Hydrophobic forces between protein molecules in aqueous solutions of concentrated electrolyte

Protein-protein interactions have been measured for a mutant (D101F) lysozyme and for native lysozyme in concentrated solutions of ammonium sulfate at pH 7 and sodium chloride at pH 4.5. In the mutant lysozyme, a surface aspartate residue has been replaced with a hydrophobic phenylalanine residue. The protein-protein interactions of D101F lysozyme are more attractive than those of native lysozyme for all conditions studied. The salt-induced attraction is correlated with a solvation potential of mean force given by the work required to desolvate the part of the protein surfaces that is buried by the protein-protein interaction. This work is proportional to the aqueous surface-tension increment of the salt and the fractional non-polar surface coverage of the protein. Experimental measurements of osmotic second virial coefficients validate a proposed potential of mean force that ascribes the salt-induced attraction between protein molecules to an enhancement of the hydrophobic attraction. This model provides a first approximation for predicting the protein-protein potential of mean force in concentrated aqueous electrolyte solutions; this potential is useful for determining solution conditions favorable for protein crystallization.     

Mapping the transition state of the WW domain beta-sheet

The folding kinetics of a three-stranded antiparallel beta-sheet (WW domain) have been measured by temperature jump relaxation. Folding and activation free energies were determined as a function of temperature for both the wild-type and the mutant domain, W39F, which modifies the beta(2)-beta(3) hydrophobic interface. The folding rate decreases at higher temperatures as a result of the increase in the activation free energy for folding. Phi-Values were obtained for thermal perturbations allowing the primary features of the folding free energy surface to be determined. The results of this analysis indicate a significant shift from an "early" (Phi(T)=0. 4) to a "late" (Phi(T)=0.8) transition state with increasing temperature. The temperature-dependent Phi-value analysis of the wild-type WW domain and of its more stable W39F hydrophobic cluster mutant reveals little participation of residue 39 in the transition state at lower temperature. As the temperature is raised, hydrophobic interactions at the beta(2)-beta(3) interface gain importance in the transition state and the barrier height of the wild-type, which contains the larger tryptophan residue, increases more slowly than the barrier height of the mutant.     

Altering the mouth of a hydrophobic pocket. Structure and kinetics of human carbonic anhydrase II mutants at residue Val-121

Eleven amino acid substitutions at Val-121 of human carbonic anhydrase II including Gly, Ala, Ser, Leu, Ile, Lys, and Arg, were constructed by site-directed mutagenesis. This residue is at the mouth of the hydrophobic pocket in the enzyme active site. The CO2 hydrase activity and the p-nitrophenyl esterase activity of these CAII variants correlate with the hydrophobicity of the residue, suggesting that the hydrophobic character of this residue is important for catalysis. The effects of these mutations on the steady-state kinetics for CO2 hydration occur mainly in kcat/Km and Km, consistent with involvement of this residue in CO2 association. The Val-121----Ala mutant, which exhibits about one-third normal CO2 hydrase activity, has been studied by x-ray crystallographic methods. No significant changes in the mutant enzyme conformation are evident relative to the wild-type enzyme. Since Val-121 is at the mouth of the hydrophobic pocket, its substitution by the methyl side chain of alanine makes the pocket mouth significantly wider than that of the wild-type enzyme. Hence, although a moderately wide (and deep) pocket is important for substrate association, a wider mouth to this pocket does not seriously compromise the catalytic approach of CO2 toward nucleophilic zinc-bound hydroxide.     

The structure and function of omega loop A replacements in cytochrome c.

