Effect of single amino acid substitutions at the same position on stability of a two-domain protein

In order to elucidate the role of individual amino acid residues on the conformational stability of a protein, the stabilities of the wild-type tryptophan synthase α-subunit from Escherichia coil and its five mutant proteins substituted by single amino acid residues at the same position 49 were compared. The five mutant proteins have glutamine, methionine, valine, serine, or tyrosine in place of glutamic acid of the wild-type protein at position 49. Denaturation of these proteins, which consist of two domains, by guanidine hydrochloride can be analyzed as a two-step process. We obtained the equilibrium constants between the native and the denatured forms and between the native and the stable intermediate forms for the above six proteins in the absence of denaturant at three pH values.     

Comparison of CD spectra in the aromatic region on a series of variant proteins substituted at a unique position of tryptophan synthase alpha-subunit

CD spectra in the aromatic region of a series of the mutant alpha-subunits of tryptophan synthase from Escherichia coli, substituted at position 49 buried in the interior of the molecule, were measured at pH 7.0 and 25 degrees C. These measurements were taken to gain information on conformational change produced by single amino acid substitutions. The CD spectra of the mutant proteins, substituted by Tyr or Trp residue in place of Glu residue at position 49, showed more intense positive bands due to one additional Tyr or Trp residue at position 49. The CD spectra of other mutant proteins also differed from that of the wild-type protein, despite the fact that the substituted residues at position 49 were not aromatic. Using the spectrum of the wild-type protein (Glu49) as a standard, the spectra of the other mutants were classified into three major groups. For 10 mutant proteins substituted by Ile, Ala, Leu, Met, Val, Cys, Pro, Ser, His, or Gly, their CD values of bands (due to Tyr residues) decreased in comparison with those of the wild-type protein. The mutant protein substituted by Phe also belonged to this group. These substituted amino acid residues are more hydrophobic than the original residue, Glu. In the second group, three mutant proteins were substituted by Lys, Gln, or Asn, and the CD values of tyrosyl bands increased compared to those of the wild-type proteins. These residues are polar. In the third group, the CD values of tyrosyl bands of two mutant proteins substituted by Asp or Thr were similar to those of the wild-type protein, except for one band at 276.5 nm. These results suggested that the changes in the CD spectra for the mutant proteins were affected by the hydrophobicity of the residues at position 49.     

Proton nuclear magnetic resonance studies on the wild-type and single amino acid substituted tryptophan synthase alpha-subunits

In order to elucidate the effect of single amino acid substitutions on the conformation of the tryptophan synthase alpha-subunit from Escherichia coli in solution, 1H NMR spectra of the wild-type and mutant proteins were measured at various pHs. Two of the four His C2-proton resonances of the alpha-subunit were assigned to two His residues at positions 92 and 146 by using a mutant protein with Thr substituted for the His at position 92. The replacement did not affect the conformation of the protein significantly. The proton resonances of all the Tyr residues in the aromatic region could be picked up from other resonance peaks, employing the wild-type alpha-subunit deuterated at all of the Phe residues. On comparison of the spectra of the wild-type protein with those of the mutant protein with Met substituted for the Glu at position 49, it was concluded that the substitution affects only the residues close to the substituted residue at acidic pH but that a larger part of the protein is affected at alkaline pH. NOE experiments showed that the five Tyr residues, four of which are located in the proximity of position 49, are close to one another. The present results are discussed in the light of the conformational stability of the protein.     

Sequences of the vesicular stomatitis virus matrix protein involved in binding to nucleocapsids.

