Positive contribution of hydration structure on the surface of human lysozyme to the conformational stability

Water molecules make a hydration structure with the network of hydrogen bonds, covering on the surface of proteins. To quantitatively estimate the contribution of the hydration structure to protein stability, a series of hydrophilic mutant human lysozymes (Val to Ser, Tyr, Asp, Asn, and Arg) modified at three different positions on the surface, which are located in the alpha-helix (Val-110), the beta-sheet (Val-2), and the loop (Val-74), were constructed. Their thermodynamic parameters of denaturation and crystal structures were examined by calorimetry and by x-ray crystallography at 100 K, respectively. The introduced polar residues made hydrogen bonds with protein atoms and/or water molecules, sometimes changing the hydration structure around the mutation site. Changes in the stability of the mutant proteins can be evaluated by a unique equation that considers the conformational changes resulting from the substitutions. Using this analysis, the relationship between the changes in the stabilities and the hydration structures for mutant human lysozymes substituted on the surface could be quantitatively estimated. The analysis indicated that the hydration structure on protein surface plays an important role in determining the conformational stability of the protein.     

Contribution of amino acid substitutions at two different interior positions to the conformational stability of human lysozyme.

To elucidate correlative relationships between structural change and thermodynamic stability in proteins, a series of mutant human lysozymes modified at two buried positions (Ile56 and Ile59) were examined. Their thermodynamic parameters of denaturation and crystal structures were studied by calorimetry and X-ray crystallography. The mutants at positions 56 and 59 exhibited different responses to a series of amino acid substitutions. The changes in stability due to substitutions showed a linear correlation with changes in hydrophobicity of substituted residues, having different slopes at each mutation site. However, the stability of each mutant was found to be represented by a unique equation involving physical properties calculated from mutant structures. By fitting present and previous stability data for mutant human lysozymes substituted at various positions to the equation, the magnitudes of the hydrophobicity of a carbon atom and the hydrophobicity of nitrogen and neutral oxygen atoms were found to be 0.178 and -0.013 kJ/mol.A(2), respectively. It was also found that the contribution of a hydrogen bond with a length of 3.0 A to protein stability was 5.1 kJ/mol and the entropy loss of newly introduction of a water molecules was 7.8 kJ/mol.     

Contribution of intra- and intermolecular hydrogen bonds to the conformational stability of human lysozyme(,).

In globular proteins, there are intermolecular hydrogen bonds between protein and water molecules, and between water molecules, which are bound with the proteins, in addition to intramolecular hydrogen bonds. To estimate the contribution of these hydrogen bonds to the conformational stability of a protein, the thermodynamic parameters for denaturation and the crystal structures of five Thr to Val and five Thr to Ala mutant human lysozymes were determined. The denaturation Gibbs energy (DeltaG) of Thr to Val and Thr to Ala mutant proteins was changed from 4.0 to -5.6 kJ/mol and from 1.6 to -6.3 kJ/mol, respectively, compared with that of the wild-type protein. The contribution of hydrogen bonds to the stability (DeltaDeltaG(HB)) of the Thr and other mutant human lysozymes previously reported was extracted from the observed stability changes (DeltaDeltaG) with correction for changes in hydrophobicity and side chain conformational entropy between the wild-type and mutant structures. The estimation of the DeltaDeltaG(HB) values of all mutant proteins after removal of hydrogen bonds, including protein-water hydrogen bonds, indicates a favorable contribution of the intra- and intermolecular hydrogen bonds to the protein stability. The net contribution of an intramolecular hydrogen bond (DeltaG(HB[pp])), an intermolecular one between protein and ordered water molecules (DeltaG(HB[pw])), and an intermolecular one between ordered water molecules (DeltaG(HB[ww])) could be estimated to be 8. 5, 5.2, and 5.0 kJ/mol, respectively, for a 3 A long hydrogen bond. This result shows the different contributions to protein stability of intra- and intermolecular hydrogen bonds. The entropic cost due to the introduction of a water molecule (DeltaG(H)()2(O)) could be also estimated to be about 8 kJ/mol.     

Electrostatic stabilization of a thermophilic cold shock protein.

The cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus differs from its mesophilic homolog Bs-CspB from Bacillus subtilis by 15.8 kJ mol(-1) in the Gibbs free energy of denaturation (DeltaG(D)). The two proteins vary in sequence at 12 positions but only two of them, Arg3 and Leu66 of Bc-Csp, which replace Glu3 and Glu66 of Bs-CspB, are responsible for the additional stability of Bc-Csp. These two positions are near the ends of the protein chain, but close to each other in the three-dimensional structure. The Glu3Arg exchange alone changed the stability by more than 11 kJ mol(-1). Here, we elucidated the molecular origins of the stability difference between the two proteins by a mutational analysis. Electrostatic contributions to stability were characterized by measuring the thermodynamic stabilities of many variants as a function of salt concentration. Double and triple mutant analyses indicate that the stabilization by the Glu3Arg exchange originates from three sources. Improved hydrophobic interactions of the aliphatic moiety of Arg3 contribute about 4 kJ mol(-1). Another 4 kJ mol(-1) is gained from the relief of a pairwise electrostatic repulsion between Glu3 and Glu66, as in the mesophilic protein, and 3 kJ mol(-1) originate from a general electrostatic stabilization by the positive charge of Arg3, which is not caused by a pairwise interaction. Mutations of all potential partners for an ion pair within a radius of 10 A around Arg3 had only marginal effects on stability. The Glu3-->Arg3 charge reversal thus optimizes ionic interactions at the protein surface by both local and global effects. However, it cannot convert the coulombic repulsion with another Glu residue into a corresponding attraction. Avoidance of unfavorable coulombic repulsions is probably a much simpler route to thermostability than the creation of stabilizing surface ion pairs, which can form only at the expense of conformational entropy.     

Contribution of hydrogen bonds to the conformational stability of human lysozyme: calorimetry and X-ray analysis of six Ser --> Ala mutants.

To further examine the contribution of hydrogen bonds to the conformational stability of the human lysozyme, six Ser to Ala mutants were constructed. The thermodynamic parameters for denaturation of these six Ser mutant proteins were investigated by differential scanning calorimetry (DSC), and the crystal structures were determined by X-ray analysis. The denaturation Gibbs energy (DeltaG) of the Ser mutant proteins was changed from 2.0 to -5.7 kJ/mol, compared to that of the wild-type protein. With an analysis in which some factors that affected the stability due to mutation were considered, the contribution of hydrogen bonds to the stability (Delta DeltaGHB) was extracted on the basis of the structures of the mutant proteins. The results showed that hydrogen bonds between protein atoms and between a protein atom and a water bound with the protein molecule favorably contribute to the protein stability. The net contribution of one intramolecular hydrogen bond to protein stability (DeltaGHB) was 8.9 +/- 2.6 kJ/mol on average. However, the contribution to the protein stability of hydrogen bonds between a protein atom and a bound water molecule was smaller than that for a bond between protein atoms.     

A hyperthermophilic protein acquires function at the cost of stability

Active-site residues are not often optimized for conformational stability (activity-stability trade-offs) in proteins from organisms that grow at moderate temperature. It is unknown if the activity-stability trade-offs can be applied to proteins from hyperthermophiles. Because enzymatic activity usually increases at higher temperature and hyperthermophilic proteins need high conformational stability, they might not sacrifice the stability for their activity. This study attempts to clarify the contribution of active-site residues to the conformational stability of a hyperthermophilic protein. We therefore examined the thermodynamic stability and enzymatic activity of wild-type and active-site mutant proteins (D7N, E8A, E8Q, D105A, and D135A) of ribonuclease HII from Thermococcus kodakaraensis (Tk-RNase HII). Guanidine hydrochloride (GdnHCl)-induced denaturation was measured with circular dichroism at 220 nm, and heat-induced denaturation was studied with differential scanning calorimetry. Both GdnHCl- and heat-induced denaturation were highly reversible in these proteins. All the mutations of these active-site residues, except that of Glu8 to Gln, reduced the enzymatic activity dramatically but increased the protein stability by 7.0 to 11.1 kJ mol(-1) at 50 degrees C. The mutation of Glu8 to Gln did not seriously affect the enzymatic activity and increased the stability only by 2.5 kJ mol(-1) at 50 degrees C. These results indicate that hyperthermophilic proteins also exhibit the activity-stability trade-offs. Therefore, the architectural mechanism for hyperthermophilic proteins is equivalent to that for proteins at normal temperature.     

pH-induced equilibrium unfolding of apomyoglobin: Substitutions at conserved Trp14 and Met131 and non-conserved Val17 positions