The structural and functional consequences of replacing omega-loop A (residues 18-32) in yeast iso-1-cytochrome c with the corresponding loop of Rhodospirillum rubrum cytochrome c2 have been examined. The three-dimensional structure of this loop replacement mutant RepA2 cytochrome c, and a second mutant RepA2(Val 20) cytochrome c in which residue 20 was back substituted to valine, were determined using X-ray diffraction techniques. A change in the molecular packing is evident in the RepA2 mutant protein, which has a phenylalanine at position 20, a residue considerably larger than the valine found in wild-type yeast iso-1-cytochrome c. The side chain of Phe 20 is redirected toward the molecular surface, altering the packing of this region of omega-loop A with the hydrophobic core of the protein. In the RepA2(Val 20) structure, omega-loop A contains a valine at position 20, which restores the original wild-type packing arrangement of the hydrophobic core. Also, as a result of omega-loop A replacement, residue 26 is changed from a histidine to asparagine, which results in displacements of the main-chain atoms near residue 44 to which residue 26 is hydrogen bonded. In vivo studies of the growth rate of the mutant strains on nonfermentable media indicate that the RepA2(Val 20) cytochrome c behaves much like the wild-type yeast iso-1 protein, whereas the stability and function of the RepA2 cytochrome c showed a temperature dependence. The midpoint reduction potential measured by cyclic voltammetry of the RepA2 mutant is 271 mV at 25 degrees C. This is 19 mV less than the wild-type and RepA2(Val 20) proteins (290 mV) and may result from disruption of the hydrophobic packing in the heme pocket and increased mobility of omega-loop A in RepA2 cytochrome c. The temperature dependence of the reduction potential is also greatly enhanced in the RepA2 protein.     

Parameter relation rows: a query system for protein structure function relationships

In the course of molecular modeling or mutant prediction one often wants quick answers to questions such as: 'Are there any residues in a beta-strand that point into an internal cavity, and are highly mutable?' 'Are there large polar residues in a helix that make a contact with a hydrophobic residue in a sheet, and don't make the maximal number of hydrogen bonds?' or 'Which hydrophobic residues are in a helix with a large hydrophobic moment, and make a contact with a co-factor, but at the same time still have a large accessible surface?'. I describe here a method to get answers to these kinds of questions in a very quick and easy manner. The method described is partly based on the principles used in the design of relational databases, and its mode of operation is similar to the query methods used in a relational database environment. Although designed for aiding in molecular modeling, its applicability is much more general. The method has been implemented as part of a large molecular modeling package which copes with the numerous problems in systematic handling of protein structures, e.g. residue numbering. This also implies that many normal tools such as graphical analyses, I/O facilities, etc. are available on-line.     

Importance of hydrophobic interaction between a SoxB-type cytochrome c oxidase with its natural substrate cytochrome c-551 and its mutants

Cytochrome c-551, the electron donor of SoxB-type cytochrome c oxidase in thermophilic bacilli, can be over-expressed in Bacillus thermodenitrificans cells by tranformation with pSTEc551. Several mutant cytochromes c-551 were prepared by site-directed mutagenesis to this expression plasmid. Among them, several Lys residues were changed to Ala/Ser, and we found that these mutant cytochromes retained their activity as substrates, although their K(m) values were 0.04-0.12 microM, depending on the site replaced. In contrast, the C19A mutant cytochrome, which was produced in Brevibacillus choshinensis as a secretion protein, lost its activity as a substrate, suggesting that the fatty acyl-glyceryl residue covalently bound to the cysteine residue of the wild-type c-551 plays a very important role in the activity. The importance of the hydrophobic fatty acid residue for the binding of cytochrome c-551 to the oxidase was also shown by the loss of substrate activity in deacylated cytochrome c-551. These results show the importance of the hydrophobic interaction between this cytochrome and SoxB-type oxidase, despite the fact that the importance of an electrostatic interaction between cytochrome c and mitochondrial cytochrome aa(3) oxidase has already been established.     

In a staphylococcal nuclease mutant the side-chain of a lysine replacing valine 66 is fully buried in the hydrophobic core

The crystal structure of the staphylococcal nuclease mutant V66K, in which valine 66 is replaced by lysine, has been solved at 1.97 A resolution. Unlike lysine residues in previously reported protein structures, this residue appears to bury its side-chain in the hydrophobic core without salt bridging, hydrogen bonding or other forms of electrostatic stabilization. Solution studies of the free energy of denaturation, delta GH2O, show marked pH dependence and clearly indicate that the lysine residue must be deprotonated in the folded state. V66K is highly unstable at neutral pH but only modestly less stable than the wild-type protein at high pH. The pH dependence of stability for V66K, in combination with similar measurements for the wild-type protein, allowed determination of the pKa values of the lysine in both the denatured and native forms. The epsilon-amine of this residue has a pKa value in the denatured state of 10.2, but in the native state it must be 6.4 or lower. The epsilon-amine is thus deprotonated in the folded molecule. These values enabled an estimation of the epsilon-amine's relative change in free energy of solvation between solvent and the protein interior at 5.1 kcal/mol or greater. This implies that the value of the dielectric constant of the protein interior must be less than 12.8. Lysine is usually found with the methylene groups of its side-chain partly buried but is nevertheless considered a hydrophilic surface residue. It would appear that the high pKa value of lysine, which gives it a positive charge at physiological pH, is the primary reason for its almost exclusive confinement to the surface proteins. When deprotonated, this amino acid type can be fully incorporated into the hydrophobic core.     

