Folding Of Lysozyme Research Articles

Folding of human lysozyme in vivo by the formation of an alternative disulfide bond.

The mutant h-lysozyme, W64CC65A, with Trp64 and Cys65 replaced by Cys and Ala, respectively, was secreted by yeast and purified. Peptide mapping confirmed that W64CC65A contained a nonnative Cys64-Cys81 bond and three native disulfide bonds. The mutant had 2% of the lytic activity of the wild-type lysozyme. The midpoint concentration of the guanidine hydrochloride denaturation curve, the [D]1/2, was 2.7 M for W64CC65A at pH 3.0 and 25 degrees C, whereas the [D]1/2 for the wild-type h-lysozyme was 2.9 M. These results show that the W64CC65A protein is a compactly folded molecule. Our previous results, using the mutant C81A, indicate that Cys81 is not required for correct folding and activity, whereas Cys65 is indispensable (Taniyama, Y., Yamamoto, Y., Kuroki, R., and Kikuchi, M. (1990) J. Biol. Chem. 65, 7570-7575). Cys64 substituted for Cys65 in W64CC65A, even though the distance between the alpha-carbons at positions 64 and 81 in the wild-type h-lysozyme is not favorable for forming a disulfide bond. Unlike C81A, the mutant W64CC65/81A, which has the additional substitution of Ala for Cys81, did not fold. These results suggest that the absence of both the Cys64-Cys81 bond and the amino acid residue Trp64 caused the misfolding or destabilization of W64CC65/81A in vivo. It is proposed that the formation of the alternative bond, Cys64-Cys81 is important for the folding of W64CC65A in vivo.

Folding and function of a T4 lysozyme containing 10 consecutive alanines illustrate the redundancy of information in an amino acid sequence.

Single and multiple Xaa----Ala substitutions were constructed in the alpha-helix comprising residues 39-50 in bacteriophage T4 lysozyme. The variant with alanines at 10 consecutive positions (A40-49) folds normally and has activity essentially the same as wild type, although it is less stable. The crystal structure of this polyalanine mutant displays no significant change in the main-chain atoms of the helix when compared with the wild-type structure. The individual substitutions of the solvent-exposed residues Asn-40, Ser-44, and Glu-45 with alanine tend to increase the thermostability of the protein, whereas replacements of the buried or partially buried residues Lys-43 and Leu-46 are destabilizing. The melting temperature of the lysozyme in which Lys-43 and Leu-46 are retained and positions 40, 44, 45, 47, and 48 are substituted with alanine (i.e., A40-42/44-45/47-49) is increased by 3.1 degrees C relative to wild type at pH 3.0, but reduced by 1.6 degrees C at pH 6.7. In the case of the charged amino acids Glu-45 and Lys-48, the changes in melting temperature indicate that the putative salt bridge between these two residues contributes essentially nothing to the stability of the protein. The results clearly demonstrate that there is considerable redundancy in the sequence information in the polypeptide chain; not every amino acid is essential for folding. Also, further evidence is provided that the replacement of fully solvent-exposed residues within alpha-helices with alanines may be a general way to increase protein stability. The general approach may permit a simplification of the protein folding problem by retaining only amino acids proven to be essential for folding and replacing the remainder with alanine.

Crystallization and Preliminary X-Ray Structure Analysis of Pigeon Egg-White Lysozyme1

Calcium binding lysozyme from pigeon egg-white was crystallized by the hanging drop vapor diffusion technique using ammonium sulphate as a precipitant. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), and have unit cell dimensions of a = 34.2 A, b = 34.8 A, and c = 99.4 A. One asymmetric unit contains one molecule of the pigeon lysozyme. The crystals diffract X-rays at least to 2.0 A resolution and are suitable for high resolution structure analysis. The diffraction data up to 3.0 A resolution were collected with a diffraction image processor, DIP100, using a Fuji imaging plate as an area detector. The structure was solved by the molecular replacement technique and refined to an R factor of 0.216. Least-squares fitting of the main-chains of pigeon egg-white lysozyme with those of chicken egg-white lysozyme and baboon alpha-lactalbumin showed that the main-chain folding of pigeon lysozyme is more similar to that of chicken lysozyme than that of alpha-lactalbumin. The largest differences between the pigeon and chicken lysozymes are in the surface loop regions.

