Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Protein Stability”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1991

Protein stability and protein folding.

Proteins show only marginal free energies of stabilization. Mutative adaptations to extremes of physical conditions (high temperature, pressure and salt concentration) tend to maintain 'corresponding states' regarding overall topology, flexibility and hydration. Since enhanced stability requires only minute local changes in the structure of a given protein, general strategies of adaptation cannot be established. Apart from alterations at the protein level, extrinsic factors such as ions, cofactors or specific ligands may serve to enhance in vivo and in vitro protein stability. Protein folding and association reflect the hierarchy of protein structure, with the formation of secondary/supersecondary structure, subdomains/domains and structured monomers as consecutive steps. The process requires highly specified environmental conditions; e.g. active mesophilic or halophilic proteins cannot be expressed in thermophilic and non-halophilic hosts. On the other hand, a given protein may tolerate extreme sequence variability without substantially altering its three-dimensional structure and stability. Significant rate-determining steps in the overall reaction, that is, formation of disulphide bridges, proline isomerization and oligomerization, are catalysed by specific enzymes or directed by 'helper proteins' (protein disulphide isomerase, peptidyl-prolyl cis-trans isomerase and chaperones). Physiological stress conditions, (site-directed) mutations, and in vitro studies may be used to unravel the significance of the three 'shuffling reactions'.

Amino Acid Isomerases↗
PubMed1990

Strength and co-operativity of contributions of surface salt bridges to protein stability.

Many of the interactions that stabilize proteins are co-operative and cannot be reduced to a sum of pairwise interactions. Such interactions may be analysed by protein engineering methods using multiple thermodynamic cycles comprising wild-type protein and all combinations of mutants in the interacting residues. There is a triad of charged residues on the surface of barnase, comprising residues Asp8, Asp12 and Arg110, that interact by forming two exposed salt bridges. The three residues have been mutated to alanine to give all the single, double and triple mutants. The free energies of unfolding of wild-type and the seven mutant proteins have been determined and the results analysed to give the contributions of the residues in the two salt bridges to protein stability. It is possible to isolate the energies of forming the salt bridges relative to the solvation of the separated ions by water. In the intact triad, the apparent contribution to the stabilization energy of the protein of the salt bridge between Asp12 and Arg110 is -1.25 kcal mol-1, whereas that of the salt bridge between Asp8 with Arg110 is -0.98 kcal mol-1. The strengths of the two salt bridges are coupled: the energy of each is reduced by 0.77 kcal mol-1 when the other is absent. The salt-linked triad, relative to alanine residues at the same positions, does not contribute to the stability of the protein since the favourable interactions of the salt bridges are more than offset by other electrostatic and non-electrostatic energy terms. Salt-linked triads occur in other proteins, for example, haemoglobin, where the energy of only the salt-bridge term is important and so the coupling of salt bridges could be of general importance to the stability and function of proteins.

Alanine↗
PubMed1992

Interaction of CPa-1 with the manganese-stabilizing protein of photosystem II: identification of domains on CPa-1 which are shielded from N-hydroxysuccinimide biotinylation by the manganese-stabilizing protein.

The structural organization of photosystem II proteins has been investigated by use of the amino group-labeling reagent N-hydroxysuccinimidobiotin (NHS-biotin) and calcium chloride-washed photosystem II membranes. We have previously shown that the presence of the extrinsic, manganese-stabilizing protein on photosystem II membranes prevents the modification of lysyl residues located on the chlorophyll protein CPa-1 (CP-47) by NHS-biotin [Bricker, T. M., Odom, W. R., & Queirolo, C. B. (1988) FEBS Lett. 231, 111-117]. Upon removal of the manganese-stabilizing protein by calcium chloride-washing, CPa-1 can be specifically modified by treatment with NHS-biotin. Preparative quantities of biotinylated CPa-1 were subjected to chemical cleavage with cyanogen bromide. Two major biotinylated peptides were identified with apparent molecular masses of 11.8 and 15.7 kDa. N-terminal sequence analysis of these peptides indicated that the 11.8-kDa peptide was 232G-330M and that the 15.7-kDa peptide was 360P-508V. The 15.7-kDa CNBr peptide was subjected to limited tryptic digestion. The two smallest tryptic fragments identified migrated at apparent molecular masses of 9.1 (nonbiotinylated) and 7.5 kDa (biotinylated). N-terminal sequence analysis and examination of the predicted amino acid sequences of these peptides suggest that the 9.1-kDa fragment was 422R-508V and that the 7.5-kDa fragment was 360P-421A. These results strongly suggest that two NHS-biotinylated domains, 304K-321K and 389K-419K, become exposed on CPa-1 when the manganese-stabilizing protein is removed by CaCl2 treatment. Both of these domains lie in the large extrinsic loop E of CPa-1.