A number of residues in globins family are well conserved but are not directly involved in the primary oxygen-carrying function of these proteins. A possible role for these conserved, non-functional residues has been suggested in promoting a rapid and correct folding process to the native tertiary structure. To test this hypothesis, we have studied pH-induced equilibrium unfolding of mutant apomyoglobins with substitutions of the conserved residues Trp14 and Met131, which are not involved in the function of myoglobin, by various amino acids. This allowed estimating their impact on the stability of various conformational states of the proteins and selecting conditions for a folding kinetics study. The results obtained from circular dichroism, tryptophan fluorescence, and differential scanning microcalorimetry for these mutant proteins were compared with those for the wild type protein and for a mutant with the non-conserved Val17 substituted by Ala. In the native folded state, all of the mutant apoproteins have a compact globular structure, but are destabilized in comparison to the wild type protein. The pH-induced denaturation of the mutant proteins occurs through the formation of a molten globule-like intermediate similar to that of the wild type protein. Thermodynamic parameters for all of the proteins were calculated using the three state model. Stability of equilibrium intermediates at pH ~4.0 was shown to be slightly affected by the mutations. Thus, all of the above substitutions influence the stability of the native state of these proteins. The cooperativity of conformational transitions and the exposed to solvent protein surface were also changed, but not for the substitution at Val17.     

Packing and hydrophobicity effects on protein folding and stability: effects of beta-branched amino acids, valine and isoleucine, on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers.

The aim of this study was to examine the differences between hydrophobicity and packing effects in specifying the three-dimensional structure and stability of proteins when mutating hydrophobes in the hydrophobic core. In DNA-binding proteins (leucine zippers), Leu residues are conserved at positions "d," and beta-branched amino acids, Ile and Val, often occur at positions "a" in the hydrophobic core. In order to discern what effect this selective distribution of hydrophobes has on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers, three Val or three Ile residues were simultaneously substituted for Leu at either positions "a" (9, 16, and 23) or "d" (12, 19, and 26) in both chains of a model coiled coil. The stability of the resulting coiled coils was monitored by CD in the presence of Gdn.HCl. The results of the mutations of Ile to Val at either positions "a" or "d" in the reduced or oxidized coiled coils showed a significant hydrophobic effect with the additional methylene group in Ile stabilizing the coiled coil (delta delta G values range from 0.45 to 0.88 kcal/mol/mutation). The results of mutations of Leu to Ile or Val at positions "a" in the reduced or oxidized coiled coils showed a significant packing effect in stabilizing the coiled coil (delta delta G values range from 0.59 to 1.03 kcal/mol/mutation). Our results also indicate the subtle control hydrophobic packing can have not only on protein stability but on the conformation adopted by the amphipathic alpha-helices. These structural findings correlate with the observation that in DNA-binding proteins, the conserved Leu residues at positions "d" are generally less tolerant of amino acid substitutions than the hydrophobic residues at positions "a."     

Conformational stability of adrenodoxin mutant proteins.

Adrenodoxin and the mutants at the positions T54, H56, D76, Y82, and C95, as well as the deletion mutants 4-114 and 4-108, were studied by high-sensitivity scanning microcalorimetry, limited proteolysis, and absorption spectroscopy. The mutants show thermal transition temperatures ranging from 46 to 56 degrees C, enthalpy changes from 250 to 370 kJ/mol, and heat capacity change delta Cp = 7.28 +/- 0.67 kJ/mol/K, except H56R. The amino acid replacement H56R produces substantial local changes in the region around positions 56 and Y82, as indicated by reduced heat capacity change (delta Cp = 4.29 +/- 0.37 kJ/mol/K) and enhanced fluorescence. Deletion mutant 4-108 is apparently more stable than the wild type, as judged by higher specific denaturation enthalpy and resistance toward proteolytic degradation. No simple correlation between conformational stability and functional properties could be found.     

Filling small, empty protein cavities: structural and energetic consequences

Most proteins contain small cavities that can be filled by replacing cavity-lining residues by larger ones. Since shortening mutations in hydrophobic cores tend to destabilize proteins, it is expected that cavity-filling mutations may conversely increase protein stability. We have filled three small cavities in apoflavodoxin and determined by NMR and equilibrium unfolding analysis their impact in protein structure and stability. The smallest cavity (14 A3) has been filled, at two different positions, with a variety of residues and, in all cases, the mutant proteins are locally unfolded, their structure and energetics resembling those of an equilibrium intermediate of the thermal unfolding of the wild-type protein. In contrast, two slightly larger cavities of 20 A3 and 21 A3 have been filled with Val to Ile or Val to Leu mutations and the mutants preserve both the native fold and the equilibrium unfolding mechanism. From the known relationship, observed in shortening mutations, between stability changes and the differential hydrophobicity of the exchanged residues and the volume of the cavities, the filling of these apoflavodoxin cavities is expected to stabilize the protein by approximately 1.5 kcal mol(-1). However, both urea and thermal denaturation analysis reveal much more modest stabilizations, ranging from 0.0 kcal mol(-1) to 0.6 kcal mol(-1), which reflects that the accommodation of single extra methyl groups in small cavities requires some rearrangement, necessarily destabilizing, that lowers the expected theoretical stabilization. As the size of these cavities is representative of that of the typical small, empty cavities found in most proteins, it seems unlikely that filling this type of cavities will give rise to large stabilizations.     