Behavior of cysteine mutants of human lysozyme in de novo synthesis and in vivo secretion

To investigate the mechanism of disulfide-bond-coupled de novo folding of human lysozyme, we have constructed 23 mutant enzymes in which cysteine residue(s) were replaced by alanine(s). The mutant genes were translated in vitro in a system composed of rabbit reticulocyte lysate, canine pancreatic microsomal vesicles and oxidized glutathione. This system allows the formation of intramolecular disulfide bonds in translation products translocated into the microsomal lumen. The mobilities of the translation products were analyzed by SDS/PAGE in nonreducing conditions. Some mutant lysozymes were found to form a compact conformation with native-like mobility in the presence of SDS. The de novo formation of the SDS-resistant compact conformation of each mutant correlated well with its efficiency of secretion by Saccharomyces cerevisiae. Our results suggest that the de novo synthesized products reflect the conformational states in vivo to some extent, and that the formation of SDS-resistant compact conformation can be regarded as a necessary condition for allowing lysozyme to be secreted. In addition, the analysis of a mutant C116A (Cys116----Ala) under different oxidative conditions suggests two distinct pathways for the disulfide-bond-coupled formation of the compact conformation.

Evidence for intramolecular disulfide bond shuffling in the folding of mutant human lysozyme.

Our previous results using the Saccharomyces cerevisiae secretion system suggest that intramolecular exchange of disulfide bonds occurs in the folding pathway of human lysozyme in vivo (Taniyama, Y., Yamamoto, Y., Kuroki, R., and Kikuchi, M. (1990) J. Biol. Chem. 265, 7570-7575). Here we report on the results of introducing an artificial disulfide bond in mutants with 2 cysteine residues substituting for Ala83 and Asp91. The mutant (C83/91) protein was not detected in the culture medium of the yeast, probably because of incorrect folding. Thereupon, 2 cysteine residues Cys77 and Cys95 were replaced with Ala in the mutant C83/91, because a native disulfide bond Cys77-Cys95 was found not necessary for correct folding in vivo (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). The resultant mutant (AC83/91) was secreted as two proteins (AC83/91-a and AC83/91-b) with different specific activities. Amino acid and peptide mapping analyses showed that two glutathiones appeared to be attached to the thiol groups of the cysteine residues introduced into AC83/91-a and that four disulfide bonds including an artificial disulfide bond existed in the AC83/91-b molecule. The presence of cysteine residues modified with glutathione may indicate that the non-native disulfide bond Cys83-Cys91 is not so easily formed as a native disulfide bond. These results suggest that the introduction of Cys83 and Cys91 may act to suppress the process of native disulfide bond formation through disulfide bond interchange in the folding of human lysozyme.

Effect of Chemical Modifications of Tryptophan Residues on the Folding of Reduced Hen Egg-White Lysozyme1

The effects of chemical modifications of Trp62 and Trp108 on the folding of hen egg-white lysozyme from the reduced form were investigated by means of the sulfhydryl-disulfide interchange reaction at pH 8 and 40 degrees C. The folding of reduced lysozyme was monitored by following the recovery of the original activity. Under the conditions employed, the apparent first-order rate constant for the folding of reduced lysozyme was not changed by the modifications of both Trp62 and Trp108 and the folding was completed within 30 min. However, the extent of the correct folding was changed by the modification of Trp62 but not by that of Trp108. Native and oxindolealanine108 lysozymes recovered 80 and 81% of their original activities after 30-min refolding, respectively, but Trp62-modified lysozymes recovered their activities to a lesser extent than native and oxindolealanine108 lysozymes. The recovered activities of Trp62-modified lysozymes after 30-min refolding were 63% for oxindolealanine62 lysozyme, 65% for delta 1-carboxamidomethylthiotryptophan62 lysozyme, and 52% for delta 1-carboxymethylthiotryptophan62 lysozyme. These results suggest that Trp62 is important for preventing the misfolding of reduced lysozyme, but that neither Trp62 nor Trp108 is involved in the rate-determining step (the slowest step) in the folding pathway. A decrease in the hydrophobic nature of Trp62 seems to increase the misfolding and thus to decrease the extent of the correct folding of reduced lysozyme. A mechanism for the involvement of Trp62 in the folding pathway of reduced lysozyme is proposed.

Evidence for difference in the roles of two cysteine residues involved in disulfide bond formation in the folding of human lysozyme.

Human lysozyme is made up of 130 amino acid residues and has four disulfide bonds at Cys6-Cys128, Cys30-Cys116, Cys65-Cys81, and Cys77-Cys95. Our previous results using the Saccharomyces cerevisiae secretion system indicate that the individual disulfide bonds of human lysozyme have different functions in the correct in vivo folding and enzymatic activity of the protein (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). In this paper, we report the results of experiments that were focused on the roles of Cys65 and Cys81 in the folding of human lysozyme protein in yeast. A mutant protein (C81A), in which Cys81 was replaced with Ala, had almost the same enzymatic activity and conformation as those of the native enzyme. On the other hand, another mutant (C65A), in which Cys65 was replaced with Ala, was not found to fold correctly. These results indicate that Cys81 is not a requisite for both correct folding and activity, whereas Cys65 is indispensable. The mutant protein C81A is seen to contain a new, non-native disulfide bond at Cys65-Cys77. The possible occurrence of disulfide bond interchange during our mapping experiments cannot be ruled out by the experimental techniques presently available, but characterization of other mutant proteins and computer analysis suggest that the intramolecular exchange of disulfide bonds is present in the folding pathway of human lysozyme in vivo.

