the optimal mutation for cysteines in the hydrophobic core of the protein is more challenging. In this work, the stability of selected Cys mutants of 14-3-3ζ was predicted by free-energy calculations and the obtained data were compared with experimentally determined stabilities. Both the computational predictions as well as the experimental validation point at a significant destabilization of mutants C94A and C94S. This destabilization could be attributed to the formation of hydrophobic cavities and a polar solvation of a hydrophilic side chain. A L12E, M78K double mutant was further studied in terms of its reduced dimerization propensity. In contrast to naïve expectations, this double mutant did not lead to the formation of strong salt bridges, which was rationalized in terms of a preferred solvation of the ionic species. Again, experiments agreed with the calculations by confirming the monomerization of the double mutants. Overall, the simulation data is in good agreement with experiments and offers additional insight into the stability and dimerization of this important family of regulatory proteins. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Concentration-dependent changes in apparent diffusion coefficients as indicator for colloidal stability of protein solutions.

PubMed

Bauer, Katharina Christin; Göbel, Mathias; Schwab, Marie-Luise; Schermeyer, Marie-Therese; Hubbuch, Jürgen

2016-09-10

The colloidal stability of a protein solution during downstream processing, formulation, and storage is a key issue for the biopharmaceutical production process. Thus, knowledge about colloidal solution characteristics, such as the tendency to form aggregates or high viscosity, at various processing conditions is of interest. This work correlates changes in the apparent diffusion coefficient as a parameter of protein interactions with observed protein aggregation and dynamic viscosity of the respective protein samples. For this purpose, the diffusion coefficient, the protein phase behavior, and the dynamic viscosity in various systems containing the model proteins α-lactalbumin, lysozyme, and glucose oxidase were studied. Each of these experiments revealed a wide range of variations in protein interactions depending on protein type, protein concentration, pH, and the NaCl concentration. All these variations showed to be mirrored by changes in the apparent diffusion coefficient in the respective samples. Whereas stable samples with relatively low viscosity showed an almost linear dependence, the deviation from the concentration-dependent linearity indicated both an increase in the sample viscosity and probability of protein aggregation. This deviation of the apparent diffusion coefficient from concentration-dependent linearity was independent of protein type and solution properties for this study. Thus, this single parameter shows the potential to act as a prognostic tool for colloidal stability of protein solutions. Copyright © 2016 Elsevier B.V. All rights reserved.

A specific role for the ZipA protein in cell division: stabilization of the FtsZ protein.

PubMed

Pazos, Manuel; Natale, Paolo; Vicente, Miguel

2013-02-01

In Escherichia coli, the cell division protein FtsZ is anchored to the cytoplasmic membrane by the action of the bitopic membrane protein ZipA and the cytoplasmic protein FtsA. Although the presence of both ZipA and FtsA is strictly indispensable for cell division, an FtsA gain-of-function mutant FtsA* (R286W) can bypass the ZipA requirement for cell division. This observation casts doubts on the role of ZipA and its need for cell division. Maxicells are nucleoid-free bacterial cells used as a whole cell in vitro system to probe protein-protein interactions without the need of protein purification. We show that ZipA protects FtsZ from the ClpXP-directed degradation observed in E. coli maxicells and that ZipA-stabilized FtsZ forms membrane-attached spiral-like structures in the bacterial cytoplasm. The overproduction of the FtsZ-binding ZipA domain is sufficient to protect FtsZ from degradation, whereas other C-terminal ZipA partial deletions lacking it are not. Individual overproduction of the proto-ring component FtsA or its gain-of-function mutant FtsA* does not result in FtsZ protection. Overproduction of FtsA or FtsA* together with ZipA does not interfere with the FtsZ protection. Moreover, neither FtsA nor FtsA* protects FtsZ when overproduced together with ZipA mutants lacking the FZB domain. We propose that ZipA protects FtsZ from degradation by ClpP by making the FtsZ site of interaction unavailable to the ClpX moiety of the ClpXP protease. This role cannot be replaced by either FtsA or FtsA*, suggesting a unique function for ZipA in proto-ring stability.

Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization

PubMed Central

Dedecker, Peter

2017-01-01

Reversibly switchable fluorescent proteins (RSFPs) enable advanced fluorescence imaging, though the performance of this imaging crucially depends on the properties of the labels. We report on the use of an existing small binding peptide, named Enhancer, to modulate the spectroscopic properties of the recently developed rsGreen series of RSFPs. Fusion constructs of Enhancer with rsGreen1 and rsGreenF revealed an increased molecular brightness and pH stability, although expression in living E. coli or HeLa cells resulted in a decrease of the overall emission. Surprisingly, Enhancer binding also increased off-switching speed and resistance to switching fatigue. Further investigation suggested that the RSFPs can interconvert between fast- and slow-switching emissive states, with the overall protein population gradually converting to the slow-switching state through irradiation. The Enhancer modulates the spectroscopic properties of both states, but also preferentially stabilizes the fast-switching state, supporting the increased fatigue resistance. This work demonstrates how the photo-physical properties of RSFPs can be influenced by their binding to other small proteins, which opens up new horizons for applications that may require such modulation. Furthermore, we provide new insights into the photoswitching kinetics that should be of general consideration when developing new RSFPs with improved or different photochromic properties. PMID:28930199

Protein secondary structure and stability determined by combining exoproteolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Villanueva, Josep; Villegas, Virtudes; Querol, Enrique; Avilés, Francesc X; Serrano, Luis

2002-09-01

In the post-genomic era, several projects focused on the massive experimental resolution of the three-dimensional structures of all the proteins of different organisms have been initiated. Simultaneously, significant progress has been made in the ab initio prediction of protein three-dimensional structure. One of the keys to the success of such a prediction is the use of local information (i.e. secondary structure). Here we describe a new limited proteolysis methodology, based on the use of unspecific exoproteases coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), to map quickly secondary structure elements of a protein from both ends, the N- and C-termini. We show that the proteolytic patterns (mass spectra series) obtained can be interpreted in the light of the conformation and local stability of the analyzed proteins, a direct correlation being observed between the predicted and the experimentally derived protein secondary structure. Further, this methodology can be easily applied to check rapidly the folding state of a protein and characterize mutational effects on protein conformation and stability. Moreover, given global stability information, this methodology allows one to locate the protein regions of increased or decreased conformational stability. All of this can be done with a small fraction of the amount of protein required by most of the other methods for conformational analysis. Thus limited exoproteolysis, together with MALDI-TOF MS, can be a useful tool to achieve quickly the elucidation of protein structure and stability. Copyright 2002 John Wiley & Sons, Ltd.

Stabilization and delivery approaches for protein and peptide pharmaceuticals: an extensive review of patents.

PubMed

Swain, Suryakanta; Mondal, Debanik; Beg, Sarwar; Patra, Chinam Niranjan; Dinda, Subas Chandra; Sruti, Jammula; Rao, Muddana Eswara Bhanoji

2013-04-01

Proteins and peptides are the building blocks of human body and act as the arsenal to combat against the invading pathogenic organisms for treatment and management of diseases. Majority of such biomacromolecules are synthesized by the human body itself. However, entry of disease causing pathogens causes misleading in the synthesis of desired proteins for antibody formation. In such alarming situations, the delivery of requisite protein and peptide from external source helps in augmenting the body's immunity. The major drawbacks underlying poor biopharmaceutical performance of high molecular weight protein and peptide drugs are due to poor oral absorption, formulation stability, degradation in the gastric milieu, susceptible to presystemic metabolism. Numerous literature recounts the application of myriad drug delivery strategies for the effective delivery of protein and peptides viz. parentral, oral, transdermal, nasal, pulmonary, rectal, buccal and ocular drug delivery systems. There are many reviews on various delivery strategies for protein and peptide pharmaceuticals, but the present review article provides a bird's eye view on various novel drug delivery systems used for enhanced delivery of protein and peptide pharmaceuticals in the light of patent literature. Apart from this, the present manuscript endeavor provides idea on possible causes and major degradation pathways responsible for poor stability of protein and peptide drugs along with recent market instances on them utilizing novel drug delivery systems.

Nogo-B Receptor stabilizes Niemann-Pick Type C2 protein and regulates intracellular cholesterol trafficking

PubMed Central

Harrison, Kenneth D.; Miao, Robert Qing; Fernandez-Hernándo, Carlos; Suárez, Yajaira; Dávalos, Alberto; Sessa, William C.

2009-01-01

Summary The Nogo-B Receptor (NgBR) is a recently identified receptor for the N-terminus of Reticulon 4B/Nogo-B. Other than its role in binding Nogo-B, little is known about the biology of NgBR. To elucidate a basic cellular role for NgBR, we performed a yeast-2-hybrid screen for interacting proteins using the C-terminal domain as bait and identified Niemann-Pick Type C2 protein (NPC2) as an NgBR-interacting protein. NPC2 protein levels are increased in the presence of NgBR and NgBR enhances NPC2 protein stability. NgBR localizes primarily to the endoplasmic reticulum (ER), and regulates the stability of nascent NPC2. RNAi-mediated disruption of NgBR or genetic deficiency in NgBR leads to a decrease in NPC2 levels, increased intracellular cholesterol accumulation and a loss of sterol sensing, all hallmarks of an NPC2 mutation. These data identify NgBR as an NPC2-interacting protein and provide evidence of a role for NgBR in intracellular cholesterol trafficking. PMID:19723497

Can Solution Supersaturation Affect Protein Crystal Quality?

NASA Technical Reports Server (NTRS)

Gorti, Sridhar

2013-01-01

The formation of large protein crystals of "high quality" is considered a characteristic manifestation of microgravity. The physical processes that predict the formation of large, high quality protein crystals in the microgravity environment of space are considered rooted in the existence of a "depletion zone" in the vicinity of crystal. Namely, it is considered reasonable that crystal quality suffers in earth-grown crystals as a result of the incorporation of large aggregates, micro-crystals and/or large molecular weight "impurities", processes which are aided by density driven convective flow or mixing at the crystal-liquid interface. Sedimentation and density driven convection produce unfavorable solution conditions in the vicinity of the crystal surface, which promotes rapid crystal growth to the detriment of crystal size and quality. In this effort, we shall further present the hypothesis that the solution supersaturatoin at the crystal surface determines the growth mechanism, or mode, by which protein crystals grow. It is further hypothesized that protein crystal quality is affected by the mechanism or mode of crystal growth. Hence the formation of a depletion zone in microgravity environment is beneficial due to inhibition of impurity incorporatoin as well as preventing a kinetic roughening transition. It should be noted that for many proteins the magnitude of neither protein crystal growth rates nor solution supersaturation are predictors of a kinetic roughening transition. That is, the kinetic roughening transition supersaturation must be dtermined for each individual protein.

Stability and in vitro digestibility of emulsions containing lecithin and whey proteins.

PubMed

Mantovani, Raphaela Araujo; Cavallieri, Ângelo Luiz Fazani; Netto, Flavia Maria; Cunha, Rosiane Lopes

2013-09-01

The effect of pH and high-pressure homogenization on the properties of oil-in-water (O/W) emulsions stabilized by lecithin and/or whey proteins (WPI) was evaluated. For this purpose, emulsions were characterized by visual analysis, droplet size distribution, zeta potential, electrophoresis, rheological measurements and their response to in vitro digestion. Lecithin emulsions were stable even after 7 days of storage and WPI emulsions were unstable only at pH values close to the isoelectric point (pI) of proteins. Systems containing the mixture of lecithin and WPI showed high kinetic instability at pH 3, which was attributed to the electrostatic interaction between the emulsifiers oppositely charged at this pH value. At pH 5.5 and 7, the mixture led to reduction of the droplet size with enhanced emulsion stability compared to the systems with WPI or lecithin. The stability of WPI emulsions after the addition of lecithin, especially at pH 5.5, was associated with the increase of droplet surface charge density. The in vitro digestion evaluation showed that WPI emulsion was more stable against gastrointestinal conditions.

Stability Mechanisms of Laccase Isoforms using a Modified FoldX Protocol Applicable to Widely Different Proteins.