Sequential hydration of dry proteins: a direct difference IR investigation of sequence homologs lysozyme and alpha- lactalbumin

Direct difference ir spectra are presented as a function of hydration for lysozyme and α-lactalbumin, and detailed sequential hydration molecular events identified. Despite the strong sequence homology between the two proteins, and their expected conformational similarity, the hydration behaviour of the polar groups is different for the two proteins. Using a Hill-type analysis, we conclude that the acid groups ionize and hydrate rapidly and noncooperatively in both proteins, consistent with the known (lysozyme) and postulated (α-lactalbumin) surface chemistry. The polar group hydration shows a clear cooperativity, which is quantitatively different in the two proteins. Complementary work suggests this cooperativity relates to a hydration-induced “loosening up” of the lysozyme conformation at about 55 mol water/mol protein. α-Lactalbumin appears to “open up” more easily for hydration than does lysozyme, consistent with its lower stability against thermal and acid denaturation.     

Contribution of polar groups in the interior of a protein to the conformational stability

It has been generally believed that polar residues are usually located on the surface of protein structures. However, there are many polar groups in the interior of the structures in reality. To evaluate the contribution of such buried polar groups to the conformational stability of a protein, nonpolar to polar mutations (L8T, A9S, A32S, I56T, I59T, I59S, A92S, V93T, A96S, V99T, and V100T) in the interior of a human lysozyme were examined. The thermodynamic parameters for denaturation were determined using a differential scanning calorimeter, and the crystal structures were analyzed by X-ray crystallography. If a polar group had a heavy energy cost to be buried, a mutant protein would be remarkably destabilized. However, the stability (Delta G) of the Ala to Ser and Val to Thr mutant human lysozymes was comparable to that of the wild-type protein, suggesting a low-energy penalty of buried polar groups. The structural analysis showed that all polar side chains introduced in the mutant proteins were able to find their hydrogen bond partners, which are ubiquitous in protein structures. The empirical structure-based calculation of stability change (Delta Delta G) [Takano et al. (1999) Biochemistry 38, 12698--12708] revealed that the mutant proteins decreased the hydrophobic effect contributing to the stability (Delta G(HP)), but this destabilization was recovered by the hydrogen bonds newly introduced. The present study shows the favorable contribution of polar groups with hydrogen bonds in the interior of protein molecules to the conformational stability.     

Stability and binding properties of wild-type and c17s mutated human sterol carrier protein 2.

The temperature- and solvent-induced denaturation of both the SCP2 wild-type and the mutated protein c71s were studied by CD measurements at 222 nm. The temperature-induced transition curves were deconvoluted according to a two-state mechanism resulting in a transition temperature of 70.5 degrees C and 59.9 degrees C for the wild-type and the c71s, respectively, with corresponding values of the van't Hoff enthalpies of 183 and 164 kJ/mol. Stability parameters characterizing the guanidine hydrochloride denaturation curves were also calculated on the basis of a two-state transition. The transitions of the wild-type occurs at 0.82 M GdnHCl and that of the c71s mutant at 0.55 M GdnHCl. These differences in the half denaturation concentration of GdnHCl reflect already the significant stability differences between the two proteins. A quantitative measure are the Gibbs energies DeltaG(0)(D)(buffer) at 25 degrees C of 15.5 kJ/mol for the wild-type and 8.0 kJ/mol for the mutant. We characterized also the alkyl chain binding properties of the two proteins by measuring the interaction parameters for the complex formation with 1-O-Decanyl-beta-D-glucoside using isothermal titration microcalorimetry. The dissociation constants, K(d), for wild-type SCP2 are 335 microM at 25 degrees C and 1.3 mM at 35 degrees C. The corresponding binding enthalpies, DeltaH(b), are -21. 5 kJ/mol at 25 degrees C and 72.2 kJ/mol at 35 degrees C. The parameters for the c71s mutant at 25 degrees C are K(d)=413 microM and DeltaH(b)=16.6 kJ/mol. These results suggest that both SCP2 wild-type and the c71s mutant bind the hydrophobic compound with moderate affinity.     

