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For the stabilization of a protein against irreversible denaturation caused by rapid reaction of the unfolded form of the protein (kinetic stabilization), the free energy change of activation for unfolding should be increased. First, we demonstrated that this strategy was effective to stabilize a protein against protease digestion. For kinetic stabilization, it is important to stabilize a protein at a site where the local structures are largely unfolded in the transition state for unfolding. We developed a method to find such sites by comparison of the thermodynamic stabilities and the unfolding rate constants between unmodified and modified proteins. Application of this method to analyze the transition state of hen-egg lysozyme using some chemically modified derivatives is also described. Moreover, it was confirmed that the protease digestion method is superior to the relaxation method for estimation of the unfolding rate constant. Namely, the protease digestion method may be useful in analyzing the transition state of protein unfolding.

By employing various synthetic substrates, as well as soluble denatured protein substrate (TAP-lysozyme) and its derivatives, endopeptidase activity of cathepsin C, dipeptidyl aminopeptidase I [EC 3.4.14.1], from bovine spleen was investigated. Cathepsin C efficiently degraded Z-Phe-Arg-MCA, Pro-Phe-Arg-MCA, and Suc-Leu-Leu-Val-Tyr-MCA. This endopeptidase activity required sulfhydryl reagents and halide ions, as in the case of the dipeptidyl aminopeptidase (DAP) activity. We confirmed that this endopeptidase activity is due to cathepsin C itself based on the results on gel-filtration and anion-exchange chromatographies, comparative studies of the inhibitory effects of leupeptin and E-64 on this activity and those of cathepsins B and L, and further the competitive inhibitions by mutual substrates for the DAP and endopeptidase activities of cathepsin C. We also found that cathepsin C endopeptidase activity towards TAP-lysozyme and its N-alpha-acetylated tryptic peptides showed marked dependence on sulfhydryl reagents and chloride ion. Thus, we concluded that cathepsin C has endopeptidase activity as well as DAP activity. The binding energy between the enzyme and the amino acid side chains of the substrate may be as important for the endopeptidase activity as is the electrostatic interaction between the enzyme and the free alpha-amino group of the substrate for the DAP activity.

A method was developed to evaluate the association constant at physiological pH (pH 7.5) between a lysozyme and the Fab fragment derived from anti-lysozyme monoclonal antibody 37-7, which was immobilized to the adsorbent for HPLC. Comparison of the association constants between lysozymes and the immobilized Fab fragment indicated that mAb 37-7 recognized the prominently exposed regions (hills and ridges) around His15 of hen lysozyme, but His15 itself was not directly involved in the binding with mAb 37-7. Moreover, the epitope was confirmed by the reactivity of His15 with monoiodoacetic acid in the presence of mAb 37-7. The association constant of 15-carboxymethylated histidine lysozyme (15CM lysozyme) with the immobilized Fab fragment was smaller by one-seventh than that of 15-carboxamidated histidine lysozyme, though the side chains introduced were almost identical in size. From the pH titration of 15CM lysozyme with 13C-enriched carboxyl group by use of 13C-NMR, the pKa of the introduced carboxyl group was evaluated to be 5.06. Since the carboxyl group was fully ionized under the conditions of measurement (pH 7.5), electrostatic repulsion was found to disturb severely the association between mAb 37-7 and hen lysozyme. Moreover, it was demonstrated that, because of the high reproducibility of measurement, the immobilized Fab fragment could detect subtle differences in the surface structure of lysozymes.

Three mutant lysozymes where the Asp101-Gly102 sequence of lysozyme was converted to Asp101-Pro102, Gly101-Pro102 and Pro101-Gly102 were prepared to investigate the effect of proline residues on the stabilization of proteins. The free energy changes of lysozymes for the unfolding in aqueous solution at pH 5.5 and 35 degrees C were 10.0, 10.1, 11.0 and 7.7 kcal/mol for wild type, Asp101Pro102, Gly101Pro102 and Pro101Gly102 lysozyme respectively. When the energy level in the unfolded state of wild type lysozyme was fixed at a standard level, the energy levels in the folded state of Asp101Pro102 and Pro101Gly102 lysozymes were found to be higher than that of wild type lysozyme on the basis of delta GD(H2O) and entropy losses of their polypeptide chains in the unfolded state. The presence of some strain in the folded state of these lysozymes was supported by both the calculation of conformational energy for a trans-L-prolyl residue [Schimmel, P.R. and Flory, P.J. (1968) J. Mol. Biol., 34, 105-120] and the analysis of structures of energy-minimized mutant lysozymes. Therefore, it is concluded that the formation of the Gly-Pro sequence is effective in avoiding possible strain in the folded state of a protein caused by the introduction of proline residue(s).