Amino Acid Sequence↗
PubMed1992

Structural organization of proteins on the oxidizing side of photosystem II. Two molecules of the 33-kDa manganese-stabilizing proteins per reaction center.

The 33-kDa manganese-stabilizing protein stabilizes the manganese cluster in the oxygen-evolving complex. There has been, however, a considerable amount of controversy concerning the stoichiometry of this photosystem II (PS II) component. In this paper, we have verified the extinction coefficient of the manganese-stabilizing protein by amino acid analysis, determined the manganese content of oxygen-evolving photosystem II membranes and reaction center complex using inductively coupled plasma spectrometry, and determined immunologically the amount of the manganese-stabilizing protein associated with photosystem II. Oxygen-evolving photosystem II membranes and reaction center complex preparations contained 258 +/- 11 and 67 +/- 3 chlorophyll, respectively, per tetranuclear manganese cluster. Immunoquantification of the manganese-stabilizing protein using mouse polyclonal antibodies on "Western blots" demonstrated the presence of 2.1 +/- 0.2 and 2.0 +/- 0.3 molecules of the manganese-stabilizing protein/tetranuclear manganese cluster in oxygen-evolving PS II membranes and highly purified PS II reaction center complex, respectively. Since the manganese-stabilizing protein co-migrated with the D2 protein in our electrophoretic system, accurate immunoquantification required the inclusion of CaCl2-washed PS II membrane proteins or reaction center complex proteins in the manganese-stabilizing protein standards to compensate for the possible masking effect of the D2 protein on the binding of the manganese-stabilizing protein to Immobilon-P membranes. Failure to include these additional protein components in the manganese-stabilizing protein standards leads to a marked underestimation of the amount of the manganese-stabilizing protein associated with these photosystem II preparations.

Chloroplasts↗
PubMed1991

Is the hydrophobic effect stabilizing or destabilizing in proteins? The contribution of disulphide bonds to protein stability.

It has been recently concluded that the hydrophobic effect, hitherto regarded as a major driving force in the folding of proteins, destabilizes the folded state relative to the unfolded state. We summarize the properties of the hydrophobic effect obtained from solvent transfer experiments and show that the recent conclusion is an artifact of crosslinking in the unfolded state, caused by disulphide bonds, metals or cofactors. We show that, for the proteins in the data set, crosslinks surprisingly destabilize folded structures entropically, but stabilize them enthalpically to a greater extent. We also calculate non-polar surface areas of these unfolded proteins. These surface areas are decreased by crosslinks. The unfolded state of proteins lacking constraints, such as myoglobin, is well approximated by a mixture of residues containing alpha-helical and beta-sheet dihedral angles. Surface areas of unfolded proteins cannot be obtained by summing the surface areas of individual residues, since this ignores any unavoidable side-chain-side-chain interactions.

Disulfides↗
PubMed1989

Modification of protein stability by introduction of disulfide bridges and prolines: geometric criteria for mutation sites.

We define geometrical parameters to characterize disulfide bridges using x-ray crystal structure data on small molecules and use them to suggest replacements of amino acids by cysteines in order to introduce disulfide bridges to increase thermal stability in proteins. We also define geometric parameters to identify target amino acids for replacements by prolines in order to conserve desired structural attributes in the vicinity of disulfide mutations leading to further structural and thermal stability of proteins. The geometric criteria are applied to the serine protease, subtilisin, to model stereochemically favorable disulfide mutants without altering the active site geometry, implying conservation of native biological activity.

Binding Sites↗
PubMed1985

Dissecting the roles of individual interactions in protein stability: lessons from a circularized protein.