PubMed

Christensen, Niels J; Kepp, Kasper P

2013-07-09

A recent computational protocol that accurately predicts and rationalizes protein multisite mutant stabilities has been extended to handle widely different isoforms of laccases. We apply the protocol to four isoenzymes of Trametes versicolor laccase (TvL) with variable lengths (498-503 residues) and thermostability (Topt ∼ 45-80 °C) and with 67-77% sequence identity. The extended protocol uses (i) statistical averaging, (ii) a molecular-dynamics-validated "compromise" homology model to minimize bias that causes proteins close in sequence to a structural template to be too stable due to having the benefits of the better sampled template (typically from a crystal structure), (iii) correction for hysteresis that favors the input template to overdestabilize, and (iv) a preparative protocol to provide robust input sequences of equal length. The computed ΔΔG values are in good agreement with the major trends in experimental stabilities; that is, the approach may be applicable for fast estimates of the relative stabilities of proteins with as little as 70% identity, something that is currently extremely challenging. The computed stability changes associated with variations are Gaussian-distributed, in good agreement with experimental distributions of stability effects from mutation. The residues causing the differential stability of the four isoforms are consistent with a range of compiled laccase wild type data, suggesting that we may have identified general drivers of laccase stability. Several sites near Cu, notably 79, 241, and 245, or near substrate, mainly 265, are identified that contribute to stability-function trade-offs, of relevance to the search for new proficient and stable variants of these important industrial enzymes.

Nature and consequences of protein-protein interactions in high protein concentration solutions.

PubMed

Saluja, Atul; Kalonia, Devendra S

2008-06-24

High protein concentration solutions are becoming increasingly important in the pharmaceutical industry. The solution behavior of proteins at high concentrations can markedly differ from that predicted based on dilute solution analysis due to thermodynamic non-ideality in these solutions. The non-ideality observed in these systems is related to the protein-protein interactions (PPI). Different types of forces play a key role in determining the overall nature and extent of these PPI and their relative contributions are affected by solute and solvent properties. However, individual contributions of these forces to the solution properties of concentrated protein solutions are not fully understood. The role of PPI, driven by these intermolecular forces, in governing solution rheology and physical stability of high protein concentration solutions is discussed from the point of view of pharmaceutical product development. Investigation of protein self-association and aggregation in concentrated protein solutions is crucial for ensuring the safety and efficacy of the final product for the duration of the desired product shelf life. Understanding rheology of high concentration protein solutions is critical for addressing issues during product manufacture and administration of final formulation to the patient. To this end, analysis of solution viscoelastic character can also provide an insight into the nature of PPI affecting solution rheology.

Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants.

PubMed

Zheng, Xuan; Dong, Shuangshuang; Zheng, Jie; Li, Duanhua; Li, Feng; Luo, Zhongli

2014-01-01

G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration. Copyright © 2014 Elsevier Inc. All rights reserved.

Size-Dependent Protein-Nanoparticle Interactions in Citrate-Stabilized Gold Nanoparticles: The Emergence of the Protein Corona.

PubMed

Piella, Jordi; Bastús, Neus G; Puntes, Víctor

2017-01-18

Surface modifications of highly monodisperse citrate-stabilized gold nanoparticles (AuNPs) with sizes ranging from 3.5 to 150 nm after their exposure to cell culture media supplemented with fetal bovine serum were studied and characterized by the combined use of UV-vis spectroscopy, dynamic light scattering, and zeta potential measurements. In all the tested AuNPs, a dynamic process of protein adsorption was observed, evolving toward the formation of an irreversible hard protein coating known as Protein Corona. Interestingly, the thickness and density of this protein coating were strongly dependent on the particle size, making it possible to identify different transition regimes as the size of the particles increased: (i) NP-protein complexes (or incomplete corona), (ii) the formation of a near-single dense protein corona layer, and (iii) the formation of a multilayer corona. In addition, the different temporal patterns in the evolution of the protein coating came about more quickly for small particles than for the larger ones, further revealing the significant role that size plays in the kinetics of this process. Since the biological identity of the NPs is ultimately determined by the protein corona and different NP-biological interactions take place at different time scales, these results are relevant to biological and toxicological studies.

Heat shock protein 70.1 (Hsp70.1) affects neuronal cell fate by regulating lysosomal acid sphingomyelinase.

PubMed

Zhu, Hong; Yoshimoto, Tanihiro; Yamashima, Tetsumori

2014-10-03

The inducible expression of heat shock protein 70.1 (Hsp70.1) plays cytoprotective roles in its molecular chaperone function. Binding of Hsp70 to an endolysosomal phospholipid, bis(monoacylglycero)phosphate (BMP), has been recently shown to stabilize lysosomal membranes by enhancing acid sphingomyelinase (ASM) activity in cancer cells. Using the monkey experimental paradigm, we have reported that calpain-mediated cleavage of oxidized Hsp70.1 causes neurodegeneration in the hippocampal cornu ammonis 1 (CA1), whereas expression of Hsp70.1 in the motor cortex without calpain activation contributes to neuroprotection. However, the molecular mechanisms of the lysosomal destabilization/stabilization determining neuronal cell fate have not been elucidated. To elucidate whether regulation of lysosomal ASM could affect the neuronal fate, we analyzed Hsp70.1-BMP binding and ASM activity by comparing the motor cortex and the CA1. We show that Hsp70.1 being localized at the lysosomal membrane, lysosomal lipid BMP levels, and the lipid binding domain of Hsp70.1 are crucial for Hsp70.1-BMP binding. In the postischemic motor cortex, Hsp70.1 being localized at the lysosomal membrane could bind to BMP without calpain activation and decreased BMP levels, resulting in increasing ASM activity and lysosomal stability. However, in the postischemic CA1, calpain activation and a concomitant decrease in the lysosomal membrane localization of Hsp70.1 and BMP levels may diminish Hsp70.1-BMP binding, resulting in decreased ASM activity and lysosomal rupture with leakage of cathepsin B into the cytosol. A TUNEL assay revealed the differential neuronal vulnerability between the CA1 and the motor cortex. These results suggest that regulation of ASM activation in vivo by Hsp70.1-BMP affects lysosomal stability and neuronal survival or death after ischemia/reperfusion. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Tryptophan to Glycine mutation in the position 116 leads to protein aggregation and decreases the stability of the LITAF protein.

PubMed

Kumar, Chundi Vinay; Swetha, Rayapadi G; Ramaiah, Sudha; Anbarasu, Anand

2015-01-01

Mutations in the gene-encoding vesicle lipopolysaccharide-induced tumor necrosis factor (LITAF) protein cause Charcot-Marie-Tooth type 1C (CMT1C) disease, a neurological disorder. The LITAF gene is mapped to chromosome number 16 and can be found at cytogenetic location 16p13 of the chromosome. CMT1C-linked small integral membrane protein of lysosome/late endosome mutants are loss-of-function mutants that act in a dominant negative manner to impair endosomal trafficking, leading to prolonged extracellular signal-regulated kinases 1/2 signaling downstream of ErbB activation. Mutation W116G in the LITAF decreases the stability of the protein and also interrupts the functioning of gene. We have analyzed the single nucleotide polymorphism (SNP) results of 28 nsSNPs obtained from dbSNP. We also carried out multiple molecular dynamics simulations of 200 ns and obtained results of root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent-accessible surface area, H-bond, and principal component analysis to check and prove the stability of both the wild type and the mutant. The protein was then checked for its aggregation and the results showed loss of helix. The loss of helix leads to the instability of the protein.

Stability of cardamom (Elettaria cardamomum) essential oil in microcapsules made of whey protein isolate, guar gum, and carrageenan.

PubMed

Mehyar, Ghadeer F; Al-Ismail, Khalid M; Al-Isamil, Khalid M; Al-Ghizzawi, Hana'a M; Holley, Richard A

2014-10-01

The effects of microencapsulating cardamom essential oil (CEO) in whey protein isolate (WPI) alone and combined with guar gum (GG) and carrageen (CG) on microencapsulation efficiency, oil chemical stability, and microcapsule structure were investigated. Freeze-dried microcapsules were prepared from emulsions containing (w/w): 15% and 30% WPI; 0.1% GG, and 0.2% CG as wall materials with CEO (at 10% of polymer concentration) as core material, and physical properties and chemical stability were compared. Bulk density of microcapsules was highest in WPI without GG or CG and in 30% WPI + GG microcapsules, and was more affected by moisture content (r = -0.6) than by mean particle diameter (d43 ; r = -0.2) and span (r = 0.1). Microcapsules containing only WPI had the highest entrapped oil (7.5%) and microencapsulation efficiency (98.5%). The concentrations of 1,8-cineole and d-limonene were used as indicators for microcapsule chemical stability since they were the main components of CEO. Microcapsules retained higher (P ≤ 0.05) concentrations of both components than non-microencapsulated CEO during 16 wk storage at 20 ºC, but higher loss of both components was noted at 35 ºC. Microencapsulated d-limonene was reduced faster than 1,8-cineole regardless of temperature. The 30% WPI and 30% WPI + GG microcapsules retained CEO best throughout storage at both storage temperatures. Scanning electron micrographs revealed that WPI microcapsules had smooth surfaces, were relatively homogenous and regular in shape, whereas GG and CG addition increased visual surface porosity and reduced shape regularity. It was concluded that the best formulation for encapsulating CEO was 30% WPI. Encapsulating cardamom essential oil in whey protein isolate alone or combined with guar gum produced dried powders that effectively retained and chemically stabilized CEO, and therefore enhanced its handling and storability. © 2014 Institute of Food Technologists®

Double-Stranded RNA-Binding Protein Regulates Vascular Endothelial Growth Factor mRNA Stability, Translation, and Breast Cancer Angiogenesis▿

PubMed Central

Vumbaca, Frank; Phoenix, Kathryn N.; Rodriguez-Pinto, Daniel; Han, David K.; Claffey, Kevin P.

2008-01-01

Vascular endothelial growth factor (VEGF) is a key angiogenic factor expressed under restricted nutrient and oxygen conditions in most solid tumors. The expression of VEGF under hypoxic conditions requires transcription through activated hypoxia-inducible factor 1 (HIF-1), increased mRNA stability, and facilitated translation. This study identified double-stranded RNA-binding protein 76/NF90 (DRBP76/NF90), a specific isoform of the DRBP family, as a VEGF mRNA-binding protein which plays a key role in VEGF mRNA stability and protein synthesis under hypoxia. The DRBP76/NF90 protein binds to a human VEGF 3′ untranslated mRNA stability element. RNA interference targeting the DRBP76/NF90 isoform limited hypoxia-inducible VEGF mRNA and protein expression with no change in HIF-1-dependent transcriptional activity. Stable repression of DRBP76/NF90 in MDA-MB-435 breast cancer cells demonstrated reduced polysome-associated VEGF mRNA levels under hypoxic conditions and reduced mRNA stability. Transient overexpression of the DRBP76/NF90 protein increased both VEGF mRNA and protein levels synthesized under normoxic and hypoxic conditions. Cells with stable repression of the DRBP76/NF90 isoform showed reduced tumorigenic and angiogenic potential in an orthotopic breast tumor model. These data demonstrate that the DRBP76/NF90 isoform facilitates VEGF expression by promoting VEGF mRNA loading onto polysomes and translation under hypoxic conditions, thus promoting breast cancer growth and angiogenesis in vivo. PMID:18039850

Complementarity of stability patches at the interfaces of protein complexes: Implication for the structural organization of energetic hot spots.

PubMed

Kuttner, Yosef Y; Engel, Stanislav

2018-02-01

A rational design of protein complexes with defined functionalities and of drugs aimed at disrupting protein-protein interactions requires fundamental understanding of the mechanisms underlying the formation of specific protein complexes. Efforts to develop efficient small-molecule or protein-based binders often exploit energetic hot spots on protein surfaces, namely, the interfacial residues that provide most of the binding free energy in the complex. The molecular basis underlying the unusually high energy contribution of the hot spots remains obscure, and its elucidation would facilitate the design of interface-targeted drugs. To study the nature of the energetic hot spots, we analyzed the backbone dynamic properties of contact surfaces in several protein complexes. We demonstrate that, in most complexes, the backbone dynamic landscapes of interacting surfaces form complementary "stability patches," in which static areas from the opposing surfaces superimpose, and that these areas are predominantly located near the geometric center of the interface. We propose that a diminished enthalpy-entropy compensation effect augments the degree to which residues positioned within the complementary stability patches contribute to complex affinity, thereby giving rise to the energetic hot spots. These findings offer new insights into the nature of energetic hot spots and the role that backbone dynamics play in facilitating intermolecular recognition. Mapping the interfacial stability patches may provide guidance for protein engineering approaches aimed at improving the stability of protein complexes and could facilitate the design of ligands that target complex interfaces. © 2017 Wiley Periodicals, Inc.