To examine the effect of the conformational stability of the CH2 domain on aggregation and peptide cleavage of a humanized IgG1, we carried out size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses of incubated sample solutions. By comparing the residual percentage of monomer after incubation at 60 and 80°C at various pH levels, we found that aggregation and peptide cleavage of the humanized IgG1 occurred during long incubation at 60°C under acidic conditions. Next, we confirmed cleavage of the Asp272-Pro273 peptide bond in the CH2 domain. Comparison of the cleavage rates of the IgG1 monomer and a peptide containing the same Asp-Pro sequence revealed that the conformational stability of the CH2 domain retards cleavage of the Asp272-Pro273 peptide bond at 60°C and pH 4.0. The finding of aggregation and peptide cleavage of the humanized IgG1 after long incubation at 60°C under acidic conditions was supported by another finding: there were lower unfolding temperatures of the CH2 domain at pH 4.0 and 5.0. We conclude that the conformational stability of the CH2 domain is closely related to aggregation and peptide cleavage of the humanized IgG1 under acidic conditions. We also found that the 2-[N-morpholino] ethane sulfonate buffer inhibits aggregation of the IgG1 at pH 4.0–5.0 and 7.0–8.0.

On irradiation, diepoxy[15]annulenone 1 and monobromo-diepoxy[15]annulenone 2 undergo an intriguing rearrangement, whereby all of the ether and carbonyl oxygens of 1 and 2 can travel freely and change their positions in the rings to give 15 position isomers in all.

Proteins are usually in an equilibrium between the folded and the unfolded state. Therefore, for the stabilization of proteins against reversible denaturation, the free energy change for the unfolding should be increased by stabilizing the folded state by lowering the energy level of the folded state or by destabilizing the unfolded state by raising the energy level of the unfolded state. On the other hand, various processes can be coupled with the unfolded state of proteins. For example, protease digestion of proteins at physiological temperature may be one of such processes. The process would lead to an irreversible denaturation. For the stabilization of proteins against the irreversible denaturation coupled with the unfolded state, a kinetic stabilization is important, that is, the activation free energy for the unfolding should be increased. Methods for the kinetic stabilization were discussed. Finally, the irreversible chemical deterioration of proteins was considered.

The "right-sided" and "left-sided" substrate binding modes at the lower saccharide binding subsites (D-F sites) of chicken lysozyme were investigated by utilizing mutant lysozymes secreted from yeast. We constructed the following mutant lysozymes; "left-sided" substitution of Asn46 to Asp, deletion of Thr47, and insertion of Gly between Thr47 and Asp48 and "right-sided" substitution of Asn37 to Gly. Analyses of their activities and substrate binding abilities showed that Asn46 and Thr47 are involved in the initial enzyme-substrate complex and Asn37 is involved in the transition state. These results support an earlier proposal that interactions between substrate and residues at the left side of lysozyme stabilize a catalytically inactive enzyme-substrate complex, while interactions between substrate and residues at the right side stabilize the catalytically active complex [Pincus, M. R., & Scheraga, H. A. (1979) Macromolecules 12, 633-644]. These results are also consistent with the proposed kinetic mechanism for lysozyme reaction that the rearrangement of an initial enzyme-substrate complex (beta-complex) to another complex (gamma-complex) is required for catalytic hydrolysis [Banerjee S. K., Holler, E., Hess, G. P., & Rupley, J. A. (1975) J. Biol. Chem. 250, 4355-4367].

Each of 3A-O-(2-naphthalenesulfonyl)-6X-O-(mesitylenesulfonyl)-α-cyclodextrins (X B, C, E, or F), which was prepared by the reaction of 3A-O-(2-naphthalenesulfonyl)-α-cyclodextrin with mesitylenesulfonyl chloride, was structurally determined through combination of chemical derivation and enzymatic hydrolysis.

Trp108 of chicken lysozyme is in van der Waals contact with Glu35, one of two catalytic carboxyl groups. The role of Trp108 in lysozyme function and stability was investigated by using mutant lysozymes secreted from yeast. By the replacement of Trp108 with less hydrophobic residues, Tyr (W108Y lysozyme) and Gln (W108Q lysozyme), the activity, saccharide binding ability, stability, and pKa of Glu35 were all decreased with a decrease in the hydrophobicity of residue 108. Namely, at pH 5.5 and 40 degrees C, the activities of W108Y and W108Q lysozymes against glycol chitin were 17.3 and 1.6% of that of wild-type lysozyme, and their dissociation constants for the binding of a trimer of N-acetyl-D-glucosamine were 7.4 and 309 times larger than that of wild-type lysozyme, respectively. For the reversible unfolding at pH 3.5 and 30 degrees C, W108Y and W108Q lysozymes were less stable than wild-type lysozyme by 1.4 and 3.6 kcal/mol, respectively. As for the pKa of Glu35, the values for W108Y and W108Q lysozymes were found to be lower than that for wild-type lysozyme by 0.2 and by 0.6 pKa unit, respectively. The pKa of Glu35 in lysozyme was also decreased from 6.1 to 5.4 by the presence of 1-3 M guanidine hydrochloride, or to 5.5 by the substitution of Asn for Asp52, another catalytic carboxyl group. Thus, both the hydrophobicity of Trp108 and the electrostatic interaction with Asp52 are equally responsible for the abnormally high pKa (6.1) of Glu35, compared with that (4.4) of a normal glutamic acid residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Micrococcus luteus (M. lysodeikticus) labeled with Remazol brilliant blue R (blue ML) was prepared as a novel substrate for the colorimetric assay of lysozyme. The treatment of the labeled substrate with lysozyme resulted in the release of soluble blue products which can be easily measured spectrophotometrically at 600 nm. The blue color was most efficiently released at pH 7 and ionic strength of 0.2 on incubation with hen lysozyme at 40 degrees C. A new colorimetric method for the assay of lysozyme using this substrate was developed. The assay system gave a linear dose-response curve, and as little as 0.1 microgram of human lysozyme (1 microgram/ml, 100 microliters) can be detected. The present method is more convenient and reproducible than the conventional lysozyme assay with bacterial cells. Application of the system to the determination of lysozyme in human serum is described.