A circular form of bovine pancreatic trypsin inhibitor (BPTI) has been prepared by introducing a peptide bond between the N- and C-termini, which are in close proximity in the native conformation. The pathway and energetics of the disulphide-coupled folding transition of the circular protein have been studied using methods applied previously to the unmodified protein. The cross-link between the termini was found not to significantly stabilize the native state in spite of the expected reduction in entropy of the unfolded protein. This unexpected result has led to a reexamination of the stabilization expected from a cross-link, considering effects on the native, as well as unfolded, states of the protein. The greatest stabilization is expected when the cross-linked groups are held rigidly in the native protein in the optimum orientation for forming the cross-link. Similar analyses, utilizing thermodynamic cycles, can be applied to other interactions that stabilize native proteins, including disulphide bonds, salt bridges, and hydrogen bonds and to modifications to the protein that remove them. In general, the contribution of an individual interaction to the stability of the native state depends on the extent to which the interaction is favored in the native conformation, which can vary greatly depending on the local environment of the interacting groups.

Animals↗
PubMed1992

Oxygen evolution in the absence of the 33-kilodalton manganese-stabilizing protein.

There has been a considerable amount of controversy concerning the ability of photosystem II to evolve oxygen in the absence of the 33-kDa, manganese-stabilizing protein. Early reports indicated that some capacity for oxygen evolution existed in manganese-stabilizing protein-depleted membranes while more recent studies have suggested that the observed oxygen evolution activity arose from residual manganese-stabilizing protein present in the salt-washed preparations. In this paper, it is conclusively demonstrated that significant rates of steady-state oxygen evolution are observed in oxygen-evolving photosystem II membranes in the absence of detectable quantities of the manganese-stabilizing protein. More then 99% of the manganese-stabilizing protein was removed by either one CaCl2 or two NaCl-urea washes. The amount of manganese-stabilizing protein removed was quantified immunologically using mouse polyclonal antibodies. Oxygen evolution rates of 115-140 mumol of O2 (mg of Chl)-1 h-1 were observed in the NaCl-urea-washed preparations. These rates represent about 24% of the rate observed in untreated membranes [450-600 mumol of O2 (mg of Chl)-1 h-1]. Somewhat lower, although still significant rates were observed in the CaCl2-washed preparations. Optimal rates of oxygen-evolving activity in NaCl-urea-washed membranes which are devoid of the manganese-stabilizing protein required high concentrations of calcium and chloride.

Kinetics↗
PubMed2026

Systematic differences in protein stability underlie species-specific developmental tempo.

Human embryonic development proceeds more slowly than in mice. The segmentation clock offers a tractable model for studying species-specific developmental tempo, as its oscillation period in human induced presomitic mesoderm (iPSM) cells is approximately twice that of mouse. While the core clock gene HES7 exhibits slower protein degradation in human cells, it remains unclear whether such cross-species differences in protein stability reflect a general principle. Here, we perform a dynamic stable isotope labeling of amino acids in cell culture (SILAC)-based proteomic analysis of ∼5,000 proteins in human and mouse iPSM, and we uncover a broad trend of slower protein degradation in human cells, regardless of subcellular localization or degradation pathways. Moreover, inhibition of glycolysis in mouse iPSM partially phenocopies the human protein stability profile, and modulation of protein stability alters the tempo of both the segmentation clock and cellular differentiation. Our findings establish protein stability, with systematic differences across species, as a key mediator linking metabolism to developmental tempo.

Animals↗
PubMed1991

Protein stabilization by engineered metal chelation.

A ligand can shift a protein's folding/unfolding equilibrium by binding with higher affinity to the native state. A metal-chelating site consisting of two histidines separated by three residues (His-X3-His) engineered into an alpha-helix provides a general and easily-implemented means for protein stabilization by this mechanism. We have tested this approach with the iso-1-cytochrome c of Saccharomyces cerevisiae substituted with histidine at positions 4 and 8 in its N-terminal alpha-helix. One mM Cu(II) complexed to iminodiacetate stabilizes the cytochrome c variant by ca. 1 kcal/mol, as determined by guanidinium chloride-induced unfolding. The protein's folding/unfolding equilibrium is shifted by a free energy equal to that calculated from the metal ion's preferential binding to the native protein. Given the ubiquity of surface alpha-helices and the additional possibility of inserting di-histidine chelating sites into turns and beta-structures, we conclude that this is a useful method for protein stabilization.