The role of a clinically important mutation in the fold and RNA-binding properties of KH motifs

We have investigated the role in the fold and RNA-binding properties of the KH modules of a hydrophobic to asparagine mutation of clinical importance in the fragile X syndrome. The mutation involves a well-conserved hydrophobic residue close to the N terminus of the second helix of the KH fold (alpha2(3) position). The effect of the mutation has been long debated: Although the mutant has been shown to disrupt the three-dimensional fold of several KH domains, the residue seems also to be directly involved in RNA binding, the main function of the KH module. Here we have used the KH3 of Nova-1, whose structure is known both in isolation and in an RNA complex, to study in detail the role of the alpha2(3) position. A detailed comparison of Nova KH3 structure with its RNA/KH complex and with other KH structures suggests a dual role for the alpha2(3) residue, which is involved both in stabilizing the hydrophobic core and in RNA contacts. We further show by nuclear magnetic resonance (NMR) studies in solution that L447 of Nova-1 in position alpha2(3) is in exchange in the absence of RNA, and becomes locked in a more rigid conformation only upon formation of an RNA complex. This implies that position alpha2(3) functions as a "gate" in the mechanism of RNA recognition of KH motifs based on the rigidification of the fold upon RNA binding.     

A functional prolipoprotein signal peptide with a deletion of four amino acid residues from the hydrophobic region

The deletion of several codons within the signal sequence coding region of the Escherichia coli lipoprotein gene has been accomplished by oligonucleotide-directed site-specific mutagenesis. The deletion of the Leu-13 residue in a mutant in which two glycine residues had previously been deleted from the hydrophobic region (Inouye, S., Vlasuk, G., Hsiung, H., and Inouye, M. (1984) J. Biol. Chem. 259, 3729-3733) was found to cause the accumulation of the unmodified form of the protein in the cytoplasm and cytoplasmic membrane. This mutation also caused a cessation in cell growth within 15 min after synthesis of the mutant protein was induced. A deletion of the Val-7 residue was capable of suppressing the effect of the Leu-13 deletion when both are present. However, by itself the Val-7 deletion appeared to have little effect on the glycine mutant. The ability of the signal sequence to mediate the secretion of the protein after the deletion of 4 residues from the hydrophobic region demonstrates a surprising degree of flexibility in the length of this region. The deletion mutations were also found to have an unusual effect on the rate of synthesis of lipoprotein.     

The structural consequences of exchanging tryptophan and tyrosine residues in B. stearothermophilus lactate dehydrogenase.

A mutant Bacillus stearothermophilus lactate dehydrogenase has been prepared in which all three tryptophan residues in the wild-type enzyme have been replaced by tyrosines. In addition, a tyrosine residue has been mutated to a tryptophan, which acts as a fluorescence probe to monitor protein folding. The mutant enzyme crystallizes in the same crystal form as the wild-type. The crystal structure of the mutant has been determined at 2.8 A resolution. Solution studies have suggested that there is little effect upon the mutant enzyme as judged by its kinetic properties. Comparison of the crystal structures of the mutant and wild-type enzymes confirms this conclusion, and reveals that alterations in structure in the region of these mutations are of a similar magnitude to those observed throughout the structure, and are not significant when compared with the errors in atomic positions expected for a structure at this resolution.     