Effects of monohydric alcohols and polyols on the thermal stability of a protein

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

Murakami, Shota; Kinoshita, Masahiro, E-mail: kinoshit@iae.kyoto-u.ac.jp

2016-03-28

The thermal stability of a protein is lowered by the addition of a monohydric alcohol, and this effect becomes larger as the size of hydrophobic group in an alcohol molecule increases. By contrast, it is enhanced by the addition of a polyol possessing two or more hydroxyl groups per molecule, and this effect becomes larger as the number of hydroxyl groups increases. Here, we show that all of these experimental observations can be reproduced even in a quantitative sense by rigid-body models focused on the entropic effect originating from the translational displacement of solvent molecules. The solvent is either puremore » water or water-cosolvent solution. Three monohydric alcohols and five polyols are considered as cosolvents. In the rigid-body models, a protein is a fused hard spheres accounting for the polyatomic structure in the atomic detail, and the solvent is formed by hard spheres or a binary mixture of hard spheres with different diameters. The effective diameter of cosolvent molecules and the packing fractions of water and cosolvent, which are crucially important parameters, are carefully estimated using the experimental data of properties such as the density of solid crystal of cosolvent, parameters in the pertinent cosolvent-cosolvent interaction potential, and density of water-cosolvent solution. We employ the morphometric approach combined with the integral equation theory, which is best suited to the physical interpretation of the calculation result. It is argued that the degree of solvent crowding in the bulk is the key factor. When it is made more serious by the cosolvent addition, the solvent-entropy gain upon protein folding is magnified, leading to the enhanced thermal stability. When it is made less serious, the opposite is true. The mechanism of the effects of monohydric alcohols and polyols is physically the same as that of sugars. However, when the rigid-body models are employed for the effect of urea, its addition is predicted to enhance

Effects of monohydric alcohols and polyols on the thermal stability of a protein

NASA Astrophysics Data System (ADS)

Murakami, Shota; Kinoshita, Masahiro

2016-03-01

The thermal stability of a protein is lowered by the addition of a monohydric alcohol, and this effect becomes larger as the size of hydrophobic group in an alcohol molecule increases. By contrast, it is enhanced by the addition of a polyol possessing two or more hydroxyl groups per molecule, and this effect becomes larger as the number of hydroxyl groups increases. Here, we show that all of these experimental observations can be reproduced even in a quantitative sense by rigid-body models focused on the entropic effect originating from the translational displacement of solvent molecules. The solvent is either pure water or water-cosolvent solution. Three monohydric alcohols and five polyols are considered as cosolvents. In the rigid-body models, a protein is a fused hard spheres accounting for the polyatomic structure in the atomic detail, and the solvent is formed by hard spheres or a binary mixture of hard spheres with different diameters. The effective diameter of cosolvent molecules and the packing fractions of water and cosolvent, which are crucially important parameters, are carefully estimated using the experimental data of properties such as the density of solid crystal of cosolvent, parameters in the pertinent cosolvent-cosolvent interaction potential, and density of water-cosolvent solution. We employ the morphometric approach combined with the integral equation theory, which is best suited to the physical interpretation of the calculation result. It is argued that the degree of solvent crowding in the bulk is the key factor. When it is made more serious by the cosolvent addition, the solvent-entropy gain upon protein folding is magnified, leading to the enhanced thermal stability. When it is made less serious, the opposite is true. The mechanism of the effects of monohydric alcohols and polyols is physically the same as that of sugars. However, when the rigid-body models are employed for the effect of urea, its addition is predicted to enhance the

Protein Stabilization and Enzyme Activation in Ionic Liquids: Specific Ion Effects

PubMed Central

Zhao, Hua

2015-01-01

There are still debates on whether the hydration of ions perturbs the water structure, and what is the degree of such disturbance; therefore, the origin of Hofmeister effect on protein stabilization continues being questioned. For this reason, it is suggested to use the ‘specific ion effect’ instead of other misleading terms such as Hofmeister effect, Hofmeister series, lyotropic effect, and lyotropic series. In this review, we firstly discuss the controversial aspect of inorganic ion effects on water structures, and several possible contributors to the specific ion effect of protein stability. Due to recent overwhelming attraction of ionic liquids (ILs) as benign solvents in many enzymatic reactions, we further evaluate the structural properties and molecular-level interactions in neat ILs and their aqueous solutions. Next, we systematically compare the specific ion effects of ILs on enzyme stability and activity, and conclude that (a) the specificity of many enzymatic systems in diluted aqueous IL solutions is roughly in line with the traditional Hofmeister series albeit some exceptions; (b) however, the specificity follows a different track in concentrated or neat ILs because other factors (such as hydrogen-bond basicity, nucelophilicity, and hydrophobicity, etc) are playing leading roles. In addition, we demonstrate some examples of biocatalytic reactions in IL systems that are guided by the empirical specificity rule. PMID:26949281

Enhancement of Peroxidase Stability Against Oxidative Self-Inactivation by Co-immobilization with a Redox-Active Protein in Mesoporous Silicon and Silica Microparticles

NASA Astrophysics Data System (ADS)

Sahare, P.; Ayala, M.; Vazquez-Duhalt, R.; Pal, U.; Loni, A.; Canham, L. T.; Osorio, I.; Agarwal, V.

2016-09-01

The study of the stability enhancement of a peroxidase immobilized onto mesoporous silicon/silica microparticles is presented. Peroxidases tend to get inactivated in the presence of hydrogen peroxide, their essential co-substrate, following an auto-inactivation mechanism. In order to minimize this inactivation, a second protein was co-immobilized to act as an electron acceptor and thus increase the stability against self-oxidation of peroxidase. Two heme proteins were immobilized into the microparticles: a fungal commercial peroxidase and cytochrome c from equine heart. Two types of biocatalysts were prepared: one with only covalently immobilized peroxidase (one-protein system) and another based on covalent co-immobilization of peroxidase and cytochrome c (two-protein system), both immobilized by using carbodiimide chemistry. The amount of immobilized protein was estimated spectrophotometrically, and the characterization of the biocatalyst support matrix was performed using Brunauer-Emmett-Teller (BET), scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX), and Fourier transform infrared (FTIR) analyses. Stability studies show that co-immobilization with the two-protein system enhances the oxidative stability of peroxidase almost four times with respect to the one-protein system. Thermal stability analysis shows that the immobilization of peroxidase in derivatized porous silicon microparticles does not protect the protein from thermal denaturation, whereas biogenic silica microparticles confer significant thermal stabilization.

Structural basis for the enhanced stability of protein model compounds and peptide backbone unit in ammonium ionic liquids.

PubMed

Vasantha, T; Attri, Pankaj; Venkatesu, Pannuru; Devi, R S Rama

2012-10-04

Protein folding/unfolding is a fascinating study in the presence of cosolvents, which protect/disrupt the native structure of protein, respectively. The structure and stability of proteins and their functional groups may be modulated by the addition of cosolvents. Ionic liquids (ILs) are finding a vast array of applications as novel cosolvents for a wide variety of biochemical processes that include protein folding. Here, the systematic and quantitative apparent transfer free energies (ΔG'(tr)) of protein model compounds from water to ILs through solubility measurements as a function of IL concentration at 25 °C have been exploited to quantify and interpret biomolecular interactions between model compounds of glycine peptides (GPs) with ammonium based ILs. The investigated aqueous systems consist of zwitterionic glycine peptides: glycine (Gly), diglycine (Gly(2)), triglycine (Gly(3)), tetraglycine (Gly(4)), and cyclic glycylglycine (c(GG)) in the presence of six ILs such as diethylammonium acetate (DEAA), diethylammonium hydrogen sulfate (DEAS), triethylammonium acetate (TEAA), triethylammonium hydrogen sulfate (TEAS), triethylammonium dihydrogen phosphate (TEAP), and trimethylammonium acetate (TMAA). We have observed positive values of ΔG'(tr) for GPs from water to ILs, indicating that interactions between ILs and GPs are unfavorable, which leads to stabilization of the structure of model protein compounds. Moreover, our experimental data ΔG'(tr) is used to obtain transfer free energies (Δg'(tr)) of the peptide backbone unit (or glycyl unit) (-CH(2)C═ONH-), which is the most numerous group in globular proteins, from water to IL solutions. To obtain the mechanism events of the ILs' role in enhancing the stability of the model compounds, we have further obtained m-values for GPs from solubility limits. These results explicitly elucidate that all alkyl ammonium ILs act as stabilizers for model compounds through the exclusion of ILs from model compounds of

Improving the accuracy of protein stability predictions with multistate design using a variety of backbone ensembles.

PubMed

Davey, James A; Chica, Roberto A

2014-05-01

Multistate computational protein design (MSD) with backbone ensembles approximating conformational flexibility can predict higher quality sequences than single-state design with a single fixed backbone. However, it is currently unclear what characteristics of backbone ensembles are required for the accurate prediction of protein sequence stability. In this study, we aimed to improve the accuracy of protein stability predictions made with MSD by using a variety of backbone ensembles to recapitulate the experimentally measured stability of 85 Streptococcal protein G domain β1 sequences. Ensembles tested here include an NMR ensemble as well as those generated by molecular dynamics (MD) simulations, by Backrub motions, and by PertMin, a new method that we developed involving the perturbation of atomic coordinates followed by energy minimization. MSD with the PertMin ensembles resulted in the most accurate predictions by providing the highest number of stable sequences in the top 25, and by correctly binning sequences as stable or unstable with the highest success rate (≈90%) and the lowest number of false positives. The performance of PertMin ensembles is due to the fact that their members closely resemble the input crystal structure and have low potential energy. Conversely, the NMR ensemble as well as those generated by MD simulations at 500 or 1000 K reduced prediction accuracy due to their low structural similarity to the crystal structure. The ensembles tested herein thus represent on- or off-target models of the native protein fold and could be used in future studies to design for desired properties other than stability. Copyright © 2013 Wiley Periodicals, Inc.

The von Hippel-Lindau protein sensitizes renal carcinoma cells to apoptotic stimuli through stabilization of BIM(EL).

PubMed

Guo, Y; Schoell, M C; Freeman, R S

2009-04-23

von Hippel-Lindau (VHL) disease is caused by germ-line mutations in the VHL tumor suppressor gene and is the most common cause of inherited renal cell carcinoma (RCC). Mutations in the VHL gene also occur in a large majority of sporadic cases of clear-cell RCC, which have high intrinsic resistance to chemotherapy and radiotherapy. Here we show that VHL-deficient RCC cells express lower levels of the proapoptotic Bcl-2 family protein BIM(EL) and are more resistant to etoposide and UV radiation-induced death compared to the same cells stably expressing the wild-type VHL protein (pVHL). Reintroducing pVHL into VHL-null cells increased the half-life of BIM(EL) protein without affecting its mRNA expression, and overexpressing pVHL inhibited BIM(EL) polyubiquitination. Suppressing pVHL expression with RNA interference resulted in a decrease in BIM(EL) protein and a corresponding decrease in the sensitivity of RCC cells to apoptotic stimuli. Directly inhibiting BIM(EL) expression in pVHL-expressing RCC cells caused a similar decrease in cell death. These results demonstrate that pVHL acts to promote BIM(EL) protein stability in RCC cells, and that destabilization of BIM(EL) in the absence of pVHL contributes to the increased resistance of VHL-null RCC cells to certain apoptotic stimuli.

Influence of protein-pectin electrostatic interaction on the foam stability mechanism.

