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PubMed2001

Tyrosine hydrogen bonds make a large contribution to protein stability.

The aim of this study was to gain a better understanding of the contribution of hydrogen bonds by tyrosine -OH groups to protein stability. The amino acid sequences of RNases Sa and Sa3 are 69 % identical and each contains eight Tyr residues with seven at equivalent structural positions. We have measured the stability of the 16 tyrosine to phenylalanine mutants. For two equivalent mutants, the stability increases by 0.3 kcal/mol (RNase Sa Y30F) and 0.5 kcal/mol (RNase Sa3 Y33F) (1 kcal=4.184 kJ). For all of the other mutants, the stability decreases with the greatest decrease being 3.6 kcal/mol for RNase Sa Y52F. Seven of the 16 tyrosine residues form intramolecular hydrogen bonds and the average decrease in stability for these is 2.0(+/-1.0) kcal/mol. For the nine tyrosine residues that do not form intramolecular hydrogen bonds, the average decrease in stability is 0.4(+/-0.6) kcal/mol. Thus, most tyrosine -OH groups contribute favorably to protein stability even if they do not form intramolecular hydrogen bonds. Generally, the stability changes for equivalent positions in the two proteins are remarkably similar. Crystal structures were determined for two of the tyrosine to phenylalanine mutants of RNase Sa: Y80F (1.2 A), and Y86F (1.7 A). The structures are very similar to that of wild-type RNase Sa, and the hydrogen bonding partners of the tyrosine residues always form intermolecular hydrogen bonds to water in the mutants. These results provide further evidence that the hydrogen bonding and van der Waals interactions of polar groups in the tightly packed interior of folded proteins are more favorable than similar interactions with water in the unfolded protein, and that polar group burial makes a substantial contribution to protein stability.

Amino Acid Substitution↗
PubMed1988

Contribution of hydrophobic interactions to protein stability.

A major factor in the folding of proteins is the burying of hydrophobic side chains. A specific example is the packing of alpha-helices on beta-sheets by interdigitation of nonpolar side chains. The contributions of these interactions to the energetics of protein stability may be measured by simple protein engineering experiments. We have used site-directed mutagenesis to truncate hydrophobic side chains at an alpha-helix/beta-sheet interface in the small ribonuclease from Bacillus amyloliquefaciens (barnase). The decreases in stability of the mutant proteins were measured by their susceptibility to urea denaturation. Creation of a cavity the size of a -CH2-group destabilizes the enzyme by 1.1 kcal mol-1, and a cavity the size of three such groups by 4.0 kcal mol-1.

Bacillus↗
PubMed1996

How Hofmeister ion interactions affect protein stability.

Model compound studies in the literature show how Hofmeister ion interactions affect protein stability. Although model compound results are typically obtained as salting-out constants, they can be used to find out how the interactions affect protein stability. The null point in the Hofmeister series, which divides protein denaturants from stabilizers, arises from opposite interactions with different classes of groups: Hofmeister ions salt out nonpolar groups and salt in the peptide group. Theories of how Hofmeister ion interactions work need to begin by explaining the mechanisms of these two classes of interactions. Salting-out nonpolar groups has been explained by the cavity model, but its use is controversial. When applied to model compound data, the cavity model 1) uses surface tension increments to predict the observed values of the salting-out constants, within a factor of 3, and 2) predicts that the salting-out constant should increase with the number of carbon atoms in the aliphatic side chain of an amino acid, as observed. The mechanism of interaction between Hofmeister ions and the peptide group is not well understood, and it is controversial whether this interaction is ion-specific, or whether it is nonspecific and the apparent specificity resides in interactions with nearby nonpolar groups. A nonspecific salting-in interaction is known to occur between simple ions and dipolar molecules; it depends on ionic strength, not on position in the Hofmeister series. A theory by Kirkwood predicts the strength of this interaction and indicates that it depends on the first power of the ionic strength. Ions interact with proteins in various ways besides the Hofmeister ion interactions discussed here, especially by charge interactions. Much of what is known about these interactions comes from studies by Serge Timasheff and his co-workers. A general model, suitable for analyzing diverse ion-protein interactions, is provided by the two-domain model of Record and co-workers.