Tapes japonica lysozyme (TJL), which belongs to the invertebrate-type lysozyme family, has a unique dimer formation. The residues, which include catalytic residues (glutamate 18 and aspartate 30), at the dimer interface form electrostatic interactions. Our previous study suggested that increasing the NaCl concentration switched TJL from a dimer to monomer structure, which increased TJL activity. Therefore, conversion from the dimeric to the monomeric structure is crucial for the TJL activity. In the present study, to further understand the effect of NaCl on TJL dimer formation, we examined the protein concentration and pH dependence of TJL activity in the presence or absence of 500 mM NaCl. TJL activity was suppressed at the high protein concentration. And the optimum pH of TJL activity was decreased in the absence of NaCl. These dependencies confirm the presence of electrostatic interactions between molecules of TJL in the dimeric form in an aqueous solution.

We found that rat cathepsin H showed strong transacylation activity under physiological conditions. It is a feature of cathepsin H to utilize amino acid amides not only as acyl-acceptors but also as acyl-donors in the reaction. The pH-dependence of the transacylation activity was distinct from those of other papain-superfamily proteases. The alkaline limb (pKapp = 7.5) could be regarded as the pKa of the alpha-amino group of the acyl-donor, which was also involved in the original amino-peptidase activity. The acidic limb (pKapp = 5.8) was suggested to be involved in the deacylation step, where amino acid amide attacked the acyl-intermediate as a nucleophile in place of water in the hydrolysis. Although the N alpha-deprotonated acyl-acceptor, which is supposed to govern the nucleophilic attack, has a small population in the acidic pH range (above pH5), the transacylation was detectable even at the acidic pH-range because of the high S1'-site binding ability and suitable nucleophilicity of the acyl-acceptor. In the transacylation between various amino acid amides, the S1 and S1' site appeared to prefer hydrophobic residues without and regardless of a branch at beta-carbon, respectively. From these results and the sequence homology in the papain superfamily, we concluded that the reaction was governed by the acyl-donor having a protonated amino group, the acyl-acceptor having a deprotonated amino group and the remarkable hydrophobic character (especially favoring tryptophan amide) of the S1' site, presumably reflecting the good conservation of Trp177 in papain-superfamily proteases.

Each of 6A-O-(p-nitrobenzenesulfonyl)-6X-O-(β-naphthalenesulfonyl)-β-cyclodextrins (X= B-G) was prepared by the reaction of 6-O-(β -naphthalenesulfonyl)-β -cyclodextrin with p-nitrobenzenesulfonyl chloride, isolated, and structurally determined through selective chemical conversion.

Dipeptidylcarboxypeptidase, endopeptidase, and carboxypeptidase activities of rat liver cathepsin B were investigated using soluble denatured protein substrates, reduced and S-(3-trimethylammonio)propylated proteins and their derivatives. It was found that the soluble denatured proteins were degraded mainly by the dipeptidylcarboxypeptidase activity and in a few cases by the endopeptidase and carboxypeptidase activities. The eipeptidylcarboxypeptidase activity showed broad substrate specificity with broad pH optimum at 4-6. A peptide having the alpha-carboxyl group amidated with methylamine could also be a good substrate for this activity. These results suggest that this activity is dependent not upon the dissociated alpha-carboxyl group at the P2' site but upon the hydrogen-bonding abilities of the alpha-imino moiety and the protonated or amidated alpha-carboxyl moiety at P2'. On the other hand, the endopeptidase and carboxypeptidase activities were observed in a few cases, suggesting that special amino acid sequences in the substrates are responsible for these activities. These activities showed sharp pH optima at 6 and seemed to prefer basic amino acid residues at P1 site. Therefore, we suppose that cathepsin B has a carboxyl group with a pKa of about 5.5 at the S1 subsite which more effectively interacts with a positive charge at the P1 site of the substrate at pH 6 than at pH 5. Based on these results, a model of the binding subsites of this enzyme is proposed.