PubMed

Sadahira, Mitie S; Lopes, Fernanda C Rezende; Rodrigues, Maria I; Netto, Flavia M

2014-03-15

This study aimed at evaluating the effect of three independent variables: biopolymer concentration (egg white proteins and pectin) (2.0-4.0%, w/w); protein:pectin ratio (15:1-55:1); and temperature (70-80 °C), at pH 3.0, using a central composite design on the foaming properties (overrun, drainage and bubble growth rate). Foams produced with protein:pectin ratio 15:1 showed the lowest bubble growth rate and the greatest drainage, whereas protein:pectin ratio 55:1 presented the lowest drainage. Complexes obtained with protein:pectin ratio 15:1 were close to electroneutrality and showed larger size (95.91 ± 8.19 μm) than those obtained with protein:pectin ratio 55:1 (45.92 ± 3.47 μm) not electrically neutral. Larger particles seemed to build an interfacial viscoelastic network at the air-water interface with reduced gas permeability, leading to greater stability concerning the disproportionation. Soluble complexes of smaller sizes increased viscosity leading to a low drainage of liquid and inhibiting the bubbles coalescence. Copyright © 2013 Elsevier Ltd. All rights reserved.

In-frame seven amino-acid duplication in AIP arose over the last 3000 years, disrupts protein interaction and stability and is associated with gigantism

PubMed Central

Salvatori, Roberto; Radian, Serban; Diekmann, Yoan; Iacovazzo, Donato; David, Alessia; Gabrovska, Plamena; Grassi, Giorgia; Bussell, Anna-Marie; Stals, Karen; Weber, Astrid; Quinton, Richard; Crowne, Elizabeth C; Corazzini, Valentina; Metherell, Lou; Kearney, Tara; Du Plessis, Daniel; Sinha, Ajay Kumar; Baborie, Atik; Lecoq, Anne-Lise; Chanson, Philippe; Ansorge, Olaf; Ellard, Sian; Trainer, Peter J; Balding, David; Thomas, Mark G

2017-01-01

Objective Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene are associated with pituitary adenoma, acromegaly and gigantism. Identical alleles in unrelated pedigrees could be inherited from a common ancestor or result from recurrent mutation events. Design and methods Observational, inferential and experimental study, including: AIP mutation testing; reconstruction of 14 AIP-region (8.3 Mbp) haplotypes; coalescent-based approximate Bayesian estimation of the time to most recent common ancestor (tMRCA) of the derived allele; forward population simulations to estimate current number of allele carriers; proposal of mutation mechanism; protein structure predictions; co-immunoprecipitation and cycloheximide chase experiments. Results Nine European-origin, unrelated c.805_825dup-positive pedigrees (four familial, five sporadic from the UK, USA and France) included 16 affected (nine gigantism/four acromegaly/two non-functioning pituitary adenoma patients and one prospectively diagnosed acromegaly patient) and nine unaffected carriers. All pedigrees shared a 2.79 Mbp haploblock around AIP with additional haploblocks privately shared between subsets of the pedigrees, indicating the existence of an evolutionarily recent common ancestor, the ‘English founder’, with an estimated median tMRCA of 47 generations (corresponding to 1175 years) with a confidence interval (9–113 generations, equivalent to 225–2825 years). The mutation occurred in a small tandem repeat region predisposed to slipped strand mispairing. The resulting seven amino-acid duplication disrupts interaction with HSP90 and leads to a marked reduction in protein stability. Conclusions The c.805_825dup allele, originating from a common ancestor, associates with a severe clinical phenotype and a high frequency of gigantism. The mutation is likely to be the result of slipped strand mispairing and affects protein–protein interactions and AIP protein stability. PMID:28634279

On the effect of hydrostatic pressure on the conformational stability of globular proteins.

PubMed

Graziano, Giuseppe

2015-12-01

The model developed for cold denaturation (Graziano, PCCP 2010, 12, 14245-14252) is extended to rationalize the dependence of protein conformational stability upon hydrostatic pressure, at room temperature. A pressure- volume work is associated with the process of cavity creation for the need to enlarge the liquid volume against hydrostatic pressure. This contribution destabilizes the native state that has a molecular volume slightly larger than the denatured state due to voids existing in the protein core. Therefore, there is a hydrostatic pressure value at which the pressure-volume contribution plus the conformational entropy loss of the polypeptide chain are able to overwhelm the stabilizing gain in translational entropy of water molecules, due to the decrease in water accessible surface area upon folding, causing denaturation. © 2015 Wiley Periodicals, Inc.

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

The Extracellular Matrix Protein TGFBI Induces Microtubule Stabilization and Sensitizes Ovarian Cancers to Paclitaxel

PubMed Central

Ahmed, Ahmed Ashour; Mills, Anthony D.; Ibrahim, Ashraf E.K.; Temple, Jillian; Blenkiron, Cherie; Vias, Maria; Massie, Charlie E.; Iyer, N. Gopalakrishna; McGeoch, Adam; Crawford, Robin; Nicke, Barbara; Downward, Julian; Swanton, Charles; Bell, Stephen D.; Earl, Helena M.; Laskey, Ronald A.; Caldas, Carlos; Brenton, James D.

2007-01-01

Summary The extracellular matrix (ECM) can induce chemotherapy resistance via AKT-mediated inhibition of apoptosis. Here, we show that loss of the ECM protein TGFBI (transforming growth factor beta induced) is sufficient to induce specific resistance to paclitaxel and mitotic spindle abnormalities in ovarian cancer cells. Paclitaxel-resistant cells treated with recombinant TGFBI protein show integrin-dependent restoration of paclitaxel sensitivity via FAK- and Rho-dependent stabilization of microtubules. Immunohistochemical staining for TGFBI in paclitaxel-treated ovarian cancers from a prospective clinical trial showed that morphological changes of paclitaxel-induced cytotoxicity were restricted to areas of strong expression of TGFBI. These data show that ECM can mediate taxane sensitivity by modulating microtubule stability. PMID:18068629

The extracellular matrix protein TGFBI induces microtubule stabilization and sensitizes ovarian cancers to paclitaxel.

PubMed

Ahmed, Ahmed Ashour; Mills, Anthony D; Ibrahim, Ashraf E K; Temple, Jillian; Blenkiron, Cherie; Vias, Maria; Massie, Charlie E; Iyer, N Gopalakrishna; McGeoch, Adam; Crawford, Robin; Nicke, Barbara; Downward, Julian; Swanton, Charles; Bell, Stephen D; Earl, Helena M; Laskey, Ronald A; Caldas, Carlos; Brenton, James D

2007-12-01

The extracellular matrix (ECM) can induce chemotherapy resistance via AKT-mediated inhibition of apoptosis. Here, we show that loss of the ECM protein TGFBI (transforming growth factor beta induced) is sufficient to induce specific resistance to paclitaxel and mitotic spindle abnormalities in ovarian cancer cells. Paclitaxel-resistant cells treated with recombinant TGFBI protein show integrin-dependent restoration of paclitaxel sensitivity via FAK- and Rho-dependent stabilization of microtubules. Immunohistochemical staining for TGFBI in paclitaxel-treated ovarian cancers from a prospective clinical trial showed that morphological changes of paclitaxel-induced cytotoxicity were restricted to areas of strong expression of TGFBI. These data show that ECM can mediate taxane sensitivity by modulating microtubule stability.

Application of a high-throughput relative chemical stability assay to screen therapeutic protein formulations by assessment of conformational stability and correlation to aggregation propensity.

PubMed

Rizzo, Joseph M; Shi, Shuai; Li, Yunsong; Semple, Andrew; Esposito, Jessica J; Yu, Shenjiang; Richardson, Daisy; Antochshuk, Valentyn; Shameem, Mohammed

2015-05-01

In this study, an automated high-throughput relative chemical stability (RCS) assay was developed in which various therapeutic proteins were assessed to determine stability based on the resistance to denaturation post introduction to a chaotrope titration. Detection mechanisms of both intrinsic fluorescence and near UV circular dichroism (near-UV CD) are demonstrated. Assay robustness was investigated by comparing multiple independent assays and achieving r(2) values >0.95 for curve overlays. The complete reversibility of the assay was demonstrated by intrinsic fluorescence, near-UV CD, and biologic potency. To highlight the method utility, we compared the RCS assay with differential scanning calorimetry and dynamic scanning fluorimetry methodologies. Utilizing C1/2 values obtained from the RCS assay, formulation rank-ordering of 12 different mAb formulations was performed. The prediction of long-term stability on protein aggregation is obtained by demonstrating a good correlation with an r(2) of 0.83 between RCS and empirical aggregation propensity data. RCS promises to be an extremely useful tool to aid in candidate formulation development efforts based on the complete reversibility of the method to allow for multiple assessments without protein loss and the strong correlation between the C1/2 data obtained and accelerated stability under stressed conditions. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Skp1 prolyl 4-hydroxylase of dictyostelium mediates glycosylation-independent and -dependent responses to O2 without affecting Skp1 stability.

PubMed

Zhang, Dongmei; van der Wel, Hanke; Johnson, Jennifer M; West, Christopher M

2012-01-13

Cytoplasmic prolyl 4-hydroxylases (PHDs) have a primary role in O(2) sensing in animals via modification of the transcriptional factor subunit HIFα, resulting in its polyubiquitination by the E3(VHL)ubiquitin (Ub) ligase and degradation in the 26 S proteasome. Previously thought to be restricted to animals, a homolog (P4H1) of HIFα-type PHDs is expressed in the social amoeba Dictyostelium where it also exhibits characteristics of an O(2) sensor for development. Dictyostelium lacks HIFα, and P4H1 modifies a different protein, Skp1, an adaptor of the SCF class of E3-Ub ligases related to the E3(VHL)Ub ligase that targets animal HIFα. Normally, the HO-Skp1 product of the P4H1 reaction is capped by a GlcNAc sugar that can be subsequently extended to a pentasaccharide by novel glycosyltransferases. To analyze the role of glycosylation, the Skp1 GlcNAc-transferase locus gnt1 was modified with a missense mutation to block catalysis or a stop codon to truncate the protein. Despite the accumulation of the hydroxylated form of Skp1, Skp1 was not destabilized based on metabolic labeling. However, hydroxylation alone allowed for partial correction of the high O(2) requirement of P4H1-null cells, therefore revealing both glycosylation-independent and glycosylation-dependent roles for hydroxylation. Genetic complementation of the latter function required an enzymatically active form of Gnt1. Because the effect of the gnt1 deficiency depended on P4H1, and Skp1 was the only protein labeled when the GlcNAc-transferase was restored to mutant extracts, Skp1 apparently mediates the cellular functions of both P4H1 and Gnt1. Although Skp1 stability itself is not affected by hydroxylation, its modification may affect the stability of targets of Skp1-dependent Ub ligases.

Physical and molecular bases of protein thermal stability and cold adaptation.

PubMed

Pucci, Fabrizio; Rooman, Marianne

2017-02-01

The molecular bases of thermal and cold stability and adaptation, which allow proteins to remain folded and functional in the temperature ranges in which their host organisms live and grow, are still only partially elucidated. Indeed, both experimental and computational studies fail to yield a fully precise and global physical picture, essentially because all effects are context-dependent and thus quite intricate to unravel. We present a snapshot of the current state of knowledge of this highly complex and challenging issue, whose resolution would enable large-scale rational protein design. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

Plant Protein and Animal Proteins: Do They Differentially Affect Cardiovascular Disease Risk?12

PubMed Central

Richter, Chesney K; Skulas-Ray, Ann C; Champagne, Catherine M; Kris-Etherton, Penny M

2015-01-01

Proteins from plant-based compared with animal-based food sources may have different effects on cardiovascular disease (CVD) risk factors. Numerous epidemiologic and intervention studies have evaluated their respective health benefits; however, it is difficult to isolate the role of plant or animal protein on CVD risk. This review evaluates the current evidence from observational and intervention studies, focusing on the specific protein-providing foods and populations studied. Dietary protein is derived from many food sources, and each provides a different composite of nonprotein compounds that can also affect CVD risk factors. Increasing the consumption of protein-rich foods also typically results in lower intakes of other nutrients, which may simultaneously influence outcomes. Given these complexities, blanket statements about plant or animal protein may be too general, and greater consideration of the specific protein food sources and the background diet is required. The potential mechanisms responsible for any specific effects of plant and animal protein are similarly multifaceted and include the amino acid content of particular foods, contributions from other nonprotein compounds provided concomitantly by the whole food, and interactions with the gut microbiome. Evidence to date is inconclusive, and additional studies are needed to further advance our understanding of the complexity of plant protein vs. animal protein comparisons. Nonetheless, current evidence supports the idea that CVD risk can be reduced by a dietary pattern that provides more plant sources of protein compared with the typical American diet and also includes animal-based protein foods that are unprocessed and low in saturated fat. PMID:26567196

Stability of milk fat globule membrane proteins toward human enzymatic gastrointestinal digestion.