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.     

Effect of cavity-modulating mutations on the stability of Escherichia coli ribonuclease HI.

The size of the cavity around Ser68 of Escherichia coli ribonuclease HI was modulated by amino acid substitutions to examine the effects on the stability of the enzyme. Five mutant proteins, Ser68----Gly, Ser68----Ala, Ser68----Thr, Ser68----Val and Ser68----Leu, were constructed. Each of the mutant proteins exhibited at least 40% of the enzyme activity of the wild-type protein. The stabilities of the mutant proteins were determined from urea-denaturation and thermal-denaturation curves. Among the five mutations, only the Ser----Val mutation resulted in an increase in the stability of the enzyme. The melting temperature, tm, at pH 3.0 of the mutant protein Ser68----Val was increased by 1.9 degrees C. Its free-energy change of unfolding in the absence of urea, delta G(H2O), and the midpoint of the denaturation curve, [D]1/2, were also increased by 5.4 kJ/mol and 0.18 M, respectively. The increase in the stability of the enzyme is probably due to the filling of the cavity space around Ser68 by valine. However, the mutation of Ser68 to glycine or leucine residues resulted in a considerable decrease in stability. In these cases, some conformational changes occur, as suggested by the CD and 1H-NMR spectra of these mutant proteins.     

High-pressure studies of aggregation of recombinant human interleukin-1 receptor antagonist: thermodynamics, kinetics, and application to accelerated formulation studies

Recombinant human interleukin-1 receptor antagonist (IL-1ra) in aqueous solutions unfolds and aggregates when subjected to hydrostatic pressures greater than about 180 MPa. This study examined the mechanism and thermodynamics of pressure-induced unfolding and aggregation of IL-1ra. The activation free energy for growth of aggregates (DeltaG-/+(aggregation)) was found to be 37 +/- 3 kJ/mol, whereas the activation volume (DeltaV-/+(aggregation)) was -120 +/- 20 mL/mol. These values compare closely with equilibrium values for denaturation: The free energy for denaturation, DeltaG(denaturation), was 20 +/- 5 kJ/mol, whereas the partial specific volume change for denaturation, DeltaV(denaturation), was -110 +/- 30 mL/mol. When IL-1ra begins to denature at pressures near 140 MPa, cysteines that are normally buried in the native state become exposed. Under oxidizing conditions, this results in the formation of covalently cross-linked aggregates containing nonnative, intermolecular disulfide bonds. The apparent activation free energy for nucleation of aggregates, DeltaG-/+(nuc), was 42 +/- 4 kJ/mol, and the activation volume for nucleation, DeltaV-/+(nuc),was -175 +/- 37 mL/mol, suggesting that a highly solvent-exposed conformation is needed for nucleation. We hypothesize that the large specific volume of IL-1ra, 0.752 +/- 0.004 mL/g, coupled with its relatively low conformational stability, leads to its susceptibility to denaturation at relatively low pressures. The positive partial specific adiabatic compressibility of IL-1ra, 4.5 +/- 0.7 +/- 10(-12) cm2/dyn, suggests that a significant component of the DeltaV(denaturation) is attributable to the elimination of solvent-free cavities. Lastly, we propose that hydrostatic pressure is a useful variable to conduct accelerated formulation studies of therapeutic proteins.     

Conformational stability of legume lectins reflect their different modes of quaternary association: solvent denaturation studies on concanavalin A and winged bean acidic agglutinin

Thermodynamic parameters associated with the unfolding of the legume lectin, WBA II, were determined by isothermal denaturation. The analysis of isothermal denaturation data provided values for conformational stability and heat capacity for WBA II unfolding. To explore the role of intersubunit contact in stability, we carried out similar studies under identical conditions on Concanavalin A, a legume lectin of nearly similar size, buried hydrophobic surface area and tertiary structure to that of WBA II but with a different oligomerization pattern. Both proteins showed a reversible two-state unfolding with guanidine hydrochloride. As expected, the change in heat capacity upon unfolding was similar for both proteins at 3.5 and 3.7 kcal mol(-1) K(-1) for Concanavalin A and WBA II, respectively. Although the deltaG(H20) at the maximum stability of both proteins is around 16 kcal/mol, Concanavalin A exhibits greater stability at higher temperatures. The T(g) obtained for Concanavalin A and WBA II were 21 degrees C apart at 87.2 and 66.6 degrees C, respectively. The higher conformational stability at higher temperatures and the T(g) of Concanavalin A as compared to that of WBA II are largely due to substantial differences in the degree of subunit contact in these dimeric proteins. Ionic interactions and hydrogen bonding between the monomers of the two proteins also seem to play a significant role in the observed stability differences between these two proteins.     