We prepared the lysozyme derivative in which the beta-carboxyl group of Asp101 was modified with alpha-O-methyl N-glycylglucosaminide as an amide by means of the carbodimide reaction (alpha-MGG lysozyme). Since Asp101 residue is located at the edge of the active site cleft, a 1H-NMR study was carried out for this derivative in order to investigate the interaction between the introduced substituent and the active site cleft. It was confirmed that the alpha-MGG moiety sat in the active site cleft in alpha-MGG lysozyme from the reduction of line broadening of the NH-proton of Trp63 located in the active site cleft, the remarkable chemical shift change of the methyl group of the alpha-MGG moiety upon adding a trimer of N-acetyl-D-glucosamine [(NAG)3], and the NOE between the C6-proton resonance of Trp63 and the methyl resonance of the alpha-MGG moiety. Furthermore, alpha-MGG lysozyme had increased thermal stability compared with native lysozyme. Therefore, it was concluded that the alpha-MGG moiety covalently attached to Asp101 interacted with the active site cleft to increase the thermal stability of lysozyme.

Sixty-nine rat cathepsin L-susceptible peptide bonds were analyzed employing various peptide substrates. The proteolytic specificities of rat cathepsin L and papain were compared and the results are discussed in relation to differences in amino acid residues around their binding sites. The specificity of cathepsin L, which is characterized by a remarkable preference for hydrophobic amino acids at the P2 site of the scissile peptide bonds, was analogous to that of papain as a whole. This analogous specificity suggests that the binding sites of the two proteases are analogous, as expected from their homologous amino acid sequences. However, there is a slight difference in the preference for S3 site between them. That is, cathepsin L showed a greater preference for bulky and hydrophobic amino acids at the S3 site than did papain. Based on the computer-graphically deduced structure of the binding sites of cathepsin L, the preferences for hydrophobic amino acids at the S2 site and for bulky and hydrophobic amino acids at the S3 site of the protease are supposed to be related to the compensating amino acid substitutions at the S2 site (V133A and V157L) and the reduction in size at the S3 site (Y61Q and Y67L), respectively. The discussion of the effect of the amino acid substitutions on the proteolytic activities of cathepsin L and papain in this paper provides a basis for more advanced studies of the relationship between structure and function of proteases belonging to the papain superfamily by means of protein engineering.

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.

We found that the reaction of hen egg-white lysozyme with an equimolar amount of tetranitromethane (TNM) at pH 8.0 and room temperature yielded derivatives in which the N-C bond of Gly104 is oxidatively cleaved, and a mono-nitrotyrosine lysozyme in which Tyr23 is nitrated. This bond cleavage occurred more predominantly with a decrease in the nitration of Tyr23, when the reaction was carried out under more dilute conditions. A possible mechanism in which a phenoxyl radical of Tyr 23 (an intermediate of nitration) is involved was proposed for this oxidative bond cleavage. When lysozyme was reacted with a 10 times molar excess of TNM, in addition to a mono-nitrotyrosine lysozyme in which only Try23 is nitrated, a di-nitrotyrosine lysozyme in which Tyr20 and Tyr23 are both nitrated and a tri-nitrotyrosine lysozyme in which Tyr20, Tyr23, and Tyr53 are all nitrated were obtained. However, no other possible mono- or di-nitrotyrosine lysozymes could be isolated. Thus, it is concluded that the three tyrosine residues in lysozyme are essentially nitrated sequentially with TNM in the order of Tyr23, Tyr20, and Tyr53. Since the derivatives obtained here were all active, none of the three tyrosine residues or the residues around Gly104 are considered to be very important for the lysozyme activity.

A reaction of β-cyclodextrin with β-naphthylsulfonyl chloride in alkaline aqueous acetonitrile gave only one isomer (3 A ,3 C ,3 E -trisulfonate, 17.8%) of five 3,3,3-tri-O-sulfonyl-β-cyclodextrins. The isomer was converted to 3 A ,6 A :3 C ,6 C :3 E ,6 E -trianhydro-β-cyclodextrin, the structure of which was assigned by comparing its spectral and HPLC data of the trianhydro-β-cyclodextrin with those of all authentic 3,6:3,6:3,6-trianhydro-β-cyclodextrins prepared by the reactions of known 6-tri-O-sulfonylated β-cyclodextrins with aqueous alkali

The lysozyme (rabbit kidney lysozyme) from the homogenate of rabbit kidney (Japanese white) was purified by repeated cation-exchange chromatography on Bio-Rex 70. The amino acid sequence was determined by automated gas-phase Edman degradation of the peptides obtained from the digestion of reduced and S-carboxymethylated rabbit lysozyme with Achromobacter protease I (lysyl endopeptidase). The sequence thus determined was KIYERCELARTLKKLGLDGYKGVSLANWMCLAKWESSYNTRATNYNPGDKSTDYGIFQ INSRYWCNDGKTPRAVNACHIPCSDLLKDDITQAVACAKRVVSDPQGIRAWVAWRNHCQ NQDLTPYIRGCGV, indicating 25 amino acid substitutions from human lysozyme. The lytic activity of rabbit lysozyme against Micrococcus lysodeikticus at pH 7, ionic strength of 0.1, and 30 degrees C was found to be 190 and 60% of those of hen and human lysozymes, respectively. The lytic activity-pH profile of rabbit lysozyme was slightly different from those of hen and human lysozymes. While hen and human lysozymes had wide optimum activities at around pH 5.5-8.5, the optimum activity of rabbit lysozyme was at around pH 5.5-7.0. The high proline content (five residues per molecule compared with two prolines per molecule in hen or human lysozyme) is one of the interesting features of rabbit lysozyme. The transition temperatures for the unfolding of rabbit, human, and hen lysozymes in 3 M guanidine hydrochloride at pH 5.5 were 51.2, 45.5, and 45.4 degrees C, respectively, indicating that rabbit lysozyme is stabler than the other two lysozymes. The high proline content may be responsible for the increased stability of rabbit lysozyme.