The purpose of these experiments was to study the physical structure of the nucleocapsid-M protein complex of vesicular stomatitis virus by analysis of nucleocapsid binding by wild-type and mutant M proteins and by limited proteolysis. We used the temperature-sensitive M protein mutant tsO23 and six temperature-stable revertants of tsO23 to test the effect of sequence changes on M protein binding to the nucleocapsid as a function of NaCl concentration. The results showed that M proteins from wild-type, mutant, and three of the revertant viruses had similar NaCl titration curves, while the curve for M proteins from the other three revertants differed significantly. The altered NaCl dependence of M protein was correlated with a single amino acid substitution from Phe to Leu at position 111 compared with the original temperature-sensitive mutant and was not correlated with a substitution of Gly to Glu at position 21 in tsO23 and the revertants. To determine whether protease cleavage sites in the M protein were protected by interaction with the nucleocapsid, nucleocapsid-M protein complexes were subjected to limited proteolysis with trypsin, chymotrypsin, or Staphylococcus aureus V8 protease. The initial trypsin and chymotrypsin cleavage sites, located after amino acids 19 and 20, respectively, were as accessible to proteases when M protein was bound to the nucleocapsid as when it was purified, indicating that this region of the protein does not interact directly with the nucleocapsid. Furthermore, trypsin or chymotrypsin treatment released the M protein fragments from the nucleocapsid, presumably due to conformational changes following proteolysis. V8 protease cleaved the M protein at position 34 or 50, producing two distinct fragments. The M protein fragment produced by V8 protease cleavage at position 34 remained associated with the nucleocapsid, while the fragment produced by cleavage at position 50 was released from the nucleocapsid. These results suggest that the amino-terminal region of the M protein around amino acid 20 does not interact directly with the nucleocapsid and that conformational changes resulting from single-amino-acid substitutions at other sites in the M protein are important for this interaction.     

Effect of amino acid residues on conformational stability in eight mutant proteins variously substituted at a unique position of the tryptophan synthase alpha-subunit

To elucidate the role of individual amino acid residues in stabilizing the conformation of a protein, the stabilities of wild-type tryptophan synthase alpha-subunit from Escherichia coli and seven mutant proteins substituted by single amino acid residues at position 49, which is buried in the interior of the protein, were compared. The mutant proteins have Gln, Met, Val, Tyr, Leu, Ser, or Lys in place of Glu in the wild-type protein. The dissociation constant, pK, of the Glu residue at position 49 for the wild-type protein was determined to be 7.5 from a titration curve obtained by comparison of two-dimensional isoelectric focusing electrophoresis of the wild-type and mutant proteins. Our results indicate that 1) the conformational stabilities of the proteins studied increase linearly with hydrophobicity of the substituting residues (except Tyr), with the coefficient of this linear dependence being 2.0, 3.4, or 1.3 at pH 5.5, 7.0, or 9.0, respectively; and 2) Lys or Glu at position 49 serve as a destabilizing factor when ionized.     

Contribution of salt bridges near the surface of a protein to the conformational stability

Salt bridges play important roles in the conformational stability of proteins. However, the effect of a surface salt bridge on the stability remains controversial even today; some reports have shown little contribution of a surface salt bridge to stability, whereas others have shown a favorable contribution. In this study, to elucidate the net contribution of a surface salt bridge to the conformational stability of a protein, systematic mutant human lysozymes, containing one Glu to Gln (E7Q) and five Asp to Asn mutations (D18N, D49N, D67N, D102N, and D120N) at residues where a salt bridge is formed near the surface in the wild-type structure, were examined. The thermodynamic parameters for denaturation between pH 2.0 and 4.8 were determined by use of a differential scanning calorimeter, and the crystal structures were analyzed by X-ray crystallography. The denaturation Gibbs energy (DeltaG) of all mutant proteins was lower than that of the wild-type protein at pH 4, whereas there was little difference between them near pH 2. This is caused by the fact that the Glu and Asp residues are ionized at pH 4 but protonated at pH 2, indicating a favorable contribution of salt bridges to the wild-type structure at pH 4. Each contribution was not equivalent, but we found that the contributions correlate with the solvent inaccessibility of the salt bridges; the salt bridge contribution was small when 100% accessible, while it was about 9 kJ/mol if 100% inaccessible. This conclusion indicates how to reconcile a number of conflicting reports about role of surface salt bridges in protein stability. Furthermore, the effect of salts on surface salt bridges was also examined. In the presence of 0.2 M KCl, the stability at pH 4 decreased, and the differences in stability between the wild-type and mutant proteins were smaller than those in the absence of salts, indicating the compensation to the contribution of salt bridges with salts. Salt bridges with more than 50% accessibility did not contribute to the stability in the presence of 0.2 M KCl.     