Mutational analysis of a CBM family 5 chitin-binding domain of an alkaline chitinase from Bacillus sp. J813

Chitinase J from alkaliphilic Bacillus sp. J813 comprises a glycoside hydrolase (GH) family 18 catalytic domain (CatD), a fibronectin type III like domain, and a carbohydrate-binding module (CBM) family 5 chitin-binding domain (ChBD). It has been suggested that the ChBD binds to insoluble chitin and enhances its degradation by the CatD. To investigate the roles of two aromatic residues (Trp541 and Trp542), which are exposed on the surface of the ChBD, mutational analysis was performed. Single and double mutations of the two aromatic residues decreased binding and hydrolyzing abilities toward insoluble chitin. This result suggests that the ChBD binds to chitin by hydrophobic interactions via two surface-exposed aromatic residues. However, the double mutant, which has no such aromatic residue, bound to chitin at pH 5.2, probably by electrostatic interactions. Moreover, the ChBD bound to insoluble chitosan by electrostatic interactions.     

A proton-NMR study of a site-directed mutation (His388----Glu) in the interdomain region of yeast phosphoglycerate kinase. Implications for domain movement

Proton NMR has been used to study a site-directed mutant of yeast phosphoglycerate kinase in which the interdomain residue His388 has been replaced by a glutamine residue. Using 1H-NMR spectroscopy, it was found that 3-phosphoglycerate binding to the mutant protein induces different conformational effects to those observed for the wild-type enzyme. These differences are not only located at the 3-phosphoglycerate binding site but are also seen as long-range effects at the surface of the protein. Measurements of the Kd for 3-phosphoglycerate from the NMR experiments show that the mutant enzyme has a 30-times reduced affinity for this substrate as compared with the wild-type enzyme. These data are consistent with the suggestion that an aromatic residue at position 388 plays an important role in the proposed hinge-bending mechanism.     

The use of genetically engineered tryptophan to identify the movement of a domain of B. stearothermophilus lactate dehydrogenase with the process which limits the steady-state turnover of the enzyme

A general technique for monitoring the intramolecular motion of a protein is described. Genetic engineering is used to replace all the natural tryptophan residues with tyrosine. A single tryptophan residue is then inserted at a specific site within the protein where motion is then detected from the fluorescence characteristics of this fluorophore. This technique has been used in B. stearothermophilus lactate dehydrogenase mutant (W80Y, W150Y, W203Y, G106W) to correlate the slow closure of a surface loop of polypeptide (residues 98-110) with the maximum catalytic velocity of the enzyme.     

A single amino acid substitution deregulates a bacterial lactate dehydrogenase and stabilizes its tetrameric structure

We have engineered a variant of the lactate dehydrogenase enzyme from Bacillus stearothermophilus in which arginine-173 at the proposed regulatory site has been replaced by glutamine. Like the wild-type enzyme, this mutant undergoes a reversible, protein-concentration-dependent subunit assembly, from dimer to tetramer. However, the mutant tetramer is much more stable (by a factor of 400) than the wild type and is destabilized rather than stabilized by binding the allosteric regulator, fructose 1,6-biphosphate (Fru-1,6-P2). The mutation has not significantly changed the catalytic properties of the dimer (Kd NADH, Km pyruvate, Ki oxamate and kcat), but has weakened the binding of Fru-1,6-P2 to both the dimeric and tetrameric forms of the enzyme and has almost abolished any stimulatory effect. We conclude that the Arg-173 residue in the wild-type enzyme is directly involved in the binding of Fru-1,6-P2, is important for allosteric communication with the active site, and, in part, regulates the state of quaternary structure through a charge-repulsion mechanism.     

Thermodynamics of glutathione binding to the tyrosine 7 to phenylalanine mutant of glutathione S-transferase from Schistosoma japonicum

The binding of glutathione (GSH) to the tyrosine 7 to phenylalanine mutant of Schistosoma japonicum glutathione S-transferase (SjGST-Y7F) has been studied by isothermal titration calorimetry (ITC). At pH 6.5 and 25 °C this mutant shows a higher affinity for glutathione than wild type enzyme despite an almost complete loss of activity in the presence of 1-chloro-2,4-dinitrobenzene (CDNB) as second substrate. The enthalpy change upon binding of GSH is more negative for the mutant than for the wild type GST (SjGST). Changes in accessible solvent areas (ASA) have been calculated based on enthalpy and heat capacity changes. ASA values indicated the burial of apolar surfaces of protein and ligand upon binding. A more negative ΔC_p value has been obtained for the mutant enzyme, suggesting a more hydrophobic interaction, as may be expected from the change of a tyrosine residue to phenylalanine.     