The aggregation propensity of humanized antibody after heat treatment is evaluated in the presence of six buffer species. The comparison under equivalent pH showed high aggregation propensity on phosphate and citrate buffer. In contrast, 2-(N-Morpholino) ethane sulfonate (MES), 3-(N-Morpholino) propane sulfonate (MOPS), acetate and imidazole buffer showed lower aggregation propensity than the above two buffers. Meanwhile, unfolding temperature evaluated by differential scanning calorimetry measurement was not altered among these buffer species. The light scattering analysis suggested that heat-denatured intermediate was aggregated slightly on MES and acetate buffer. Therefore, it was found that the different aggregation propensity among buffer species was caused from the aggregation propensity of heat-denatured intermediate rather than the unfolding temperature. Furthermore, it was revealed that the aggregation dependency on buffer species is accounted for by the specific molecular interaction between buffer and IgG, rather than the ionic strength. On the contrary, on the analyses of unfolding and aggregation propensity by molecular dissection of IgG into Fab and Fc fragments, aggregation propensity of Fc fragment on MES, acetate and phosphate buffer was almost the same as whole IgG. From the above results, it was suggested that the specific interaction between buffer molecule and Fc domain of IgG was involved in the aggregation propensity of heat-denatured IgG.

The single nucleotide polymorphism interleukin-13 (IL-13) R110Q is associated with severe bronchial asthma because its lower affinity leads to the augmentation of local IL-13 concentration, resulting in an increase in the signal transduction via IL-13R. Since the mutation site does not directly bind to IL-13Ralpha2, we carried out NMR relaxation analyses of the wild-type IL-13 and IL-13-R110Q in order to examine whether the R110Q mutation affects the internal motions in IL-13 molecules. The results showed that the internal motion in the micro- to millisecond time scale on helix D, which is suggested to be important for the interaction between IL-13 and IL-13Ralpha2, is increased in IL-13-R110Q compared with that in the wild-type IL-13. It therefore appears that the difference in the internal motions on helix D between the wild-type IL-13 and IL-13-R110Q may be involved in their affinity differences with IL-13Ralpha2.

Six hydrophobic clusters involved in long-range interaction have been identified in the residual structure of reduced lysozyme at pH 2. Recently, it was found that modulation in the residual structure affected amyloid formation. In this paper, we examined the effect of the hydrophobic cluster containing W111 (cluster 5) on amyloid fibril formation of reduced lysozyme. The reduced W62G lysozyme, in which most of the hydrophobic clusters except for cluster 5 are disrupted, formed hardly any amyloid fibrils in comparison with the reduced wild-type. However, the disruption of cluster 5 by the mutation of Trp111 to Gly allowed significant amyloid fibril formation of reduced W62G lysozyme. Moreover, the extent of amyloid formation in the reduced W62G/W111G lysozyme was greater than that of the reduced wild-type lysozyme. From the above results, it became clear that cluster 5 contributed to retarding the amyloid fibrils formation of the W62G lysozyme.

The best way to introduce information about amino acid residues into calculations of protein bioinformatics was examined. That was done for predicting helical regions with a neural network. Several fundamental and instructive ways for information processing were developed and are described.

The backbone dynamics of RNase T1 in the presence of exo-guanosine 2',3'-cyclophosphorothioate (exo-cGPS isomer), which is a productive substrate, and in the presence of 3'-guanylic acid (3'GMP), which is an nonproductive substrate, were examined using (15)N nuclear magnetic resonance. Although the X-ray crystal structure suggests that the modes of binding of these substrates to the active-site cleft are very similar, the order parameters in a number of regions in RNase T1 complexed with exo-cGPS isomer were different from those with 3'GMP. Moreover, the chemical exchange in line width observed for RNase T1 complexed with exo-cGPS isomer was also different from that observed for RNase T1 complexed with 3'GMP. From these results, we concluded that the internal motions in RNase T1 complexed with a productive substrate were not always identical to those in RNase T1 complexed with a nonproductive substrate.

In previous paper, we showed that W62G mutation caused ill effects at the early stages of folding of the reduced hen lysozyme in vitro. Here, we investigated whether the single mutation brings about drastic turn to in vivo folding of lysozyme. W62G lysozyme was secreted from yeast cells and then purified with ion-exchange chromatography. From the results of gel chromatography and peptide analysis, the species with two cysteines, Cys80 and Cys94, and non-native cystine, Cys64-Cys76, was partially present in secreted product of yeast containing gene for W62G lysozyme. Thus, it was suggested that W62G mutation also affected the in vivo folding of lysozyme.