Effect of an engineered disulfide bond on the folding of T4 lysozyme at low temperatures

Equilibrium and kinetic effects on the folding of T4 lysozyme were investigated by fluorescence emission spectroscopy in cryosolvent. To study the role of disulfide cross-links in stability and folding, a comparison was made with a mutant containing an engineered disulfide bond between Cys-3 (Ile-3 in the wild type) and Cys-97, which links the C-terminal domain to the N terminus of the protein [Perry & Wetzel (1984) Science 226, 555]. In our experimental system, stability toward thermal and denaturant unfolding was increased slightly as a result of the cross-link. The corresponding reduced protein was significantly less stable than the wild type. Unfolding and refolding kinetics were carried out in 35% methanol, pH 6.8 at -15 degrees C, with guanidine hydrochloride as the denaturant. Unfolding/refolding of the wild-type and reduced enzyme showed biphasic kinetics both within and outside the denaturant-induced transition region and were consistent with the presence of a populated intermediate in folding. Double-jump refolding experiments eliminated proline isomerization as a possible cause for the biphasicity. The disulfide mutant protein, however, showed monophasic kinetics in all guanidine concentrations studied.

PH-Induced denaturation of proteins: a single salt bridge contributes 3-5 kcal/mol to the free energy of folding of T4 lysozyme

The energetics of a salt bridge formed between the side chains of aspartic acid 70 (Asp70) and histidine 31 (His31) of T4 lysozyme have been examined by nuclear magnetic resonance techniques. The pKa values of the residues in the native state are perturbed from their values in the unfolded protein such that His31 has a pKa value of 9.1 in the native state and 6.8 in the unfolded state at 10 degrees C in moderate salt. Similarly, the aspartate pKa is shifted to a value of about 0.5 in the native state from its value of 3.5-4.0 in the unfolded state. These shifts in pKa show that the salt bridge is stabilized 3-5 kcal/mol. This implies that the salt bridge stabilizes the native state by 3-5 kcal/mol as compared to the unfolded state. This is reflected in the thermodynamic stability of mutants of the protein in which Asp70, His31, or both are replaced by asparagine. These observations and consideration of the thermodynamic coupling of protonation state to folding of proteins suggest a mechanism of acid denaturation in which the unfolded state is progressively stabilized by protonation of its acid residues as pH is lowered below pH 4. The unfolded state is stabilized only if acidic groups in the folded state have lower pKa values than in the unfolded state. When the pH is sufficiently low, the acid groups of both the native and unfolded states are fully protonated, and the apparent unfolding equilibrium constant becomes pH independent. Similar arguments apply to base-induced unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)

Occurrence of S‐(1,2‐dicarboxyethyl)‐cysteine at position 77 in mutant human lysozyme secreted by Saccharomyces cerevisiae

A mutant human lysozyme P110, in which Val110 was replaced with Pro, was secreted by Saccharomyces cerevisiae; modification of the cysteine residue at position 77 was found in a purified mutant protein (P110-B) upon primary structure analysis. A peptide fragment containing 15 amino acid residues from Thr70 to Leu84 was obtained by proteolytic digestion of the protein and subsequently isolated by reverse-phase HPLC. This fragment was analyzed by high-resolution fast-atom-bombardment (FAB) mass spectrometry, which showed that 1,2-dicarboxyethyl group was attached to the thiol group of Cys77. This modification was confirmed by comparing it with a sample of chemically synthesized S-(1,2-dicarboxyethyl)-L-cysteine. It was found that the modification caused a disruption of the disulfide bond Cys77-Cys95 in the mutant molecule. These observations, plus structural considerations, suggest that Cys77 and Cys95 either remain uncrosslinked or the disulfide bond Cys77-Cys95, once formed, is opened during the final step in the folding of human lysozyme in vivo.

Role of disulfide bonds in folding and secretion of human lysozyme in saccharomycescerevisiae

We examined folding and secretion of human lysozyme using four mutants each lacking two cysteines expressed in a yeast secretion system. Our results have revealed that the formation of the disulfide bond Cys6/Cys128 in human lysozyme is a prerequisite for correct folding in vivo in yeast. Substitution of Ala for Cys77 and Cys95 gave eight-fold greater secretion of a molecule with almost the same specific activity as that of the native enzyme. Substitutions of the other cysteines gave molecules that were secreted at a lower rate and had lower specific activities than the native enzyme. These are the first findings that the individual disulfide bonds of human lysozyme have different functions in folding and secretion in vivo.

Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl-disulfide interchange.

In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coli. Since this lysozyme contained two amino acid substitutions (Ala31----Val and Asn106----Ser) in addition to an extra methionine residue at the NH2-terminus, the substituted amino acid residues were converted back to the original ones by means of oligonucleotide-directed site-specific mutagenesis and in vitro recombination. Thus, four kinds of chicken lysozyme [Met-1Val31Ser106-, Met-1Ser106-, Met-1Val31- and Met-1 (wild type)] were expressed in E. coli. From the results of folding experiments of the reduced lysozymes by sulfhydryl-disulfide interchange at pH 8.0 and 38 degrees C, followed by the specific activity measurements of the folded enzymes, the following conclusions can be drawn: (i) an extra methionine residue at the NH2-terminus reduces the folding rate but does not affect the lysozyme activity of the folded enzyme; (ii) the substitution of Asn106 by Ser decreases the activity to 58% of that of intact native lysozyme without changing the folding rate; and (iii) the substitution of Ala31 Val prohibits the correct folding of lysozyme. Since the wild type enzyme (Met-1-lysozyme) was activated in vitro without loss of specific activity, the systems described in this study (mutagenesis, overproduction, purification and folding of inactive mutant lysozymes) may be useful in the study of folding pathways, expression of biological activity and stability of lysozyme.

Experimental studies of folding kinetics and structural dynamics of small proteins.

Main points given in the above reports can be summarized as follows. Multiple unfolded forms exist for lysozyme as well as for RNase. The existence of fast- and slow-folding forms appears to be a general phenomenon; it has been confirmed for a number of globular proteins, which contain proline residues. The major slow refolding reaction of RNase A is a sequential process via structural intermediates. A rapidly formed intermediate has also been detected on the direct UF----N refolding pathway of lysozyme. The activated state for folding of lysozyme shows a conformation similar to the native protein in terms of packing of hydrophobic groups. This suggests that, in terms of compactness, the rate-limiting step occurs at a late stage of the refolding process. A protein homologous to lysozyme, alpha-lactalbumin, shows similar kinetics although alpha-lactalbumin shows an apparent equilibrium unfolding intermediate. The location of the rate-limiting step close to the native state has also been suggested for other proteins. It still remains open whether this is a general property of protein folding reactions. As shown for the unfolding of Mb, the multi-probe kinetic measurements will be a powerful tool for investigating the mechanism of folding, in particular for characterizing structural kinetic intermediates. The dynamics of local fluctuations of a well-defined part of RNase S can be monitored by NMR measurements of NH proton exchange. An increasing number of experimental and theoretical studies are focussing on the problem of protein dynamics. Application of NMR methods to protein folding should give extensive information about the structure of intermediates, which cannot be given by other techniques.

Kinetic analysis of the reversible folding reactions of small proteins: Application to the folding of lysozyme and cytochrome c

AbstractA general kinetic analysis for unimolecular three‐species models is presented. Criteria are established for each model in order to allow selection among alternative models on the basis of their amplitude behavior. These criteria are applied to the previously reported kinetic data for the folding transitions of lysozyme and cytochrome c in guanidine hydrochloride. The degree of success in applying these criteria is found to depend on the extent to which the apparent rate constants, λ1 and λ2, approach each other in certain conditions. Ikai and Tanford have presented a similar kinetic analysis, although these authors considered only the case where no intermediate kinetic species was populated initially. For one protein system, namely, ribonuclease A (RNase A), at least one stable intermediate exists after unfolding outside of the equilibrium transition zone. The criteria developed by Ikai and Tanford cannot be applied to RNase A kinetics and, therefore, a more general analysis is presented. Previously published studies of the reversible folding kinetics of lysozyme and cytochrome c are reexamined using the current amplitude analysis, and it is found that both of these folding transitions can be described by the three‐species model, used previously to describe the folding kinetics of RNase A (U1 and U2 are two forms of unfolded enzyme and N is the native species). These results suggest that this three‐species model may provide a fairly general description of the reversible folding kinetics of small proteins. As an outgrowth of the current analysis, several suggestions for experimental design in studying protein folding will be presented and discussed.

Kinetic determinism of lysozyme folding at high temperatures

Reduced, disordered hen egg lysozyme rapidly regains enzymic activity in a nonenzymic system previously reported from this laboratory. When such regenerations are carried out at high temperatures (where the native enzyme is unstable), native enzyme is formed as a transient intermediate. Thermal inactivation does not depend on irreversible changes in the protein, as shown by experiments wherein thermally inactivated regenerated lysozyme spontaneously reactivates at 37°. These findings are inconsistent with thermodynamic determinism of protein structure.