Sodium dodecyl sulfate stability of HLA-DR1 complexes correlates with burial of hydrophobic residues in pocket 1

Certain class II MHC-peptide complexes are resistant to SDS-induced dissociation. This property, which has been used as an in vivo as well as an in vitro peptide binding assay, is not understood at the molecular level. Here we have investigated the mechanistic basis of SDS stability of HLA-DR1 complexes by using a biosensor-based assay and SDS-PAGE with a combination of wild-type and mutant HLA-DR1 and variants of hemagglutinin peptide HA306-318. Experiments with wild-type DR1 along with previously published results establish that the SDS-stable complexes are formed only when the hydrophobic pocket 1 (P1) is occupied by a bulky aromatic (Trp, Phe, Tyr) or an aliphatic residue (Met, Ile, Val, Leu). To further explore whether the SDS sensitivity is primarily due to the exposed hydrophobic regions, we mutated residue beta Gly86 at the bottom of P1 to tyrosine, presumably reducing the depth of the pocket and the exposure of hydrophobic residues and increasing the contacts between subunits. In direct contrast to wild-type DR1, the peptide-free mutant DR1 exists as an alpha/beta heterodimer in SDS. Moreover, the presence of a smaller hydrophobic residue, such as alanine, as P1 anchor with no contribution from any other anchor is sufficient to enhance the SDS stability of the mutant complexes, demonstrating that the basis of SDS resistance may be localized to P1 interactions. The good correlation between SDS sensitivity and the exposure of hydrophobic residues provides a biochemical rationale for the use of this assay to investigate the maturation of class II molecules and the longevity of the complexes.     

Redesign of a four-helix bundle protein by phage display coupled with proteolysis and structural characterization by NMR and X-ray crystallography

To test whether it is practical to use phage display coupled with proteolysis for protein design, we used this approach to convert a partially unfolded four-helix bundle protein, apocytochrome b(562), to a stably folded four-helix bundle protein. Four residues expected to form a hydrophobic core were mutated. One residue was changed to Trp to provide a fluorescence probe for studying the protein's physical properties and to partially fill the void left by the heme. The other three positions were randomly mutated. In addition, another residue in the region to be redesigned was substituted with Arg to provide a specific cutting site for protease Arg-c. This library of mutants was displayed on the surface of phage and challenged with protease Arg-c to select stably folded proteins. The consensus sequence that emerged from the selection included hydrophobic residues at only one of the three positions and non-hydrophobic residues at the other two. Nevertheless, the selected proteins were thermodynamically very stable. The structure of a selected protein was characterized using multi-dimensional NMR. All four helices were formed in the structure. Further, site-directed mutagenesis was used to change one of the two non-hydrophobic residues to a hydrophobic residue, which increased the stability of the protein, indicating that the selection result was not based solely on the protein's global stability and that local structural characteristics may also govern the selection. This conclusion is supported by the crystal structure of another mutant that has two hydrophobic residues substituted for the two non-hydrophobic residues. These results suggest that the hydrophobic interactions in the core are not sufficient to dictate the selection and that the location of the cutting site of the protease also influences the selection of structures.     

Estimation of the hydrophobic effect in an antigen-antibody protein-protein interface