Differential degradation of variant medium-chain acyl-CoA dehydrogenase by the protein quality control proteases Lon and ClpXP

The coordinated activities of chaperones and proteases that supervise protein folding and degradation are important factors for deciding the fate of proteins whose folding is impaired by missense variations. We have studied the role of Lon and ClpXP proteases in handling of wild-type and a folding-impaired disease-associated variant (R28C) of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). Using an Escherichia coli model system, we co-overexpressed the MCAD variants and the respective proteases at two conditions: at 31 degrees C where R28C MCAD protein folds partially and at 37 degrees C where it misfolds and aggregates. Co-overexpression of Lon protease considerably accelerated the degradation rate of a pool of R28C variant MCAD synthesised during a 30min pulse and counteracted accumulation of aggregates at 37 degrees C, whereas increasing the amounts of ClpXP protease had no clear effect. Co-overexpression of either Lon or ClpXP protease markedly decreased the steady state levels of both wild-type and R28C mutant MCAD at 37 degrees C but not at 31 degrees C. Our results suggest that Lon is more efficient than ClpXP in elimination of non-native MCAD protein conformations, and accordingly, that Lon can recognise a broader spectrum of MCAD protein conformations.     

A set of aspartyl protease-deficient strains for improved expression of heterologous proteins in Kluyveromyces lactis

Secretion of recombinant proteins is a common strategy for heterologous protein expression using the yeast Kluyveromyces lactis. However, a common problem is degradation of a target recombinant protein by secretory pathway aspartyl proteases. In this study, we identified five putative pfam00026 aspartyl proteases encoded by the K. lactis genome. A set of selectable marker-free protease deletion mutants was constructed in the prototrophic K. lactis GG799 industrial expression strain background using a PCR-based dominant marker recycling method based on the Aspergillus nidulans acetamidase gene (amdS). Each mutant was assessed for its secretion of protease activity, its health and growth characteristics, and its ability to efficiently produce heterologous proteins. In particular, despite having a longer lag phase and slower growth compared with the other mutants, a Δyps1 mutant demonstrated marked improvement in both the yield and the quality of Gaussia princeps luciferase and the human chimeric interferon Hy3, two proteins that experienced significant proteolysis when secreted from the wild-type parent strain.     

Genetic or chemical protease inhibition causes significant changes in the Bacillus subtilis exoproteome

Bacillus subtilis is a prolific producer of enzymes and biopharmaceuticals. However, the susceptibility of heterologous proteins to degradation by (extracellular) proteases is a major limitation for use of B. subtilis as a protein cell factory. An increase in protein production levels has previously been achieved by using either protease-deficient strains or addition of protease inhibitors to B. subtilis cultures. Notably, the effects of genetic and chemical inhibition of proteases have thus far not been compared in a systematic way. In the present studies, we therefore compared the exoproteomes of cells in which extracellular proteases were genetically or chemically inactivated. The results show substantial differences in the relative abundance of various extracellular proteins. Furthermore, a comparison of the effects of genetic and/or chemical protease inhibition on the stress response triggered by (over) production of secreted proteins showed that chemical protease inhibition provoked a genuine secretion stress response. From a physiological point of view, this suggests that the deletion of protease genes is a better way to prevent product degradation than the use of protease inhibitors. Importantly however, studies with human interleukin-3 show that chemical protease inhibition can result in improved production of protease-sensitive secreted proteins even in mutant strains lacking eight extracellular proteases.     