Two exposed amino acid residues confer thermostability on a cold shock protein

Thermophilic organisms produce proteins of exceptional stability. To understand protein thermostability at the molecular level we studied a pair of cold shock proteins, one of mesophilic and one of thermophilic origin, by systematic mutagenesis. Although the two proteins differ in sequence at 12 positions, two surface-exposed residues are responsible for the increase in stability of the thermophilic protein (by 15.8 kJ mol-1 at 70 degrees C). 11.5 kJ mol-1 originate from a predominantly electrostatic contribution of Arg 3 and 5.2 kJ mol-1 from hydrophobic interactions of Leu 66 at the carboxy terminus. The mesophilic protein could be converted to a highly thermostable form by changing the Glu residues at positions 3 and 66 to Arg and Leu, respectively. The variation of surface residues may thus provide a simple and powerful approach for increasing the thermostability of a protein.     

Effect of foreign N-terminal residues on the conformational stability of human lysozyme.

To minutely understand the effect of foreign N-terminal residues on the conformational stability of human lysozyme, five mutant proteins were constructed: two had Met or Ala in place of the N-terminal Lys residue (K1M and K1A, respectively), and others had one additional residue, Met, Gly or Pro, to the N-terminal Lys residue (Met(-1), Gly(-1) and Pro(-1), respectively). The thermodynamic parameters for denaturation of these mutant proteins were examined by differential scanning calorimetry and were compared with that of the wild-type protein. Three mutants with the extra residue were significantly destabilized: the changes in unfolding Gibbs energy (DeltaDeltaG) were -9.1 to -12.2 kJ.mol-1. However, the stability of two single substitutions at the N-terminal slightly decreased; the DeltaDeltaG values were only -0.5 to -2.5 kJ.mol-1. The results indicate that human lysozyme is destabilized by an expanded N-terminal residue. The crystal structural analyses of K1M, K1A and Gly(-1) revealed that the introduction of a residue at the N-terminal of human lysozyme caused the destruction of hydrogen bond networks with ordered water molecules, resulting in the destabilization of the protein.     

Role of amino acid residues at turns in the conformational stability and folding of human lysozyme

To clarify the role of amino acid residues at turns in the conformational stability and folding of a globular protein, six mutant human lysozymes deleted or substituted at turn structures were investigated by calorimetry, GuHCl denaturation experiments, and X-ray crystal analysis. The thermodynamic properties of the mutant and wild-type human lysozymes were compared and discussed on the basis of their three-dimensional structures. For the deletion mutants, Delta47-48 and Delta101, the deleted residues are in turns on the surface and are absent in human alpha-lactalbumin, which is homologous to human lysozyme in amino acid sequence and tertiary structure. The stability of both mutants would be expected to increase due to a decrease in conformational entropy in the denatured state; however, both proteins were destabilized. The destabilizations were mainly caused by the disappearance of intramolecular hydrogen bonds. Each part deleted was recovered by the turn region like the alpha-lactalbumin structure, but there were differences in the main-chain conformation of the turn between each deletion mutant and alpha-lactalbumin even if the loop length was the same. For the point mutants, R50G, Q58G, H78G, and G37Q, the main-chain conformations of these substitution residues located in turns adopt a left-handed helical region in the wild-type structure. It is thought that the left-handed non-Gly residue has unfavorable conformational energy compared to the left-handed Gly residue. Q58G was stabilized, but the others had little effect on the stability. The structural analysis revealed that the turns could rearrange the main-chain conformation to accommodate the left-handed non-Gly residues. The present results indicate that turn structures are able to change their main-chain conformations, depending upon the side-chain features of amino acid residues on the turns. Furthermore, stopped-flow GuHCl denaturation experiments on the six mutants were performed. The effects of mutations on unfolding-refolding kinetics were significantly different among the mutant proteins. The deletion/substitutions in turns located in the alpha-domain of human lysozyme affected the refolding rate, indicating the contribution of turn structures to the folding of a globular protein.