Drug Stability↗
PubMed1996

Effects of protein stabilizing agents on thermal backbone motions: a disulfide trapping study.

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

Calcium-Binding Proteins↗
PubMedDate not supplied

Synthetic model proteins: contribution of hydrophobic residues and disulfide bonds to protein stability.

The objective of this study was to evaluate quantitatively the contribution that hydrophobic residues and disulfide bonds make to protein stability using a model protein with well-defined secondary, tertiary and quaternary structure. This de novo-designed protein consists of two identical 35-residue alpha-helical polypeptide chains arranged in a coiled-coil structure, which are stabilized by nine pairs of interchain hydrophobic interactions from leucine residues and an interchain disulfide bond. The mutant proteins differ from the Leu-protein only at positions 16 and 19 of each chain which contain either Ile, Val, Ala, Phe or Tyr instead of Leu residues. The stability of each protein was determined in its reduced and oxidized form from guanidine hydrochloride denaturation experiments using circular dichroism measurements. The Leu-, Ile-, Val- and Phe-proteins containing the disulfide bond were essentially 100% alpha-helical in benign medium (0.05 M phosphate buffer, pH 7, containing 0.1 M KCl) with transition midpoints of 5.3, 4.1, 2.9 and 2.4 M denaturant, respectively. Reduction of the disulfide bond had little effect on the Leu-protein but resulted in significant decreases in helicity of the other mutants. The order of protein stability of these analogs remains the same in the absence or presence of the disulfide bond (Leu- greater than Ile- greater than Val- greater than Phe- greater than Tyr- greater than Ala-protein). Interestingly, the more stable the protein was in the absence of the disulfide bond, the larger the contribution the disulfide bond made to protein stability. The identical mutation in the disulfide bridge protein had a much greater effect on protein stability than in the reduced protein. This suggests that the disulfide bond controls the conformation of the coiled-coil some 14-17 residues further along the polypeptide chain (the disulfide bond is between positions 2 and 2' and the closest mutation site in the coiled-coil is at position 16 and 16'). In contrast, in the absence of the disulfide bond, the coiled-coil is more flexible and can accommodate the mutations more easily by adjusting the interchain packing around the mutation sites. The helix-forming tendency of nonpolar residues was deduced in this study by comparing the alpha-helicity of the mutant polypeptides in aqueous buffer containing 50% trifluoroethanol where they exist as single-stranded helices. Hydrophobicity is an important factor in determining the alpha-helix forming tendency of aliphatic nonpolar residues in amphipathic alpha-helices.

Amino Acid Sequence↗
PubMed1995

2.0 A structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability.

BACKGROUND: Recent efforts to understand the basis of protein stability have focused attention on comparative studies of proteins from hyperthermophilic and mesophilic organisms. Most work to date has been on either oligomeric enzymes or monomers comprising more than one domain. Such studies are hampered by the need to distinguish between stabilizing interactions acting between subunits or domains from those acting within domains. In order to simplify the search for determinants of protein stability we have chosen to study the monomeric enzyme indole-3-glycerol phosphate synthase from the hyperthermophilic archaeon Sulfolobus solfataricus (sIGPS), which grows optimally at 90 degrees C. RESULTS: The 2.0 A crystal structure of sIGPS was determined and compared with the known 2.0 A structure of the IGPS domain of the bifunctional enzyme from the mesophilic bacterium Escherichia coli (eIGPS). sIGPS and eIGPS have only 30% sequence identity, but share high structural similarity. Both are single-domain (beta/alpha)8 barrel proteins, with one (eIGPS) or two (sIGPS) additional helices inserted before the first beta strand. The thermostable sIGPS has many more salt bridges than eIGPS. Several salt bridges crosslink adjacent alpha helices or participate in triple or quadruple salt-bridge clusters. The number of helix capping, dipole stabilizing and hydrophobic interactions is also increased in sIGPS. CONCLUSIONS: The higher stability of sIGPS compared with eIGPS seems to be the result of several improved interactions. These include a larger number of salt bridges, stabilization of alpha helices and strengthening of both polypeptide chain termini and solvent-exposed loops.