The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain

Enzymes of the ATP-independent Deg serine endopeptidase family are very flexible with regard to their substrate specificity. Some family members cleave only one substrate, while others act as general proteases on unfolded substrates. The proteolytic activity of Deg proteases is regulated by PDZ protein interaction domains. Here we characterized the HhoA protease from Synechocystis sp. strain PCC 6803 in vitro using several recombinant protein constructs. The proteolytic activity of HhoA was found to increase with temperature and basic pH and was stimulated by the addition of Mg(2+) or Ca(2+). We found that the single PDZ domain of HhoA played a critical role in regulating protease activity and in the assembly of a hexameric complex. Deletion of the PDZ domain strongly reduced proteolysis of a sterically challenging resorufin-labeled casein substrate, but unlabeled beta-casein was still degraded. Reconstitution of the purified HhoA with total membrane proteins isolated from Synechocystis sp. wild-type strain PCC 6803 and a DeltahhoA mutant resulted in specific degradation of selected proteins at elevated temperatures. We concluded that a single PDZ domain of HhoA plays a critical role in defining the protease activity and oligomerization state, combining the functions that are attributed to two PDZ domains in the homologous DegP protease from Escherichia coli. Based on this first enzymatic study of a Deg protease from cyanobacteria, we propose a general role for HhoA in the quality control of extracytoplasmic proteins, including membrane proteins, in Synechocystis sp. strain PCC 6803.     

Correlation of surface properties with conformational stabilities of wild-type and six mutant tryptophan synthase alpha-subunits substituted at the same position

The surface properties of wild-type and six mutant alpha-subunits of tryptophan synthase substituted at the same position, 49, which is buried in the interior, were measured by surface tension, foaming and emulsifying properties to correlate the surface properties with the stabilities. The conformational stabilities of the seven alpha-subunits differed dramatically depending on the characteristics of the substituting residues [Yutani et al. (1987) Proc. Natl. Acad. Sci., 84, 4441-4444]. The mutant proteins substituted by isoleucine and phenylalanine in place of glutamic acid at position 49 were more stable than the other proteins and showed higher surface tension and lower foaming and emulsifying properties than the wild-type and other mutant proteins. Good correlations were observed between these surface properties and values of the Gibbs free energy of unfolding in water, of the proteins. This indicates that the surface properties of the alpha-subunits of tryptophan synthase depend closely on the conformational stabilities.     

Calorimetric study of tryptophan synthase alpha-subunit and two mutant proteins

Heat-denaturation of tryptophan synthase alpha-subunit from E. coli and two mutant proteins (Glu 49 leads to Gln or Ser; called Gln 49 or Ser 49, respectively) has been studied by the scanning microcalorimetric method at various pH, in an attempt to elucidate the role of individual amino acid residues in the conformational stability of a protein. The partial specific heat capacity in the native state at 20 degrees, Cp20, has been found to be (0.43 +/- 0.02) cal . k-1 . g-1, the unfolding heat capacity change, delta dCp, (0.10 +/- 0.01) cal . K-1 . g-1, and the unfolding enthalpy value extrapolated to 110 degrees, delta dh110, (9.3 +/- 0.5) cal . g-1 for the three proteins. The value of Cp20 was larger than those found for "fully compact protein" and that of delta dh110 was smaller. Unfolding Gibbs energy, delta dG at 25 degrees for Wild-type, Gln 49, and Ser 49 were 5.8, 8.4, and 7.1 kcal . mol-1 at pH 9.3, respectively. Unfolding enthalpy, delta dH, of the three proteins seemed to be the same and equal to (23.2 +/- 1.2) kcal . mol-1 at 25 degrees. As a consequence of the same value of delta dH and the different value in delta dG, substantial differences in unfolding entropy, delta dS, were found for the three proteins. The values of delta dG for the three proteins at 25 degrees coincided with those from equilibrium methods of denaturation by guanidine hydrochloride.     