PubMed

Le, T T; Van de Wiele, T; Do, T N H; Debyser, G; Struijs, K; Devreese, B; Dewettinck, K; Van Camp, J

2012-05-01

The milk fat globule membrane (MFGM) fraction refers to the thin film of polar lipids and membrane proteins that surrounds fat globules in milk. It is its unique biochemical composition that renders MFGM with some beneficial biological activities, such as anti-adhesive effects toward pathogens. However, a prerequisite for the putative bioactivity of MFGM is its stability during gastrointestinal digestion. We, therefore, subjected MFGM material, isolated from raw milk, to an in vitro enzymatic gastrointestinal digestion. Sodium dodecyl sulfate PAGE, in combination with 2 staining methods, Coomassie Blue and periodic acid Schiff staining, was used to evaluate polypeptide patterns of the digest, whereas mass spectrometry was used to confirm the presence of specific MFGM proteins. Generally, it was observed that glycoproteins showed higher resistance to endogenous proteases compared with non-glycosylated proteins. Mucin 1 displayed the highest resistance to digestion and a considerable part of this protein was still detected at its original molecular weight after gastric and small intestine digestion. Cluster of differentiation 36 was also quite resistant to pepsin. A significant part of periodic acid Schiff 6/7 survived the gastric digestion, provided that the lipid moiety was not removed from the MFGM material. Overall, MFGM glycoproteins are generally more resistant to gastrointestinal digestion than serum milk proteins and the presence of lipids, besides glycosylation, may protect MFGM glycoproteins from gastrointestinal digestion. This gastrointestinal stability makes MFGM glycoproteins amenable to further studies in which their putative health-promoting effects can be explored. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Dietary protein level affects iridescent coloration in Anna's hummingbirds, Calypte anna

PubMed Central

Meadows, Melissa G.; Roudybush, Thomas E.; McGraw, Kevin J.

2012-01-01

SUMMARY Many animal displays involve colorful ornamental traits that signal an individual's quality as a mate or rival. Brilliant iridescent ornaments are common, but little is currently known about their production cost and signaling value. One potential cost of colorful ornaments is the acquisition of limited dietary resources that may be involved, directly or indirectly, in their production. Protein, the primary component of bird feathers and of many nanostructural components of iridescent traits, is naturally restricted in hummingbird diets (comprised mostly of sugars), suggesting that iridescent coloration may be especially challenging to produce in these animals. In this study, we experimentally investigated the effect of dietary protein availability during molt on iridescent color expression in male Anna's hummingbirds (Calypte anna). We fed captive birds either a 6% (high) or a 3% (low) protein diet and stimulated molt by plucking half the gorget and crown ornaments on each bird as well as the non-ornamental iridescent green tail feathers. We found that birds receiving more protein grew significantly more colorful crown feathers (higher red chroma and redder hue) than those fed the low-protein diet. Diet did not affect gorget coloration, but regrowth of feathers in captivity affected both gorget and crown coloration. Additionally, birds on the high-protein diet grew yellower (higher hue) green tail feathers than birds on the low-protein diet. These results indicate that iridescent ornamental feathers are sensitive to diet quality and may serve as honest signals of nutrition to mates or rivals. Further, because both ornamental and non-ornamental iridescent coloration were affected by conditions during their growth, iridescent color in these birds appears to be generally condition dependent. PMID:22837446

Dietary protein level affects iridescent coloration in Anna's hummingbirds, Calypte anna.

PubMed

Meadows, Melissa G; Roudybush, Thomas E; McGraw, Kevin J

2012-08-15

Many animal displays involve colorful ornamental traits that signal an individual's quality as a mate or rival. Brilliant iridescent ornaments are common, but little is currently known about their production cost and signaling value. One potential cost of colorful ornaments is the acquisition of limited dietary resources that may be involved, directly or indirectly, in their production. Protein, the primary component of bird feathers and of many nanostructural components of iridescent traits, is naturally restricted in hummingbird diets (comprised mostly of sugars), suggesting that iridescent coloration may be especially challenging to produce in these animals. In this study, we experimentally investigated the effect of dietary protein availability during molt on iridescent color expression in male Anna's hummingbirds (Calypte anna). We fed captive birds either a 6% (high) or a 3% (low) protein diet and stimulated molt by plucking half the gorget and crown ornaments on each bird as well as the non-ornamental iridescent green tail feathers. We found that birds receiving more protein grew significantly more colorful crown feathers (higher red chroma and redder hue) than those fed the low-protein diet. Diet did not affect gorget coloration, but regrowth of feathers in captivity affected both gorget and crown coloration. Additionally, birds on the high-protein diet grew yellower (higher hue) green tail feathers than birds on the low-protein diet. These results indicate that iridescent ornamental feathers are sensitive to diet quality and may serve as honest signals of nutrition to mates or rivals. Further, because both ornamental and non-ornamental iridescent coloration were affected by conditions during their growth, iridescent color in these birds appears to be generally condition dependent.

ATP-stabilized amorphous calcium carbonate nanospheres and their application in protein adsorption.

PubMed

Qi, Chao; Zhu, Ying-Jie; Lu, Bing-Qiang; Zhao, Xin-Yu; Zhao, Jing; Chen, Feng; Wu, Jin

2014-05-28

Calcium carbonate is a common substance found in rocks worldwide, and is the main biomineral formed in shells of marine organisms and snails, pearls and eggshells. Amorphous calcium carbonate (ACC) is the least stable polymorph of calcium carbonate, which is so unstable under normal conditions that it is difficult to be prepared in vitro because it rapidly crystallizes to form one of the more stable polymorphs in aqueous solution. Herein, we report the successful synthesis of highly stable ACC nanospheres in vitro using adenosine 5'-triphosphate disodium salt (ATP) as a stabilizer. The effect of ATP on the stability of ACC nanospheres is investigated. Our experiments show that ATP plays an unique role in the stabilization of ACC nanospheres in aqueous solution. Moreover, the as-prepared ACC nanospheres are highly stable in phosphate buffered saline for a relatively long period of time (12 days) even under relatively high concentrations of calcium and phosphate ions. The cytotoxicity tests show that the as-prepared highly stable ACC nanospheres have excellent biocompatibility. The highly stable ACC nanospheres have high protein adsorption capacity, implying that they are promising for applications in biomedical fields such as drug delivery and protein adsorption. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization

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

Rutz, Natalja; Heilbronn, Regine; Weger, Stefan, E-mail: stefan.weger@charite.de

2015-08-28

Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 inducedmore » polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability. - Highlights: • KCTD5 nuclear translocation depends upon M phase and protein oligomerization. • Identification of MCM7, ZNF711 and FAM193 as KCTD5 interaction partners. • Formation of trimeric complexes of KCTD5/cullin3 with MCM7, ZNF711 and FAM193B. • KCTD5 is not involved in polyubiquitylation of MCM7 replication factor. • The KCTD5/cullin3 complex stabilizes ZNF711 transcription factor.« less

Protein denaturants at aqueous-hydrophobic interfaces: self-consistent correlation between induced interfacial fluctuations and denaturant stability at the interface.

PubMed

Cui, Di; Ou, Shu-Ching; Patel, Sandeep

2015-01-08

The notion of direct interaction between denaturing cosolvent and protein residues has been proposed in dialogue relevant to molecular mechanisms of protein denaturation. Here we consider the correlation between free energetic stability and induced fluctuations of an aqueous-hydrophobic interface between a model hydrophobically associating protein, HFBII, and two common protein denaturants, guanidinium cation (Gdm(+)) and urea. We compute potentials of mean force along an order parameter that brings the solute molecule close to the known hydrophobic region of the protein. We assess potentials of mean force for different relative orientations between the protein and denaturant molecule. We find that in both cases of guanidinium cation and urea relative orientations of the denaturant molecule that are parallel to the local protein-water interface exhibit greater stability compared to edge-on or perpendicular orientations. This behavior has been observed for guanidinium/methylguanidinium cations at the liquid-vapor interface of water, and thus the present results further corroborate earlier findings. Further analysis of the induced fluctuations of the aqueous-hydrophobic interface upon approach of the denaturant molecule indicates that the parallel orientation, displaying a greater stability at the interface, also induces larger fluctuations of the interface compared to the perpendicular orientations. The correlation of interfacial stability and induced interface fluctuation is a recurring theme for interface-stable solutes at hydrophobic interfaces. Moreover, observed correlations between interface stability and induced fluctuations recapitulate connections to local hydration structure and patterns around solutes as evidenced by experiment (Cooper et al., J. Phys. Chem. A 2014, 118, 5657.) and high-level ab initio/DFT calculations (Baer et al., Faraday Discuss 2013, 160, 89).

Protein comparability assessments and potential applicability of high throughput biophysical methods and data visualization tools to compare physical stability profiles

PubMed Central

Alsenaidy, Mohammad A.; Jain, Nishant K.; Kim, Jae H.; Middaugh, C. Russell; Volkin, David B.

2014-01-01

In this review, some of the challenges and opportunities encountered during protein comparability assessments are summarized with an emphasis on developing new analytical approaches to better monitor higher-order protein structures. Several case studies are presented using high throughput biophysical methods to collect protein physical stability data as function of temperature, agitation, ionic strength and/or solution pH. These large data sets were then used to construct empirical phase diagrams (EPDs), radar charts, and comparative signature diagrams (CSDs) for data visualization and structural comparisons between the different proteins. Protein samples with different sizes, post-translational modifications, and inherent stability are presented: acidic fibroblast growth factor (FGF-1) mutants, different glycoforms of an IgG1 mAb prepared by deglycosylation, as well as comparisons of different formulations of an IgG1 mAb and granulocyte colony stimulating factor (GCSF). Using this approach, differences in structural integrity and conformational stability profiles were detected under stress conditions that could not be resolved by using the same techniques under ambient conditions (i.e., no stress). Thus, an evaluation of conformational stability differences may serve as an effective surrogate to monitor differences in higher-order structure between protein samples. These case studies are discussed in the context of potential utility in protein comparability studies. PMID:24659968

Protein comparability assessments and potential applicability of high throughput biophysical methods and data visualization tools to compare physical stability profiles.

PubMed

Alsenaidy, Mohammad A; Jain, Nishant K; Kim, Jae H; Middaugh, C Russell; Volkin, David B

2014-01-01

In this review, some of the challenges and opportunities encountered during protein comparability assessments are summarized with an emphasis on developing new analytical approaches to better monitor higher-order protein structures. Several case studies are presented using high throughput biophysical methods to collect protein physical stability data as function of temperature, agitation, ionic strength and/or solution pH. These large data sets were then used to construct empirical phase diagrams (EPDs), radar charts, and comparative signature diagrams (CSDs) for data visualization and structural comparisons between the different proteins. Protein samples with different sizes, post-translational modifications, and inherent stability are presented: acidic fibroblast growth factor (FGF-1) mutants, different glycoforms of an IgG1 mAb prepared by deglycosylation, as well as comparisons of different formulations of an IgG1 mAb and granulocyte colony stimulating factor (GCSF). Using this approach, differences in structural integrity and conformational stability profiles were detected under stress conditions that could not be resolved by using the same techniques under ambient conditions (i.e., no stress). Thus, an evaluation of conformational stability differences may serve as an effective surrogate to monitor differences in higher-order structure between protein samples. These case studies are discussed in the context of potential utility in protein comparability studies.