Efficient induction of self tolerance is critical for avoiding autoimmunity. The T cells specific for the well-processed and -presented (dominant) determinants of a native self protein are generally tolerized in the thymus, whereas those potentially directed against the inefficiently processed and presented (cryptic) self epitopes escape tolerance induction. We examined whether the crypticity of certain determinants of mouse lysozyme-M (ML-M) could be attributed to the nonavailability of a proteolytic site, and whether it could be reversed to immunodominance by engraftment of a novel cleavage site in the flanking region of the epitope. Using site-directed mutagenesis, we created the dibasic motif (RR or RK; R = arginine, K = lysine), a target of intracellular proteases, in the region adjoining one of the three cryptic epitopes (46–61, 66–79, or 105–119) of ML-M. Interestingly, the mutated lysozyme proteins, but not unmutated ML-M, were immunogenic in mice. The T cell response to the altered lysozyme was attributable to the efficient processing and presentation of the previously cryptic epitope, and this response was both epitope and MHC haplotype specific. In addition, the anti-self T cell response was associated with the generation of autoantibodies against self lysozyme. However, the results using one of three mutated lysozymes suggested that the naturally processed, dibasic motif-marked epitope may not always correspond precisely to the cryptic determinant within a synthetic peptide. This is the first report describing the circumvention of self tolerance owing to the targeted reversal of crypticity to dominance in vivo of a specific epitope within a native self Ag.

We investigated the activity and the internal motions of a stabilized mutant hen lysozyme (HEL) in which the residues M12 and L56 were mutated to L and F, respectively (LF mutant HEL). The result of the activity measurements against glycol chitin at various temperatures suggested that the temperature dependence of the activity of LF mutant HEL shifted to the high-temperature side compared with that of wild-type HEL. The detailed internal motions of LF mutant HEL in the absence and presence of a substrate analogue, (NAG)3, were examined by model-free analysis at 35 degrees C. The results showed that the internal motions of LF mutant HEL in the presence of (NAG)3 were drastically restricted compared with those in wild-type HEL. Our findings thus suggested that the mutation to the stabilized lysozyme restricted internal motions required for the enzymatic reaction.

We previously demonstrated that the hydrophobic clusters present in hen lysozyme under denaturing conditions were disrupted by the mutation of Trp62 to Gly (W62G). In order to examine the effects of the structure of the denatured state of W62G lysozyme on folding, we analyzed the early events in the folding of reduced W62G lysozyme in detail. From the exchange measurements of disulfide bonds using the variants containing a pair of cysteine residues (1SS), it was found that the formation of disulfide bond in the W62G1SS lysozyme was not accompanied by a prominent interaction between amino acid residues, indicating that the disruption of the hydrophobic core led to the random folding at the early stages in the process of folding of the reduced lysozyme. On the other hand, analyses of the oxidative-renaturation of reduced W62G lysozymes, as well as measurements of the extent of aggregation of the reduced and carboxy amido methylated W62G lysozyme, indicated that the formation of an aggregate is more prominent in the reduced W62G lysozyme than in the reduced wild-type lysozyme. Moreover, a lag phase was detected in the oxidative-renaturation of reduced W62G lysozyme, as based on observations of the recovery of activity. The simulation of the folding process indicated that intermediates were present at the early stages in the folding of the reduced W62G lysozyme. These results suggest that the presence of the intermediates was derived from the random folding at the early stages in the folding process of reduced W62G lysozyme due to the disruption of the structure of the denatured state. Folding thus appears to have been kinetically delayed by these processes, which then led to the significant aggregation of reduced lysozyme. Moreover, from the analysis of amyloid aggregation of the reduced lysozymes, it was suggested that the disruption of the residual structure in denatured state by W62G mutation deterred the formation of the amyloid fibrils of lysozyme.

Twenty-eight hen lysozyme variants that contained a pair of cysteines were constructed to examine the formation of the individual native and nonnative disulfide bonds. We analyzed the extent of the formation of a disulfide bond in each lysozyme variant using a redox buffer (pH 8) containing 1.0 mM reduced and 0.1 mM oxidized glutathione in the absence or presence of 6 M guanidine hydrochloride. In the presence of 6 M guanidine hydrochloride, the extent of the formation of the disulfide bond in each lysozyme variant was proportional to the distance between cysteine residues, indicating that reduced hen lysozyme under a highly denaturing condition adopted a randomly coiled structure. In aqueous solution, the formations of all disulfide bonds occurred much more easily than under a denatured condition. This finding indicated that reduced lysozyme had a somewhat compact structure. Moreover, the scattering data for the extents of the formation of the disulfide bonds among all lysozyme variants were observed. These results suggested that the nonrandom folding occurred in the early stage of the folding of reduced lysozyme, which should provide new insight into the early-stage events in the folding process of reduced lysozyme.