Structural and functional role of the amino-terminal region of porcine cytosolic aspartate aminotransferase. Catalytic and structural properties of enzyme derivatives truncated on the amino-terminal side

In porcine cytosolic aspartate aminotransferase, a dimeric enzyme, the amino-terminal region anchoring onto the neighboring subunit is linked to the adjoining floppy peptide segment (residues 12-47), an integral part of the small domain whose facile movement upon substrate binding is a striking "induced fit" feature of this enzyme. To assess the contribution by the amino-terminal region to small domain movement and protein stability, a series of enzyme derivatives truncated on the amino-terminal side (residues 1-9) was prepared by using oligonucleotide-directed in vitro mutagenesis. Deletion of residues 1-3 showed no effect on catalytic activity and heat stability. Del 1-5 mutant enzyme with an extra methionine at position 5 showed only 43% of the kappa cat value (in the overall transamination) of the wild-type enzyme. Further deletion up to residue 9 resulted in a slight decrease in kappa cat values. Del 1-9 mutant enzyme still retained a kappa cat value of 33% that of wild-type enzyme. Km values for aspartate and 2-oxoglutarate increased sharply upon deletion of residues 1-9. Accordingly, Del 1-9 mutant enzyme showed a striking decrease in the kappa cat/Km value, to only 2% of that for the wild-type enzyme. Deletion of amino-terminal residues 1-9 resulted also in a large decrease in thermostability and in an enhanced susceptibility to limited proteolysis by protease 401, which is known to cleave at Leu20 of the wild-type enzyme. These findings indicate that an increase in the conformational freedom of the floppy segment (residues 12-47) would occur upon the loss of most of the anchorage region, thereby presenting an entropic barrier to conformational changes that facilitate substrate binding with high affinity.     

Subunit interface of triosephosphate isomerase: site-directed mutagenesis and characterization of the altered enzyme

We have replaced asparagine residues at the subunit interface of yeast triosephosphate isomerase (TIM) using site-directed mutagenesis in order to elucidate the effects of substitutions on the catalytic activity and conformational stability of the enzyme. The mutant proteins were expressed in a strain of Escherichia coli lacking the bacterial isomerase and purified by ion-exchange and immunoadsorption chromatography. Single replacements of Asn-78 by either Thr or Ile residues had little effect on the enzyme's catalytic efficiency, while the single replacement Asn-78----Asp-78 and the double replacement Asn-14/Asn-78----Thr-14/Ile-78 appreciably lowered kcat for the substrate D-glyceraldehyde 3-phosphate. The isoelectric point of the mutant Asn-78----Asp-78 was equivalent to that of wild-type yeast TIM that had undergone a single, heat-induced deamidation, and this mutant enzyme was less resistant than wild-type TIM to denaturation and inactivation caused by elevated temperature, denaturants, tetrabutylammonium bromide, alkaline pH, and proteases.     

Effects of local protein stability and the geometric position of the substrate degradation tag on the efficiency of ClpXP denaturation and degradation

ClpX and related AAA+ ATPases of the Clp/Hsp100 family are able to denature native proteins. Here, we explore the role of protein stability in ClpX denaturation and subsequent ClpP degradation of model substrates bearing ssrA degradation tags at different positions. ClpXP degraded T. thermophilus RNase-H* with a C-terminal ssrA tag very efficiently, despite the very high global stability of this thermophilic protein. In fact, global thermodynamic stability appears to play little role in susceptibility to degradation, as a far less stable RNase-H*-ssrA mutant was degraded more slowly than wild type by ClpXP and a completely unfolded mutant variant was degraded less than twice as fast as the wild-type parent. When ssrA peptide tags were covalently linked to surface cysteines at positions 114 or 140 of RNase-H*, the conjugates were proteolyzed very slowly. This resistance to degradation was not caused by inaccessibility of the ssrA tag or an inability of ClpXP to degrade proteins with side-chain linked ssrA tags. Our results support a model in which ClpX denatures proteins by initially unfolding structural elements attached to the degradation tag, suggest an important role for the position of the degradation tag and direction of force application, and correlate well with the mapping of local protein stability within RNase-H* by native-state hydrogen exchange.     