Biophysical analysis of the effect of chemical modification by 4-oxononenal on the structure, stability, and function of binding immunoglobulin protein (BiP)

PubMed Central

Shah, Dinen D.; Singh, Surinder M.; Dzieciatkowska, Monika

2017-01-01

Binding immunoglobulin protein (BiP) is a molecular chaperone important for the folding of numerous proteins, which include millions of immunoglobulins in human body. It also plays a key role in the unfolded protein response (UPR) in the endoplasmic reticulum. Free radical generation is a common phenomenon that occurs in cells under healthy as well as under stress conditions such as ageing, inflammation, alcohol consumption, and smoking. These free radicals attack the cell membranes and generate highly reactive lipid peroxidation products such as 4-oxononenal (4-ONE). BiP is a key protein that is modified by 4-ONE. In this study, we probed how such chemical modification affects the biophysical properties of BiP. Upon modification, BiP shows significant tertiary structural changes with no changes in its secondary structure. The protein loses its thermodynamic stability, particularly, that of the nucleotide binding domain (NBD) where ATP binds. In terms of function, the modified BiP completely loses its ATPase activity with decreased ATP binding affinity. However, modified BiP retains its immunoglobulin binding function and its chaperone activity of suppressing non-specific protein aggregation. These results indicate that 4-ONE modification can significantly affect the structure-function of key proteins such as BiP involved in cellular pathways, and provide a molecular basis for how chemical modifications can result in the failure of quality control mechanisms inside the cell. PMID:28886061

Effect of sodium chloride on the structure and stability of spider silk's N-terminal protein domain.

PubMed

Gronau, Greta; Qin, Zhao; Buehler, Markus J

2013-03-01

A spider's ability to store silk protein solutions at high concentration is believed to be related to the protein's terminal domains. It has been suggested that a shift in salt concentration and pH can have a significant influence on the assembly process. Based on experimental data, a model has been proposed in which the N-terminal domain exists as a monomer during storage and assembles into a homodimer upon spinning. Here we perform a systematic computational study using atomistic, coarse-grained and well-tempered metadynamics simulation to understand how the NaCl concentration in the solution affects the N-terminal domain of the silk protein. Our results show that a high salt concentration, as found during storage, weakens key salt bridges between the monomers, inducing a loss in bond energy by 28.6% in a single salt bridge. As a result dimer formation is less likely as 35.5% less energy is required to unfold the dimer by mechanical force. Conversely, homodimer formation appears to be more likely at low salt concentrations as the salt bridge stays at the lower energy state. The link between salt concentration, structure and stability of the N-terminal domain provides a possible mechanism that prevents premature fiber formation during storage.

Isomeric Detergent Comparison for Membrane Protein Stability: Importance of Inter-Alkyl-Chain Distance and Alkyl Chain Length

PubMed Central

Cho, Kyung Ho; Hariharan, Parameswaran; Mortensen, Jonas S.; Du, Yang; Nielsen, Anne K.; Byrne, Bernadette; Kobilka, Brian K.; Loland, Claus J.; Guan, Lan

2017-01-01

Membrane proteins encapsulated by detergent micelles are widely used for structural study. Because of their amphipathic property, detergents have the ability to maintain protein solubility and stability in an aqueous medium. However, conventional detergents have serious limitations in their scope and utility, particularly for eukaryotic membrane proteins and membrane protein complexes. Thus, a number of new agents have been devised; some have made significant contributions to membrane protein structural studies. However, few detergent design principles are available. In this study, we prepared meta and ortho isomers of the previously reported para-substituted xylene-linked maltoside amphiphiles (XMAs), along with alkyl chain-length variation. The isomeric XMAs were assessed with three membrane proteins, and the meta isomer with a C12 alkyl chain was most effective at maintaining solubility/stability of the membrane proteins. We propose that interplay between the hydrophile–lipophile balance (HLB) and alkyl chain length is of central importance for high detergent efficacy. In addition, differences in inter-alkyl-chain distance between the isomers influence the ability of the detergents to stabilise membrane proteins. PMID:27981750

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

PubMed Central

Surya, Wahyu; Torres, Jaume

2015-01-01

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single α-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.   PMID:25867485

Sedimentation equilibrium of a small oligomer-forming membrane protein: effect of histidine protonation on pentameric stability.

PubMed

Surya, Wahyu; Torres, Jaume

2015-04-02

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single α-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.

BCAS2 interacts with HSF4 and negatively regulates its protein stability via ubiquitination.

PubMed

Liao, Shengjie; Du, Rong; Wang, Lei; Qu, Zhen; Cui, Xiukun; Li, Chang; Liu, Fei; Huang, Mi; Wang, Jiuxiang; Chen, Jiaxiang; Gao, Meng; Yu, Shanshan; Tang, Zhaohui; Li, David Wan-Cheng; Jiang, Tao; Liu, Mugen

2015-11-01

Heat shock factor 4 (HSF4) is an important transcriptional factor that plays a vital role in lens development and differentiation, but the mechanism underlying the regulation of HSF4 is ambiguous. BCAS2 was reported to be an essential subunit of pre-mRNA splicing complex. Here, we identified BCAS2 as a novel HSF4 interacting partner. High expression of BCAS2 in the lens epithelium cells and the bow region of mouse lens was detected by immunohistochemistry. In human lens epithelial cells, BCAS2 negatively regulates HSF4 protein level and transcriptional activity, whereas in BCAS2 knockdown cells, HSF4 protein stability was increased significantly. We further demonstrated that the prolonged protein half-time of HSF4 in BCAS2 knockdown cells was due to reduced ubiquitination. Moreover, we have identified the lysine 206 of HSF4 as the key residue for ubiquitination. The HSF4-K206R mutant blocked the impact of BCAS2 on HSF4 protein stability. Taken together, we identified a pathway for HSF4 degradation through the ubiquitin-proteasome system, and a novel function for BCAS2 that may act as a negative regulatory factor for modulating HSF4 protein homeostasis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Tumor protein D52 expression is post-transcriptionally regulated by T-cell intercellular antigen (TIA) 1 and TIA-related protein via mRNA stability.

PubMed

Motohashi, Hiromi; Mukudai, Yoshiki; Ito, Chihiro; Kato, Kosuke; Shimane, Toshikazu; Kondo, Seiji; Shirota, Tatsuo

2017-05-04

Although tumor protein D52 (TPD52) family proteins were first identified nearly 20 years ago, their molecular regulatory mechanisms remain unclear. Therefore, we investigated the post-transcriptional regulation of TPD52 family genes. An RNA immunoprecipitation (RIP) assay showed the potential binding ability of TPD52 family mRNAs to several RNA-binding proteins, and an RNA degradation assay revealed that TPD52 is subject to more prominent post-transcriptional regulation than are TPD53 and TPD54. We subsequently focused on the 3'-untranslated region (3'-UTR) of TPD52 as a cis -acting element in post-transcriptional gene regulation. Several deletion mutants of the 3'-UTR of TPD52 mRNA were constructed and ligated to the 3'-end of a reporter green fluorescence protein gene. An RNA degradation assay revealed that a minimal cis -acting region, located in the 78-280 region of the 5'-proximal region of the 3'-UTR, stabilized the reporter mRNA. Biotin pull-down and RIP assays revealed specific binding of the region to T-cell intracellular antigen 1 (TIA-1) and TIA-1-related protein (TIAR). Knockdown of TIA-1/TIAR decreased not only the expression, but also the stability of TPD52 mRNA; it also decreased the expression and stability of the reporter gene ligated to the 3'-end of the 78-280 fragment. Stimulation of transforming growth factor-β and epidermal growth factor decreased the binding ability of these factors, resulting in decreased mRNA stability. These results indicate that the 78-280 fragment and TIA-1/TIAR concordantly contribute to mRNA stability as a cis -acting element and trans -acting factor(s), respectively. Thus, we here report the specific interactions between these elements in the post-transcriptional regulation of the TPD52 gene. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

A novel mechanism for antiglycative action of limonene through stabilization of protein conformation.

PubMed

Joglekar, Madhav M; Panaskar, Shrimant N; Chougale, Ashok D; Kulkarni, Mahesh J; Arvindekar, Akalpita U

2013-10-01

Inhibition of protein glycation is known to ameliorate secondary complications in diabetes. In the present study antiglycative properties of limonene, a natural product, were evaluated using BSA as a model protein. AMG (aminoguanidine) was used as a positive control. Measurement of total AGEs (Advanced Glycation End-products) and specific AGEs revealed that limonene could inhibit protein glycation to the extent of 56.3% and 75.1% respectively at 50 μM concentration as against 54.4% and 82.2% by AMG at 1 mM. Congo red binding and CD (Circular Dichroism) analysis revealed inhibition of α-helix to β-sheet transition wherein 18.5% β-sheet structures were observed in glycated BSA (bovine serum albumin) as against 4.9% with limonene. Glycation of protein in the presence of urea was enhanced by 18%, while in the presence of limonene it was reduced by 23% revealing the stabilizing effect of limonene. Electrophoretic mobility was similar to the normal control and a zeta potential value of -12.1 mV as against -15.1 mV in diabetic control was observed. Inhibition of glycation in limonene treated samples was confirmed through LC-MS analysis wherein AGEs such as pentosidine, CML (N(ε)-(carboxymethyl)lysine), CEL (N(ε)-(carboxyethyl)lysine), MOLD (methylglyoxal-lysine dimer) and imidazolone observed in glycated samples were absent in limonene treated samples. PatchDock studies revealed that limonene could bind to the major glycation sites IB, IIA and IIB sub domains and AMG to the IIIA sub domain. Thus limonene is a potent protein glycation inhibitor that prevents protein glycation through a novel mechanism of stabilization of protein structure through hydrophobic interactions.

Zebrafish Meis functions to stabilize Pbx proteins and regulate hindbrain patterning.

PubMed

Waskiewicz, A J; Rikhof, H A; Hernandez, R E; Moens, C B

2001-11-01

Homeodomain-containing Hox proteins regulate segmental identity in Drosophila in concert with two partners known as Extradenticle (Exd) and Homothorax (Hth). These partners are themselves DNA-binding, homeodomain proteins, and probably function by revealing the intrinsic specificity of Hox proteins. Vertebrate orthologs of Exd and Hth, known as Pbx and Meis (named for a myeloid ecotropic leukemia virus integration site), respectively, are encoded by multigene families and are present in multimeric complexes together with vertebrate Hox proteins. Previous results have demonstrated that the zygotically encoded Pbx4/Lazarus (Lzr) protein is required for segmentation of the zebrafish hindbrain and proper expression and function of Hox genes. We demonstrate that Meis functions in the same pathway as Pbx in zebrafish hindbrain development, as expression of a dominant-negative mutant Meis results in phenotypes that are remarkably similar to that of lzr mutants. Surprisingly, expression of Meis protein partially rescues the lzr(-) phenotype. Lzr protein levels are increased in embryos overexpressing Meis and are reduced for lzr mutants that cannot bind to Meis. This implies a mechanism whereby Meis rescues lzr mutants by stabilizing maternally encoded Lzr. Our results define two functions of Meis during zebrafish hindbrain segmentation: that of a DNA-binding partner of Pbx proteins, and that of a post-transcriptional regulator of Pbx protein levels.

Counteraction of the deleterious effects of urea on structure and stability of mammalian kidney proteins by osmolytes.