I have pursued research on lysozymes for 42 years. During that time, I made Several new findings, some of them by chance. My enjoyment of the following areas is reviewed: the story of tryptophan; protease digestion mechanisms; peptide mapping with RP-HPLC; gene engineering; renaturation of protein; catalytic residues; fluctuation and function; stabilization; folding; antigenecity; tolerance; and various lysozymes.

The lysozyme of the marine bilave Tapes japonica (13.8 kDa) is a novel protein. The protein has 46% homology with the destabilase from medicinal leech that has isopeptidase activity. Based on these data, we confirmed hydrolysis activity of T. japonica lysozyme against three substrates: L-gamma-Glu-pNA, D-gamma-Glu-pNA, and epsilon-(gamma-Glu)-L-Lys. The optimal pH of chitinase and isopeptidase activity was 5.0 and 7.0, respectively. The isopeptidase activity was inhibited with serine protease inhibitor, but the lytic and chitinase activities were not. Moreover, only isopeptidase activity is decreased by lyophilization, but lytic and chitinase activities were not. We conclude that T. japonica lysozyme expresses isopeptidase and chitinase activity at different active sites.

DnaA protein binds specifically to a 9-base- pair motif called the DnaA box. Domain IV comprises 94 amino acid residues and is required for DNA binding. Using nuclear magnetic resonance analysis, we investigated the interaction between DnaA domain IV and both a DnaA box and a non-specific oligonucleotide that has a reduced affinity for DnaA. The 1H-15N HSQC spectrum of DnaA domain IV showed prominent chemical shift perturbations on six residues (Arg399, Ala404, Leu422, Asp433, Thr435 and Thr436) in the presence of the DnaA box. Through homology modeling, we located all of these residues on one side surface of the DnaA domain IV molecule. Moreover, we compared the chemical shift perturbation of the 1H-15N HSQC spectrum in the presence of the DnaA box with that in the presence of a non-specific oligonucleotide, and the results suggested that Leu422 imparts specificity in binding with the DnaA box.

A mutant lysozyme in which Arg14 and His15 were deleted together exhibited higher activity toward glycol chitin than the wild-type lysozyme. Moreover, the mutant lysozyme, which is less stable than the wild-type lysozyme by 7 degrees C, showed a shift of temperature dependence of activity to the low temperature side compared with the wild-type lysozyme [Protein Eng. 7, 743-748 (1994)]. In the free enzyme, the internal motion of the mutant lysozyme was similar to that of the wild-type. The internal motions of the wild-type and mutant lysozymes in the enzyme-substrate complex increased more than those in the free enzymes. Moreover, the increased internal motions of the substrate-complexed mutant lysozyme were greater than those of the substrate-complexed wild-type lysozyme in several residues [J. Mol. Biol. 286, 1547-1565 (1999)]. The structure of the mutant lysozyme was very similar to that of the wild-type lysozyme. Both structures were also alike in the complex of the trimer of N-acetyl-D-glucosamine. The mobility from B-factors agreed to some degree with that from order parameters in the regions showing great mobility of the protein, but this was not the case in the regions showing fast motion. However, we came to the same conclusion that the increased activity of the mutant lysozyme is due to the increase in the fluctuation of the lysozyme molecule. B-factor and order parameter do not always exhibit harmony because the time-scale of the analysis of mobility is different. However, they are not incompatible but complementary for detecting precise protein motions.

In order to examine the effect of metal binding to the polypeptide chain on the aggregation of a protein in the refolding process, we prepared a mutant hen lysozyme possessing the same Ca 2 + binding site as in human α-lactalbumin by Escherichia coli expression system (Ser - 1 CaB lysozyme). In the presence of 2 mM CaCl 2 , the refolding yield of Ser - 1 CaB lysozyme at a low protein concentration (25 μg/mL) was similar to that of the wild-type lysozyme (80%), but that at high protein concentration (200 μg/mL) decreased (15%) due to aggregation comparing to that of the wild-type lysozyme (45%). However, the refolding yield of Ser - 1 CaB lysozyme in the presence of 100 mM CaCl 2 even at a protein concentration of 200 μg/mL was 80% and was higher than that of the wild-type lysozyme. From analysis of chemical shift changes of the cross peaks in the backbone region of total correlated spectroscopy (TOCSY) spectra of a decapeptide possessing the same calcium binding site as in Ser - 1 CaB lysozyme in the presence of various concentrations of Ca 2 + , it was suggested that the dissociation constant of Ca 2 + -peptide complex was estimated to be 20-36 mM. Moreover, the solubility of the denatured Ser - 1 CaB lysozyme in the presence of 100 mM CaCl 2 was higher than that in the presence of 2 mM CaCl 2 whereas the solubility of the denatured Ser - 1 lysozyme in the presence of 100 mM CaCl 2 was not higher than that in the presence of 2 mM CaCl 2 . Therefore, it was concluded that the reduced lysozyme possessing the Ca 2 + binding site was efficiently folded in the presence of high concentration of Ca 2 + (100 mM) even at high protein concentration due to depression of aggregation by the binding of Ca 2 + to the polypeptide chain in Ser - 1 CaB lysozyme.