Aldehyde Oxidoreductases↗
PubMed2004

Protein sequence randomization: efficient estimation of protein stability using knowledge-based potentials.

Modifications of the amino acid sequence generally affect protein stability. Here, we use knowledge-based potentials to estimate the stability of protein structures under sequence variation. Calculations on a variety of protein scaffolds result in a clear distinction of known mutable regions from arbitrarily chosen control patches. For example, randomly changing the sequence of an antibody paratope yields a significantly lower number of destabilized mutants as compared to the randomization of comparable regions on the protein surface. The technique is computationally efficient and can be used to screen protein structures for regions that are amenable to molecular tinkering by preserving the stability of the mutated proteins.

Amino Acid Sequence↗
PubMed2004

Construction of stabilized proteins by combinatorial consensus mutagenesis.

We constructed stabilized variants of beta-lactamase (BLA) from Enterobacter cloacae by combinatorial recruitment of consensus mutations. By aligning the sequences of 38 BLA homologs, we identified 29 positions where the E.cloacae gene differs from the consensus sequence of lactamases and constructed combinatorial libraries using mixtures of mutagenic oligonucleotides encompassing all 29 positions. Screening of 90 random isolates from these libraries identified 15 variants with significantly increased thermostability. The stability of these isolates suggest that all tested mutations make additive contributions to protein stability. A statistical analysis of sequence and stability data identified 11 mutations that made stabilizing contributions and eight mutations that destabilized the protein. A second-generation library recombining these 11 stabilizing mutations led to the identification of BLA variants that showed further stabilization. The most stable variant had a mid-point of thermal denaturation (Tm) that was 9.1 degrees C higher than the starting molecule and contained eight consensus mutations. Incubation of three stabilized BLA variants with several proteases showed that all tested isolates have significantly increased resistance to proteolysis. Our data demonstrate that combinatorial consensus mutagenesis (CCM) allows the rapid generation of protein variants with improved thermal and proteolytic stability.

Amino Acid Sequence↗
PubMed2006

Comparative study of protein stabilization in white wine using zirconia and bentonite: physicochemical and wine sensory analysis.

A semi-industrial application of the continuous stabilization of white wine protein using a column packed with zirconia was studied and compared to the traditional bentonite treatment using a Macabeu white wine. Physicochemical and wine sensory properties were evaluated using a rating system and triangle tests. Continuous protein stabilization was analyzed in three residence times, and the equivalent of 300 BV of wine was used for both treatments. Wine protein content was reduced by 21%, 40%, and 42% using the continuous process with residence times of 7.5, 15, and 30 min, respectively, and by 61.4% using the bentonite treatment. The wines obtained from the packed column were protein stable up to 25, 75, and 175 BV for residence times of 7.5, 15, and 30 min, respectively. The amount of polyphenol removed was less than 10%, and similar amounts were removed from the wine regardless of residence time, while 20.6% of polyphenol was removed using bentonite. The physicochemical and sensory properties of wine treated with bentonite were similar to those of wine treated with zirconia.

Adsorption↗
PubMed2002

Lipopolysaccharide-induced methylation of HuR, an mRNA-stabilizing protein, by CARM1. Coactivator-associated arginine methyltransferase.

The RNA-binding protein HuR stabilizes labile mRNAs carrying AU-rich instability elements. This mRNA stabilization can be induced by hypoxia, lipopolysaccharide, and UV light. The mechanism by which these stimuli activate HuR is unclear and might be related to post-translational modification of this protein. Here we show that HuR can be methylated on arginine. However, HuR is not a substrate for PRMT1, the most prominent protein-arginine methyltransferase in mammalian cells, which methylates a number of heterogeneous nuclear ribonucleoproteins. Instead, HuR is specifically methylated by coactivator-associated arginine methyltransferase 1 (CARM1), a protein-arginine methyltransferase previously shown to serve as a transcriptional coactivator. By analyzing methylation of specific HuR arginine-to-lysine mutants and by sequencing radioactively methylated HuR peptides, Arg(217) was identified as the major HuR methylation site. Arg(217) is located in the hinge region between the second and third of the three HuR RNA recognition motif domains. Antibodies against a methylated HuR peptide were used to demonstrate in vivo methylation of HuR. HuR methylation increased in cells that overexpressed CARM1. Importantly, lipopolysaccharide stimulation of macrophages, which leads to HuR-mediated stabilization of tumor necrosis factor alpha mRNA in these cells, caused increased methylation of endogenous HuR. Thus, CARM1, which plays a role in transcriptional activation through histone H3 methylation, may also play a role in post-transcriptional gene regulation by methylating HuR.