PubMed

Dar, Mohammad Aasif; Wahiduzzaman; Islam, Asimul; Hassan, Md Imtaiyaz; Ahmad, Faizan

2018-02-01

Owing to the urine concentrating mechanism of kidney cells, urea concentration is very high (3.0-5.0M) in mammalian kidneys which may denature many kidney proteins. Methylamines are known to counteract the deleterious effects of urea on structure, stability and function of proteins at 2:1 molar ratio of urea to methylamines. It is known that mammalian kidney cells also contain stabilizing osmolytes, non-methylamines (myo-inositol and sorbitol). A question arises: Do these non-methylmine osmolytes have ability to counteract the deleterious effects of urea on kidney proteins? To answer this question, we took two kidney proteins, namely, sheep serum albumin and Human carbonic anhydrase II. We measured their thermodynamic stability (ΔG 0 N↔D , the Gibbs free energy change in absence of GdmCl (guanidinium chloride) associated with the equilibrium, native (N) state↔denatured (D) state) from the GdmCl-induced denaturation curves in the presence of different concentrations of urea and each kidney osmolyte individually and in combination. For both proteins, we observed that (i) glycine betaine and myo-inositol provide perfect counteraction at 2:1 molar ratio of urea to osmolyte, i.e., denaturing effect of 2M urea is 100% neutralized by 1M of glycine betaine (or myo-inositol), and (ii) sorbitol fails to refold urea denatured proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Selection for Protein Kinetic Stability Connects Denaturation Temperatures to Organismal Temperatures and Provides Clues to Archaean Life

PubMed Central

Romero-Romero, M. Luisa; Risso, Valeria A.; Martinez-Rodriguez, Sergio; Gaucher, Eric A.; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

2016-01-01

The relationship between the denaturation temperatures of proteins (Tm values) and the living temperatures of their host organisms (environmental temperatures: TENV values) is poorly understood. Since different proteins in the same organism may show widely different Tm’s, no simple universal relationship between Tm and TENV should hold, other than Tm≥TENV. Yet, when analyzing a set of homologous proteins from different hosts, Tm’s are oftentimes found to correlate with TENV’s but this correlation is shifted upward on the Tm axis. Supporting this trend, we recently reported Tm’s for resurrected Precambrian thioredoxins that mirror a proposed environmental cooling over long geological time, while remaining a shocking ~50°C above the proposed ancestral ocean temperatures. Here, we show that natural selection for protein kinetic stability (denaturation rate) can produce a Tm↔TENV correlation with a large upward shift in Tm. A model for protein stability evolution suggests a link between the Tm shift and the in vivo lifetime of a protein and, more specifically, allows us to estimate ancestral environmental temperatures from experimental denaturation rates for resurrected Precambrian thioredoxins. The TENV values thus obtained match the proposed ancestral ocean cooling, support comparatively high Archaean temperatures, and are consistent with a recent proposal for the environmental temperature (above 75°C) that hosted the last universal common ancestor. More generally, this work provides a framework for understanding how features of protein stability reflect the environmental temperatures of the host organisms. PMID:27253436

Folding 19 proteins to their native state and stability of large proteins from a coarse-grained model.

PubMed

Kapoor, Abhijeet; Travesset, Alex

2014-03-01

We develop an intermediate resolution model, where the backbone is modeled with atomic resolution but the side chain with a single bead, by extending our previous model (Proteins (2013) DOI: 10.1002/prot.24269) to properly include proline, preproline residues and backbone rigidity. Starting from random configurations, the model properly folds 19 proteins (including a mutant 2A3D sequence) into native states containing β sheet, α helix, and mixed α/β. As a further test, the stability of H-RAS (a 169 residue protein, critical in many signaling pathways) is investigated: The protein is stable, with excellent agreement with experimental B-factors. Despite that proteins containing only α helices fold to their native state at lower backbone rigidity, and other limitations, which we discuss thoroughly, the model provides a reliable description of the dynamics as compared with all atom simulations, but does not constrain secondary structures as it is typically the case in more coarse-grained models. Further implications are described. Copyright © 2013 Wiley Periodicals, Inc.

The Methionine-aromatic Motif Plays a Unique Role in Stabilizing Protein Structure*

PubMed Central

Valley, Christopher C.; Cembran, Alessandro; Perlmutter, Jason D.; Lewis, Andrew K.; Labello, Nicholas P.; Gao, Jiali; Sachs, Jonathan N.

2012-01-01

Of the 20 amino acids, the precise function of methionine (Met) remains among the least well understood. To establish a determining characteristic of methionine that fundamentally differentiates it from purely hydrophobic residues, we have used in vitro cellular experiments, molecular simulations, quantum calculations, and a bioinformatics screen of the Protein Data Bank. We show that approximately one-third of all known protein structures contain an energetically stabilizing Met-aromatic motif and, remarkably, that greater than 10,000 structures contain this motif more than 10 times. Critically, we show that as compared with a purely hydrophobic interaction, the Met-aromatic motif yields an additional stabilization of 1–1.5 kcal/mol. To highlight its importance and to dissect the energetic underpinnings of this motif, we have studied two clinically relevant TNF ligand-receptor complexes, namely TRAIL-DR5 and LTα-TNFR1. In both cases, we show that the motif is necessary for high affinity ligand binding as well as function. Additionally, we highlight previously overlooked instances of the motif in several disease-related Met mutations. Our results strongly suggest that the Met-aromatic motif should be exploited in the rational design of therapeutics targeting a range of proteins. PMID:22859300

Beta-Barrel Scaffold of Fluorescent Proteins: Folding, Stability and Role in Chromophore Formation

PubMed Central

Stepanenko, Olesya V.; Stepanenko, Olga V.; Kuznetsova, Irina M.; Verkhusha, Vladislav V.; Turoverov, Konstantin K.

2013-01-01

This review focuses on the current view of the interaction between the β-barrel scaffold of fluorescent proteins and their unique chromophore located in the internal helix. The chromophore originates from the polypeptide chain and its properties are influenced by the surrounding protein matrix of the β-barrel. On the other hand, it appears that a chromophore tightens the β-barrel scaffold and plays a crucial role in its stability. Furthermore, the presence of a mature chromophore causes hysteresis of protein unfolding and refolding. We survey studies measuring protein unfolding and refolding using traditional methods as well as new approaches, such as mechanical unfolding and reassembly of truncated fluorescent proteins. We also analyze models of fluorescent protein unfolding and refolding obtained through different approaches, and compare the results of protein folding in vitro to co-translational folding of a newly synthesized polypeptide chain. PMID:23351712

Mild hypothermic culture conditions affect residual host cell protein composition post-Protein A chromatography

PubMed Central

Goey, Cher Hui; Bell, David; Kontoravdi, Cleo

2018-01-01

ABSTRACT Host cell proteins (HCPs) are endogenous impurities, and their proteolytic and binding properties can compromise the integrity, and, hence, the stability and efficacy of recombinant therapeutic proteins such as monoclonal antibodies (mAbs). Nonetheless, purification of mAbs currently presents a challenge because they often co-elute with certain HCP species during the capture step of protein A affinity chromatography. A Quality-by-Design (QbD) strategy to overcome this challenge involves identifying residual HCPs and tracing their source to the harvested cell culture fluid (HCCF) and the corresponding cell culture operating parameters. Then, problematic HCPs in HCCF may be reduced by cell engineering or culture process optimization. Here, we present experimental results linking cell culture temperature and post-protein A residual HCP profile. We had previously reported that Chinese hamster ovary cell cultures conducted at standard physiological temperature and with a shift to mild hypothermia on day 5 produced HCCF of comparable product titer and HCP concentration, but with considerably different HCP composition. In this study, we show that differences in HCP variety at harvest cascaded to downstream purification where different residual HCPs were present in the two sets of samples post-protein A purification. To detect low-abundant residual HCPs, we designed a looping liquid chromatography-mass spectrometry method with continuous expansion of a preferred, exclude, and targeted peptide list. Mild hypothermic cultures produced 20% more residual HCP species, especially cell membrane proteins, distinct from the control. Critically, we identified that half of the potentially immunogenic residual HCP species were different between the two sets of samples. PMID:29381421

Mild hypothermic culture conditions affect residual host cell protein composition post-Protein A chromatography.

PubMed

Goey, Cher Hui; Bell, David; Kontoravdi, Cleo

2018-04-01

Host cell proteins (HCPs) are endogenous impurities, and their proteolytic and binding properties can compromise the integrity, and, hence, the stability and efficacy of recombinant therapeutic proteins such as monoclonal antibodies (mAbs). Nonetheless, purification of mAbs currently presents a challenge because they often co-elute with certain HCP species during the capture step of protein A affinity chromatography. A Quality-by-Design (QbD) strategy to overcome this challenge involves identifying residual HCPs and tracing their source to the harvested cell culture fluid (HCCF) and the corresponding cell culture operating parameters. Then, problematic HCPs in HCCF may be reduced by cell engineering or culture process optimization. Here, we present experimental results linking cell culture temperature and post-protein A residual HCP profile. We had previously reported that Chinese hamster ovary cell cultures conducted at standard physiological temperature and with a shift to mild hypothermia on day 5 produced HCCF of comparable product titer and HCP concentration, but with considerably different HCP composition. In this study, we show that differences in HCP variety at harvest cascaded to downstream purification where different residual HCPs were present in the two sets of samples post-protein A purification. To detect low-abundant residual HCPs, we designed a looping liquid chromatography-mass spectrometry method with continuous expansion of a preferred, exclude, and targeted peptide list. Mild hypothermic cultures produced 20% more residual HCP species, especially cell membrane proteins, distinct from the control. Critically, we identified that half of the potentially immunogenic residual HCP species were different between the two sets of samples.

Factors affecting hazardous waste solidification/stabilization: a review.

PubMed

Malviya, Rachana; Chaudhary, Rubina

2006-09-01

Solidification/stabilization is accepted as a well-established disposal technique for hazardous waste. As a result many different types of hazardous wastes are treated with different binders. The S/S products have different property from waste and binders individually. The effectiveness of S/S process is studied by physical, chemical and microstructural methods. This paper summarizes the effect of different waste stream such as heavy metals bearing sludge, filter cake, fly ash, and slag on the properties of cement and other binders. The factors affecting strength development is studied using mix designs, including metal bearing waste alters the hydration and setting time of binders. Pore structure depends on relative quantity of the constituents, cement hydration products and their reaction products with admixtures. Carbonation and additives can lead to strength improvement in waste-binder matrix.

Pickering emulsions stabilized by whey protein nanoparticles prepared by thermal cross-linking.

PubMed

Wu, Jiande; Shi, Mengxuan; Li, Wei; Zhao, Luhai; Wang, Ze; Yan, Xinzhong; Norde, Willem; Li, Yuan

2015-03-01

A Pickering (o/w) emulsion was formed and stabilized by whey protein isolate nanoparticles (WPI NPs). Those WPI NPs were prepared by thermal cross-linking of denatured WPI proteins within w/o emulsion droplets at 80°C for 15 min. During heating of w/o emulsions containing 10% (w/v) WPI proteins in the water phase, the emulsions displayed turbid-transparent-turbid phase transitions, which is ascribed to the change in the size of the protein-containing water droplets caused by thermal cross-linking between denatured protein molecules. The transparent stage indicated the formation of WPI NPs. WPI NPs of different sizes were obtained by varying the mixing speed. WPI NPs of 200-500 nm were selected to prepare o/w Pickering emulsions because of their good stability against coalescence. By Confocal Laser Scanning Microscopy, it was observed that WPI NPs were closely packed and distributed at the surface of the emulsion droplets. By measuring water contact angles of WPI NPs films, it was found that under most conditions WPI NPs present good partial wetting properties, but that at the isoelectric point (pI) and high ionic strength the particles become more hydrophobic, resulting in less stable Pickering emulsion. Thus, at pH above and below the pI of WPI NPs and low to moderate ionic strengths (1-10 mM), and with a WPI NPs concentration of 2% (w/v), a stable Pickering emulsion can be obtained. The results may provide useful information for applications of WPI NPs in environmentally friendly and food grade applications, notably in food, pharmaceutical and cosmetic products. Copyright © 2015 Elsevier B.V. All rights reserved.

Aggregation of recombinant human botulinum protein antigen serotype C in varying solution conditions: implications of conformational stability for aggregation kinetics.