Antigen-antibody complexes provide useful models for analyzing the thermodynamics of protein-protein association reactions. We have employed site-directed mutagenesis, X-ray crystallography, and isothermal titration calorimetry to investigate the role of hydrophobic interactions in stabilizing the complex between the Fv fragment of the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL. Crystal structures of six FvD1.3-HEL mutant complexes in which an interface tryptophan residue (V(L)W92) has been replaced by residues with smaller side chains (alanine, serine, valine, aspartate, histidine, and phenylalanine) were determined to resolutions between 1.75 and 2.00 A. In the wild-type complex, V(L)W92 occupies a large hydrophobic pocket on the surface of HEL and constitutes an energetic "hot spot" for antigen binding. The losses in apolar buried surface area in the mutant complexes, relative to wild-type, range from 25 (V(L)F92) to 115 A(2) (V(L)A92), with no significant shifts in the positions of protein atoms at the mutation site for any of the complexes except V(L)A92, where there is a peptide flip. The affinities of the mutant Fv fragments for HEL are 10-100-fold lower than that of the original antibody. Formation of all six mutant complexes is marked by a decrease in binding enthalpy that exceeds the decrease in binding free energy, such that the loss in enthalpy is partly offset by a compensating gain in entropy. No correlation was observed between decreases in apolar, polar, or aggregate (sum of the apolar and polar) buried surface area in the V(L)92 mutant series and changes in the enthalpy of formation. Conversely, there exist linear correlations between losses of apolar buried surface and decreases in binding free energy (R(2) = 0.937) as well as increases in the solvent portion of the entropy of binding (R(2) = 0.909). The correlation between binding free energy and apolar buried surface area corresponds to 21 cal mol(-1) A(-2) (1 cal = 4.185 J) for the effective hydrophobicity at the V(L)92 mutation site. Furthermore, the slope of the line defined by the correlation between changes in binding free energy and solvent entropy approaches unity, demonstrating that the exclusion of solvent from the binding interface is the predominant energetic factor in the formation of this protein complex. Our estimate of the hydrophobic contribution to binding at site V(L)92 in the D1.3-HEL interface is consistent with values for the hydrophobic effect derived from classical hydrocarbon solubility models. We also show how residue V(L)W92 can contribute significantly less to stabilization when buried in a more polar pocket, illustrating the dependence of the hydrophobic effect on local environment at different sites in a protein-protein interface.     

Mutational Analysis of a CBM Family 5 Chitin-Binding Domain of an Alkaline Chitinase from Bacillus sp. J813

Chitinase J from alkaliphilic Bacillus sp. J813 comprises a glycoside hydrolase (GH) family 18 catalytic domain (CatD), a fibronectin type III like domain, and a carbohydrate-binding module (CBM) family 5 chitin-binding domain (ChBD). It has been suggested that the ChBD binds to insoluble chitin and enhances its degradation by the CatD. To investigate the roles of two aromatic residues (Trp541 and Trp542), which are exposed on the surface of the ChBD, mutational analysis was performed. Single and double mutations of the two aromatic residues decreased binding and hydrolyzing abilities toward insoluble chitin. This result suggests that the ChBD binds to chitin by hydrophobic interactions via two surface-exposed aromatic residues. However, the double mutant, which has no such aromatic residue, bound to chitin at pH 5.2, probably by electrostatic interactions. Moreover, the ChBD bound to insoluble chitosan by electrostatic interactions.     

The loss in hydrophobic surface area resulting from a Leu to Val mutation at the N-terminus of the aldehyde dehydrogenase presequence prevents import of the protein into mitochondria

An apparent conservative mutation, Leu to Val, at the second residue of the rat liver mitochondrial aldehyde dehydrogenase (ALDH) presequence resulted in a precursor protein that was not imported into mitochondria. Additional mutants were made to substitute various amino acids with nonpolar side chains for Leu2. The Ile, Phe, and Trp mutants were imported to an extent similar to that of the native precursor, but the Ala mutant was imported only about one-fourth as well. It was shown that the N-terminal methionine was removed from the L2V mutant in a reaction catalyzed by methionine aminopeptidase. The N-terminal methionine of native pALDH and the other mutant presequences was blocked, presumably by acetylation. Because of the difference in co-translational modification, the L2V mutant sustained a significant loss in the available hydrophobic surface of the presequence. Import competence was restored to the L2V mutant when it was translated using a system that did not remove Met1. The removal of an Arg-Gly-Pro helix linker segment (residues 11-14) from the L2V mutant, which shifted three leucine residues toward the N-terminus, also restored import competence. These results lead to the conclusion that a minimum amount of hydrophobic surface area near the N-termini of mitochondrial presequences is an essential property to determine their ability to be imported. As a result, both electrostatic and hydrophobic components must be considered when trying to understand the interactions between precursor proteins and proteins of the mitochondrial import apparatus.