Animals↗
PubMed2001

MdmX binding to ARF affects Mdm2 protein stability and p53 transactivation.

Regulation of p53 involves a complex network of protein interactions. The primary regulator of p53 protein stability is the Mdm2 protein. ARF and MdmX are two proteins that have recently been shown to inhibit Mdm2-mediated degradation of p53 via distinct associations with Mdm2. We demonstrate here that ARF is capable of interacting with MdmX and in a manner similar to its association with Mdm2, sequestering MdmX within the nucleolus. The sequestration of MdmX by ARF results in an increase in p53 transactivation. In addition, the redistribution of MdmX by ARF requires that a nucleolar localization signal be present on MdmX. Although expression of either MdmX or ARF leads to Mdm2 stabilization, coexpression of both MdmX and ARF results in a decrease in Mdm2 protein levels. Similarly, increasing ARF protein levels in the presence of constant MdmX and Mdm2 leads to a dose-dependent decrease in Mdm2 levels. Under these conditions, ARF can synergistically reverse the ability of Mdm2 and MdmX to inhibit p53-dependent transactivation. Finally, the association and redistribution of MdmX by ARF has no effect on the protein stability of either ARF or MdmX. Taken together, these results demonstrate that the interaction between MdmX and ARF represents a novel pathway for regulating Mdm2 protein levels. Additionally, both MdmX and Mdm2, either individually or together, are capable of antagonizing the effects of the ARF tumor suppressor on p53 activity.

Amino Acid Sequence↗
PubMed2003

Actin filament-stabilizing protein tropomyosin regulates the size of dendritic fields.

Dendritic arbors of different neuronal subtypes cover distinct spatial territories, known as dendritic fields, to receive specific inputs in a nervous system. How the size of dendritic fields is determined by cell-intrinsic factors during development remains primarily unknown. To address this issue, we used the Drosophila embryonic peripheral nervous system. In each hemisegment, six dorsal cluster dendritic arborization (DA) neurons elaborate stereotypic dendritic branching patterns underneath the epidermis. Here we report the identification of loss-of-function mutations in the tropomyosin II gene (TmII) that result in expanded dendritic fields of DA neurons. Mosaic analysis with a repressible cell marker demonstrated that TmII functions in a cell-autonomous manner to control the formation of dendritic fields. Furthermore, we show that TmII genetically interacted with flamingo, a gene encoding a membrane receptor-like molecule that affects dendritic growth. TmII encodes multiple isoforms of a protein that stabilizes actin filaments. Our findings suggest that tropomyosin helps control the size of dendritic fields by regulating actin cytoskeletal dynamics.

Actin Cytoskeleton↗
PubMed1998

Effect of high molecular mobility of poly(vinyl alcohol) on protein stability of lyophilized gamma-globulin formulations.

The protein stability of lyophilized serum gamma-globulin (BGG) formulations containing poly(vinyl alcohol) (PVA) and dextran was studied in relation to the molecular mobility as determined by proton NMR. The critical temperature, Tmc, at which the Lorentzian relaxation process due to liquid polymer protons appears in these lyophilized formulations was lower than the glass transition temperature, Tg. Above Tmc, protein aggregation in the formulations was related to the Tmc according to the Williams-Landel-Ferry equation by replacing Tg with Tmc. Protein aggregation appears to occur substantially in a "rubbery-like" state even below Tg, if the formulations become microscopically liquidized above Tmc. Lyophilized BGG formulations containing PVA with a lower water content were less stable than those containing dextran with a higher water content. The difference in stability can be explained by the difference in the Tmc of these formulations. Tmc that is determined by NMR relaxation measurement appears to be a useful parameter for the characterization of protein formulations, for which the Tg cannot generally be determined by standard calorimetric techniques. Furthermore, Tmc appears to be more closely related to protein stability than does Tg.