Investigating interdomain region mutants Phe194----Leu and Phe194----Trp of yeast phosphoglycerate kinase by 1H-NMR spectroscopy.

Site-directed mutagenesis has been used to produce two mutant forms of yeast phosphoglycerate kinase in which the interdomain residue Phe194 has been replaced by a leucine or tryptophan residue. Using 1H-NMR spectroscopy, it was found that the mutations at position 194 induce both local and long-range conformational changes in the protein. It was also found that 3-phosphoglycerate binding to the mutant proteins induces somewhat different conformational effects to those observed for wild-type phosphoglycerate kinase. The affinity of mutant Phe194----Trp for 3-phosphoglycerate was found by NMR studies to be unaffected, while the affinity of Phe194----Leu mutant is reduced by about threefold relative to the wild-type enzyme. The binding of ATP at the electrostatic site of the mutant proteins is also seen to be about three times weaker for the Phe194----Leu mutant when compared to wild-type or Phe194----Leu mutant. These results are discussed in the light of the kinetic studies on the mutants which show that for Phe194----Leu mutant the Km values for both 3-phosphoglycerate and ATP, as well as the Vmax, are decreased relative to the wild-type enzyme, while for mutant Phe194----Trp, the Km values for 3-phosphoglycerate and ATP are unaffected and the Vmax is decreased when compared to wild-type enzyme. Kinetic studies in the presence of sulphate reveal that the anion activation is greater for mutant Phe194----Trp and less for mutant Phe194----Leu, relative to that observed for wild-type phosphoglycerate kinase. The NMR data, taken together with the kinetic data, are consistent with the on and off rates of 3-phosphoglycerate being affected by the mutations at position 194. It is suggested that Phe194 is important for the mobility of the interdomain region and the relative movement of the 3-phosphoglycerate binding site which allows the optimum conformation for catalysis to be attained. Apparently Trp194 reduces the mobility of the interdomain region of the protein, while Leu194 increases it.     

An essential proline in lambda repressor is required for resistance to intracellular proteolysis

Pro78 is a solvent-exposed residue at the N-terminal end of alpha-helix 5 in the DNA binding domain of lambda repressor. Random mutagenesis experiments have suggested that Pro78 is essential [Reidhaar-Olson, J.F., & Sauer, R.T. (1990) Proteins: Struct., Funct., Genet. (in press)]. To investigate the requirement for proline at this position, we constructed and studied the properties of a set of ten position 78 mutant proteins. All of these mutants have decreased intracellular activities and are expressed at significantly lower levels than wild type. Pulse-chase experiments show that the mutant proteins are rapidly degraded in the cell; the mutants examined had half-lives of 11-35 min, whereas the wild-type protein has a half-life of greater than 10 h. The rapid degradation of position 78 mutants is not suppressed by mutations that affect known Escherichia coli proteases. The Pro78----Ala mutant could be overexpressed in a dnaJ- strain and was purified. This mutant has full DNA binding activity in vitro, suggesting that its folded structure and ability to form active dimers are similar to those of wild type. The PA78 mutant (Tm = 48 degrees C) is less thermally stable than wild type (Tm = 55 degrees C). Double-mutant studies show that this instability contributes to but is not the main cause of its rapid intracellular degradation and also suggest that proteolysis proceeds from the denatured forms of proteins containing the PA78 substitution. The PA78 mutation does not appear to introduce a new cleavage site for cellular proteases, nor does the mutation enhance susceptibility to proteases such as thermolysin and trypsin in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature, organic solvent and pH stabilization of the neutral protease from Salinovibrio proteolyticus: significance of the structural calcium