Binding Sites↗
PubMed1992

Interaction of CPa-1 with the manganese-stabilizing protein of photosystem II: identification of domains cross-linked by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide.

The structural organization of photosystem II proteins has been investigated by use of the zero-length protein cross-linking reagent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and monoclonal and polyclonal antibody reagents. Photosystem II membranes were treated with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide which cross-links amino groups to carboxyl groups which are in van der Waals contact. This treatment did not affect the oxygen evolution rates of these membranes and increased the retention of oxygen evolution after CaCl2 washing. Analysis of the proteins cross-linked by this treatment indicated that two cross-linked species with apparent molecular masses of 95 and 110 kDa were formed which cross-reacted with antibodies against both the 33-kDa manganese-stabilizing protein and the chlorophyll protein CPa-1. Cleavage of the 110-kDa cross-linked species with cyanogen bromide followed by N-terminal sequence analysis was used to identify the peptide fragments of CPa-1 and the manganese-stabilizing protein which were cross-linked. Two cyanogen bromide fragments were identified with apparent molecular masses of 50 and 25 kDa. N-Terminal sequence analysis of the 50-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa fragment of CPa-1 and the intact manganese-stabilizing protein. This strongly suggests that the manganese-stabilizing protein is cross-linked to the large extrinsic loop domain of CPa-1. N-Terminal analysis of the 25-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa peptide of CPa-1 and the N-terminal 8-kDa peptide of the manganese-stabilizing protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Replacements of Pro86 in phage T4 lysozyme extend an alpha-helix but do not alter protein stability.

To investigate the relation between protein stability and the predicted stabilities of individual secondary structural elements, residue Pro86 in an alpha-helix in phage T4 lysozyme was replaced by ten different amino acids. The x-ray crystal structures of seven of the mutant lysozymes were determined at high resolution. In each case, replacement of the proline resulted in the formation of an extended alpha-helix. This involves a large conformational change in residues 81 to 83 and smaller shifts that extend 20 angstroms across the protein surface. Unexpectedly, all ten amino acid substitutions marginally reduce protein thermostability. This insensitivity of stability to the amino acid at position 86 is not simply explained by statistical and thermodynamic criteria for helical propensity. The observed conformational changes illustrate a general mechanism by which proteins can tolerate mutations.

Enzyme Stability↗
PubMed1989

Substantial increase of protein stability by multiple disulphide bonds.

Disulphide bonds can significantly stabilize the native structures of proteins. The effect is presumed to be due mainly to a decrease in the configurational chain entropy of the unfolded polypeptide. In phage T4 lysozyme, a disulphide-free enzyme, engineered disulphide mutants that crosslink residues 3-97, 9-164 and 21-142 are significantly more stable than the wild-type protein. To investigate the effect of multiple-disulphide bonds on protein stability, mutants were constructed in which two or three stabilizing disulphide bridges were combined in the same protein. Reversible thermal denaturation shows that the increase in melting temperature resulting from the individual disulphide bonds is approximately additive. The triple-disulphide variant unfolds at a temperature 23.4 degrees C higher than wild-type lysozyme. The results demonstrate that a combination of disulphide bonds, each of which contributes to stability, can achieve substantial overall improvement in the stability of a protein.

DNA Mutational Analysis↗
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↗
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↗
PubMed1992

Probing protein stability with unnatural amino acids.

Unnatural amino acid mutagenesis, in combination with molecular modeling and simulation techniques, was used to probe the effect of side chain structure on protein stability. Specific replacements at position 133 in T4 lysozyme included (i) leucine (wt), norvaline, ethylglycine, and alanine to measure the cost of stepwise removal of methyl groups from the hydrophobic core, (ii) norvaline and O-methyl serine to evaluate the effects of side chain solvation, and (iii) leucine, S,S-2-amino-4-methylhexanoic acid, and S-2-amino-3-cyclopentylpropanoic acid to measure the influence of packing density and side chain conformational entropy on protein stability. All of these factors (hydrophobicity, packing, conformational entropy, and cavity formation) significantly influence protein stability and must be considered when analyzing any structural change to proteins.

Amino Acids↗