PubMed

Bai, Shujun; Manning, Mark Cornell; Randolph, Theodore W; Carpenter, John F

2011-03-01

Solution conditions greatly affect the aggregation rate of a protein. Elucidating these influences provides insight into the critical factors governing aggregation. In this study, recombinant human botulinum protein antigen serotype C [rBoNTC (H(c))] was employed as a model protein. rBoNTC (H(c)) aggregated irreversibly during incubation at 42°C. The aggregation rate was studied as a function of solution conditions, including varying the pH from 3.5 to 8.0 and with or without 150 mM NaCl, 7.5% (w/v) trehalose, and 0.5 M urea. Some solution conditions retarded rBoNTC (H(c)) aggregation, whereas others accelerated aggregation, particularly acidic pH and addition of NaCl or urea. To better understand the mechanism by which these solution conditions influenced aggregation rates, the structure of rBoNTC (H(c)) was characterized using circular dichroism, fluorescence, and ultraviolet absorbance spectroscopies. Conformational stability was assessed from equilibrium urea-induced unfolding studies and by using differential scanning calorimetry (DSC). The activation energy of the aggregation reaction (E(a)) was estimated from an analysis of the heating-rate dependence of the thermal transition observed during DSC heating scans. Overall, for rBoNTC (H(c)), an inverse correlation was found between conformational stability and aggregation rate, as well as between the kinetic barrier to unfolding (i.e., E(a)) and aggregation rate. Copyright © 2010 Wiley-Liss, Inc.

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.

Amino acid substitutions affecting protein dynamics in eglin C do not affect heat capacity change upon unfolding.

PubMed

Gribenko, Alexey V; Keiffer, Timothy R; Makhatadze, George I

2006-08-01

The heat capacity change upon unfolding (deltaC(p)) is a thermodynamic parameter that defines the temperature dependence of the thermodynamic stability of proteins; however, physical basis of the heat capacity change is not completely understood. Although empirical surface area-based calculations can predict heat capacity changes reasonably well, accumulating evidence suggests that changes in hydration of those surfaces is not the only parameter contributing to the observed heat capacity changes upon unfolding. Because packing density in the protein interior is similar to that observed in organic crystals, we hypothesized that changes in protein dynamics resulting in increased rigidity of the protein structure might contribute to the observed heat capacity change upon unfolding. Using differential scanning calorimetry we characterized the thermodynamic behavior of a serine protease inhibitor eglin C and two eglin C variants with altered native state dynamics, as determined by NMR. We found no evidence of changes in deltaC(p) in either of the variants, suggesting that changes in rigidity do not contribute to the heat capacity change upon unfolding in this model system. Copyright 2006 Wiley-Liss, Inc.

Surgical options for lumbosacral fusion: biomechanical stability, advantage, disadvantage and affecting factors in selecting options.

PubMed

Yoshihara, Hiroyuki

2014-07-01

Numerous surgical procedures and instrumentation techniques for lumbosacral fusion (LSF) have been developed. This is probably because of its high mechanical demand and unique anatomy. Surgical options include anterior column support (ACS) and posterior stabilization procedures. Biomechanical studies have been performed to verify the stability of those options. The options have their own advantage but also disadvantage aspects. This review article reports the surgical options for lumbosacral fusion, their biomechanical stability, advantages/disadvantages, and affecting factors in option selection. Review of literature. LSF has lots of options both for ACS and posterior stabilization procedures. Combination of posterior stabilization procedures is an option. Furthermore, combinations of ACS and posterior stabilization procedures are other options. It is difficult to make a recommendation or treatment algorithm of LSF from the current literature. However, it is important to know all aspects of the options and decision-making of surgical options for LSF needs to be tailored for each patient, considering factors such as biomechanical stress and osteoporosis.

Protein Denaturants at Aqueous–Hydrophobic Interfaces: Self-Consistent Correlation between Induced Interfacial Fluctuations and Denaturant Stability at the Interface

PubMed Central

2015-01-01

The notion of direct interaction between denaturing cosolvent and protein residues has been proposed in dialogue relevant to molecular mechanisms of protein denaturation. Here we consider the correlation between free energetic stability and induced fluctuations of an aqueous–hydrophobic interface between a model hydrophobically associating protein, HFBII, and two common protein denaturants, guanidinium cation (Gdm+) and urea. We compute potentials of mean force along an order parameter that brings the solute molecule close to the known hydrophobic region of the protein. We assess potentials of mean force for different relative orientations between the protein and denaturant molecule. We find that in both cases of guanidinium cation and urea relative orientations of the denaturant molecule that are parallel to the local protein–water interface exhibit greater stability compared to edge-on or perpendicular orientations. This behavior has been observed for guanidinium/methylguanidinium cations at the liquid–vapor interface of water, and thus the present results further corroborate earlier findings. Further analysis of the induced fluctuations of the aqueous–hydrophobic interface upon approach of the denaturant molecule indicates that the parallel orientation, displaying a greater stability at the interface, also induces larger fluctuations of the interface compared to the perpendicular orientations. The correlation of interfacial stability and induced interface fluctuation is a recurring theme for interface-stable solutes at hydrophobic interfaces. Moreover, observed correlations between interface stability and induced fluctuations recapitulate connections to local hydration structure and patterns around solutes as evidenced by experiment (Cooper et al., J. Phys. Chem. A2014, 118, 5657.) and high-level ab initio/DFT calculations (Baer et al., Faraday Discuss2013, 160, 89). PMID:25536388

Rapid and Tunable Control of Protein Stability in Caenorhabditis elegans Using a Small Molecule

PubMed Central

Cho, Ukrae; Zimmerman, Stephanie M.; Chen, Ling-chun; Owen, Elliot; Kim, Jesse V.; Kim, Stuart K.; Wandless, Thomas J.

2013-01-01

Destabilizing domains are conditionally unstable protein domains that can be fused to a protein of interest resulting in degradation of the fusion protein in the absence of stabilizing ligand. These engineered protein domains enable rapid, reversible and dose-dependent control of protein expression levels in cultured cells and in vivo. To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20–25°C. This raises the possibility that our technology could be adapted for use at any temperature. We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans. These data reinforce that DD can function in virtually any organism and temperature. PMID:23991108

Antioxidant activity and emulsion-stabilizing effect of pectic enzyme treated pectin in soy protein isolate-stabilized oil/water emulsion.

PubMed

Huang, Ping-Hsiu; Lu, Hao-Te; Wang, Yuh-Tai; Wu, Ming-Chang

2011-09-14

The antioxidant activity of pectic enzyme treated pectin (PET-pectin) prepared from citrus pectin by enzymatic hydrolysis and its potential use as a stabilizer and an antioxidant for soy protein isolate (SPI)-stabilized oil in water (O/W) emulsion were investigated. Trolox equivalent antioxidant capacity (TEAC) was found to be positively associated with molecular weight (M(w)) of PET-pectin and negatively associated with degree of esterification (DE) of PET-pectin. PET-pectin (1 kDa and 11.6% DE) prepared from citrus pectin after 24 h of hydrolysis by commercial pectic enzyme produced by Aspergillus niger expressed higher α,α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging activity, TEAC, and reducing power than untreated citrus pectin (353 kDa and 60% DE). The addition of PET-pectin could increase both emulsifying activity (EA) and emulsion stability (ES) of SPI-stabilized O/W emulsion. When the SPI-stabilized lipid droplet was coated with the mixture of PET-pectin and pectin, the EA and ES of the emulsion were improved more than they were when the lipid droplet was coated with either pectin or PET-pectin alone. The amount of secondary oxidation products (thiobarbituric acid reactive substances) produced in the emulsion prepared with the mixture of SPI and PET-pectin was less than the amount produced in the emulsion prepared with either SPI or SPI/pectin. These results suggest that PET-pectin has an emulsion-stabilizing effect and lipid oxidation inhibition ability on SPI-stabilized emulsion. Therefore, PET-pectin can be used as a stabilizer as well as an antioxidant in plant origin in SPI-stabilized O/W emulsion and thus prolong the shelf life of food emulsion.

Nicotine affects protein complex rearrangement in Caenorhabditis elegans cells.

PubMed

Sobkowiak, Robert; Zielezinski, Andrzej; Karlowski, Wojciech M; Lesicki, Andrzej

2017-10-01

Nicotine may affect cell function by rearranging protein complexes. We aimed to determine nicotine-induced alterations of protein complexes in Caenorhabditis elegans (C. elegans) cells, thereby revealing links between nicotine exposure and protein complex modulation. We compared the proteomic alterations induced by low and high nicotine concentrations (0.01 mM and 1 mM) with the control (no nicotine) in vivo by using mass spectrometry (MS)-based techniques, specifically the cetyltrimethylammonium bromide (CTAB) discontinuous gel electrophoresis coupled with liquid chromatography (LC)-MS/MS and spectral counting. As a result, we identified dozens of C. elegans proteins that are present exclusively or in higher abundance in either nicotine-treated or untreated worms. Based on these results, we report a possible network that captures the key protein components of nicotine-induced protein complexes and speculate how the different protein modules relate to their distinct physiological roles. Using functional annotation of detected proteins, we hypothesize that the identified complexes can modulate the energy metabolism and level of oxidative stress. These proteins can also be involved in modulation of gene expression and may be crucial in Alzheimer's disease. The findings reported in our study reveal putative intracellular interactions of many proteins with the cytoskeleton and may contribute to the understanding of the mechanisms of nicotinic acetylcholine receptor (nAChR) signaling and trafficking in cells.

RNA-modifying proteins as anticancer drug targets.

PubMed

Boriack-Sjodin, P Ann; Ribich, Scott; Copeland, Robert A

2018-06-01

All major biological macromolecules (DNA, RNA, proteins and lipids) undergo enzyme-catalysed covalent modifications that impact their structure, function and stability. A variety of covalent modifications of RNA have been identified and demonstrated to affect RNA stability and translation to proteins; these mechanisms of translational control have been termed epitranscriptomics. Emerging data suggest that some epitranscriptomic mechanisms are altered in human cancers as well as other human diseases. In this Review, we examine the current understanding of RNA modifications with a focus on mRNA methylation, highlight their possible roles in specific cancer indications and discuss the emerging potential of RNA-modifying proteins as therapeutic targets.

General Characteristics of the Changes in the Thermal Stability of Proteins and Enzymes After the Chemical Modification of Their Functional Groups

NASA Astrophysics Data System (ADS)

Kutuzova, G. D.; Ugarova, N. N.; Berezin, Ilya V.

1984-11-01

The principal structural and physicochemical factors determining the stability of protein macromolecules in solution and the characteristics of the structure of the proteins from thermophilic microorganisms are examined. The mechanism of the changes in the thermal stability of proteins and enzymes after the chemical modification of their functional side groups and the experimental data concerning the influence of chemical modification on the thermal stability of proteins are analysed. The dependence of the stabilisation effect and of the changes in the structure of protein macromolecules on the degree of modification and on the nature of the modified groups and the groups introduced into proteins in the course of modification (their charge and hydrophobic properties) is demonstrated. The great practical value of the method of chemical modification for the preparation of stabilised forms of biocatalysts is shown in relation to specific examples. The bibliography includes 178 references.

Stabilization of model beverage cloud emulsions using protein-polysaccharide electrostatic complexes formed at the oil-water interface.

PubMed

Harnsilawat, Thepkunya; Pongsawatmanit, Rungnaphar; McClements, David J

2006-07-26

The potential of utilizing interfacial complexes, formed through the electrostatic interactions of proteins and polysaccharides at oil-water interfaces, to stabilize model beverage cloud emulsions has been examined. These interfacial complexes were formed by mixing charged polysaccharides with oil-in-water emulsions containing oppositely charged protein-coated oil droplets. Model beverage emulsions were prepared that consisted of 0.1 wt % corn oil droplets coated by beta-lactoglobulin (beta-Lg), beta-Lg/alginate, beta-Lg/iota-carrageenan, or beta-Lg/gum arabic interfacial layers (pH 3 or 4). Stable emulsions were formed when the polysaccharide concentration was sufficient to saturate the protein-coated droplets. The emulsions were subjected to variations in pH (from 3 to 7), ionic strength (from 0 to 250 mM NaCl), and thermal processing (from 30 or 90 degrees C), and the influence on their stability was determined. The emulsions containing alginate and carrageenan had the best stability to ionic strength and thermal processing. This study shows that the controlled formation of protein-polysaccharide complexes at droplet surfaces may be used to produce stable beverage emulsions, which may have important implications for industrial applications.

Rapid and Adaptable Measurement of Protein Thermal Stability by Differential Scanning Fluorimetry: Updating a Common Biochemical Laboratory Experiment