In order to clarify the impact of Ca-binding sites (Ca1 and 2) on the conformational stability of neutral proteases (NPs), we have analyzed the thermal, pH and organic solvent stability of a NP variant, V189P/A195E/G203D/A268E (Q-mutant), from Salinovibrio proteolyticus. This mutant has shown to bind calcium more tightly than the wild-type (WT) at Ca1 and to possess Ca2. Q-mutant was resisted against autolysis, thermoinactivation and pH denaturation in a Ca-dependent manner and exhibited better activity in organic solvents compared to the WT enzyme. These results imply that Ca1 and Ca2 are important for the conformational stability of NPs.     

Involvement of the chaperonin dnaK in the rapid degradation of a mutant protein in Escherichia coli.

The ability of Escherichia coli rapidly to degrade abnormal proteins is inhibited by mutations affecting any of several heat shock proteins (hsps). We therefore tested whether a short-lived mutant protein might become associated with hsps as part of its degradation. At 30 degrees C, the non-secreted mutant form of alkaline phosphatase, phoA61, is relatively stable, and very little phoA61 is found associated with the hsp dnaK. However, raising the temperature to 37 degrees C or 41 degrees C stimulated the degradation of this protein, and up to 30% of cellular phoA61 became associated with dnaK, as shown by immunoprecipitation and Western blot analysis. Also found in complexes with phoA61 were the hsps, protease La and grpE (but no groEL, or groES). The rapid degradation of phoA61 at 37 degrees C and 41 degrees C is in part by protease La, since it decreased by 50% in lon mutants. This process also requires dnaK, since deletion of this gene prevented phoA61 degradation almost completely (unless a wild-type dnaK gene was introduced). In contrast, the missense mutation, dnaK756, enhanced phoA61 degradation. The dnaK756 protein also was associated with phoA61, but this complex, unlike that containing wild-type dnaK could not be dissociated by ATP addition. Furthermore, in a grpE mutant, the degradation of phoA61 and the amount associated with dnaK increased, while in a dnaJ mutant, phoA61 degradation and its association with dnaK decreased. Thus, complex formation with dnaK appears essential for phoA61 degradation by protease La and some other cell proteases, and a failure of the dnaK to dissociate normally may accelerate proteolytic attack.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enthalpic and entropic contributions mediate the role of disulfide bonds on the conformational stability of interleukin-4

The role of disulfide bridges in the structure, stability, and folding pathways of proteins has been the subject of wide interest in the fields of protein design and engineering. However, the relative importance of entropic and enthalpic contributions for the stabilization of proteins provided by disulfides is not always clear. Here, we perform a detailed analysis of the role of disulfides in the conformational stability of human Interleukin-4 (IL4), a four-helix bundle protein. In order to evaluate the contribution of two out of the three disulfides to the structure and stability of IL4, two IL4 mutants, C3T-IL4 and C24T-IL4, were used. NMR and ANS binding experiments were compatible with altered dynamics and an increase of the nonpolar solvent-accessible surface area of the folded state of the mutant proteins. Chemical and thermal unfolding experiments followed by fluorescence and circular dichroism revealed that both mutant proteins have lower conformational stability than the wild-type protein. Transition temperatures of unfolding decreased 14 degrees C for C3T-IL4 and 10 degrees C for C24T-IL4, when compared to WT-IL4, and the conformational stability, at 25 degrees C, decreased 4.9 kcal/mol for C3T-IL4 and 3.2 kcal/mol for C24T-IL4. Interestingly, both the enthalpy and the entropy of unfolding, at the transition temperature, decreased in the mutant proteins. Moreover, a smaller change in heat capacity of unfolding was also observed for the mutants. Thus, disulfide bridges in IL4 play a critical role in maintaining the thermodynamic stability and core packing of the helix bundle.