Calorimetry, Differential Scanning↗
PubMed2003

Amino acid sequences and solution structures of manganese stabilizing protein that affect reconstitution of Photosystem II activity.

This minireview presents a summary of information available on the secondary and tertiary structure of manganese stabilizing protein (MSP) in solution, and on the identity of amino acid residues that affect binding and functional assembly of this protein into Photosystem II. New data on the secondary structure of C-terminal mutants and 90 degrees C-heated manganese stabilizing protein, along with earlier data on the secondary structure of N-terminal mutants and the tertiary structure of all modified MSP species, allow for an evaluation of models for spinach MSP secondary and tertiary structure. This summary of previous and new information better documents the natively unfolded behavior of the protein in solution. A two-step mechanism for binding of manganese stabilizing protein to Photosystem II is discussed and possible solution three-dimensional conformations of the wild-type protein and some of its unfolded mutants, are proposed.

Journal Article↗
PubMed1994

Metal-mediated protein stabilization.

Metal binding provides several useful routes to protein stabilization. Metal chelation by binding sites engineered into proteins can increase thermal stability, conformational stability and reduce proteolytic degradation. As few as two coordinating ligands, if properly positioned on the protein surface, can form chelating sites suitable for metal binding and stabilization. Thus, this approach is relatively easy to implement and can be applied to a wide range of proteins.

Amino Acid Sequence↗
PubMed1997

Mutation Val235Ala weakens binding of the 33-kDa manganese stabilizing protein of photosystem II to one of two sites.

The 33-kDa protein of the photosynthetic O2-evolving complex, also known as manganese stabilizing protein, contributes to the structural stability of the photosystem II tetranuclear Mn cluster and stimulates the water-oxidizing activity of this cluster. Quantification of extrinsic polypeptides in photosystem II has yielded data that support stoichiometries of either one or two copies of each protein per photosystem II reaction center. We recently described the cold-sensitive assembly of a mutant 33-kDa protein with a single amino acid replacement (Val235Ala) [Betts, S. D., Ross, J. R., Pichersky, E., & Yocum, C. F. (1996) Biochemistry 35, 6302-6307]. We have extended the characterization of this mutation. When photosystem II membranes depleted of the 33 kDa extrinsic protein are exposed to mixtures of wild type and Val235Ala manganese stabilizing protein, binding of the wild type protein is strongly preferred. If, however, protein containing the Val235Ala mutation is first bound to photosystem II only half of this protein (about 1 mol/mol of photosystem II reaction centers) is susceptible to displacement by the wild type protein, even after multiple exposures to the latter. These results support the conclusion that 2 mol of manganese stabilizing protein are bound per reaction center. Our data show as well that the mutant 33-kDa protein competes with the wild type protein for at least one of two binding sites on photosystem II and that the mutant protein binds tightly to only one of two sites. These results demonstrate that the two binding sites on photosystem II for the 33-kDa protein have different properties with respect to recognition and binding of this protein.

Arabidopsis↗
PubMed2005

Predicting protein stability changes from sequences using support vector machines.

MOTIVATION: The prediction of protein stability change upon mutations is key to understanding protein folding and misfolding. At present, methods are available to predict stability changes only when the atomic structure of the protein is available. Methods addressing the same task starting from the protein sequence are, however, necessary in order to complete genome annotation, especially in relation to single nucleotide polymorphisms (SNPs) and related diseases. RESULTS: We develop a method based on support vector machines that, starting from the protein sequence, predicts the sign and the value of free energy stability change upon single point mutation. We show that the accuracy of our predictor is as high as 77% in the specific task of predicting the DeltaDeltaG sign related to the corresponding protein stability. When predicting the DeltaDeltaG values, a satisfactory correlation agreement with the experimental data is also found. As a final blind benchmark, the predictor is applied to proteins with a set of disease-related SNPs, for which thermodynamic data are also known. We found that our predictions corroborate the view that disease-related mutations correspond to a decrease in protein stability. AVAILABILITY: http://gpcr2.biocomp.unibo.it/cgi/predictors/I-Mutant2.0/I-Mutant2.0.cgi

Algorithms↗