Beta-amyloid peptide 1-42 is a major peptide constituent of beta-amyloid fibrils. We investigated the role of sucrose on the deposition and the D-aspartic acid formation in an amyloidogenic peptide 1-42 under physiological conditions. From analyses using thioflavine-T fluorometric assay and electronmicroscopic spectroscopy after 60 h incubation at 37 degrees C, it was found that sucrose retarded the fibril formation in the amyloidogenic peptide. The retardation of the formation of amyloid fibrils by sucrose was suggested to be not due to viscosity but due to disturbance of the assemlby of alpha-helix containing peptides. Moreover, we showed that the formation of D-aspartyl residue, which is found in beta-amyloid fibrils from Alzheimer disease brains, in the amyloidogenic peptide was also retarded in the presence of sucrose.

Protein folding and unfolding are coupled to a range of biological phenomena, from the regulation of cellular activity to the onset of neurodegenerative diseases. Defining the nature of the conformations sampled in nonnative proteins is crucial for understanding the origins of such phenomena. We have used a combination of nuclear magnetic resonance (NMR) spectroscopy and site-directed mutagenesis to study unfolded states of the protein lysozyme. Extensive clusters of hydrophobic structure exist within the wild-type protein even under strongly denaturing conditions. These clusters involve distinct regions of the sequence but are all disrupted by a single point mutation that replaced residue Trp 62 with Gly located at the interface of the two major structural domains in the native state. Thus, nativelike structure in the denatured protein is stabilized by the involvement of Trp 62 in nonnative and long-range interactions.

We investigated whether or not the Amyloid-beta-protein (A beta) itself spontaneously generates free radicals using electron spin resonance (ESR) spectroscopy while also monitoring the aggregational state of A beta and A beta-induced cytotoxicity. The present results demonstrated a four-line spectrum in the presence of A beta25-35 with N-tert-butyl-alpha-phenylnitrone (PBN) but not in the presence of PBN alone in phosphate-buffered saline (PBS). The fact that the four-line spectrum obtained for the A beta25-35/PBN in PBS was completely abolished in the presence of the iron-chelating agent Desferal demonstrated the observed four-line spectrum to be iron-dependent. On the other hand, A beta25-35 with PBN in phosphate buffer (PB) did not produce any definite four-line spectrum. the present results showed the amyloid fibril formation of A beta25-35 in PBS to be much higher than that of A beta25-35 in PB. Moreover, A beta-induced cytotoxicity assays showed A beta incubated in PBS to be more cytotoxic than that incubated in PB. These results thus demonstrate that A beta(25-35)-associated free radical generation is strongly influenced by the aggregational state of the peptides.

We prepared two dissected fragments of hen lysozyme and examined whether or not these two fragments associated to form a native-like structure. One (Fragment I) is the peptide fragment Asn59-homoserine-105 containing Cys64-Cys80 and Cys76-Cys94. The other (Fragment II) is the peptide fragment Lys1-homoserine-58 connected by two disulfide bridges, Cys6-Cys127 and Cys30-Cys115, to the peptide fragment Asn106-Leu129. It was found that the Fragment I immobilized in the cuvette formed an equimolar complex with Fragment II (K(d) = 3.3x10(-4) M at pH 8 and 25 degrees C) by means of surface plasmon resonance. Moreover, from analyses by circular dichroism spectroscopy and ion-exchange chromatography of the mixture of Fragments I and II at pH 8 under non-reducing conditions, it was suggested that these fragments associated to give the native-like structure. However, the mutant Fragment I in which Cys64-Cys80 and Cys76-Cys94 are lacking owing to the mutation of Cys to Ala, or the mutant fragment in which Trp62 is mutated to Gly, did not form the native-like species with Fragment II, because the mutant Fragment I derived from mutant lysozymes had no local conformation due to mutations. Considering our previous results where the preferential oxidation of two inside disulfide bonds, Cys64-Cys80 and Cys76-Cys94, occurred in the refolding of the fully reduced Fragment I, we suggest that the peptide region corresponding to Fragment I is an initiation site for hen lysozyme folding.

We determined if a single amino acid substitution in a self protein causes autoantibody responses. Mouse lysozyme (ML) was used as a model self protein, and a mutant ML (F57L ML) was prepared by replacing 57Phe of ML to Leu, an approach which resulted in introducing into ML the immunogenic sequence of peptide 50-61 of hen egg lysozyme (HEL) restricted to I-A(k) MHC class II molecule. We found that F57L ML but not native ML primed HEL specific T cells and triggered ML specific autoantibody responses in B10.A and C3H mice (I-A(k), I-E(k)). Peptide regions, ML 14-69 and ML 98-130, were major epitopes of autoantibodies in both strains of mice. These findings indicate that a single amino acid substitution in self proteins can cause an autoantibody response when the mutated region is presented by MHC class II molecules and recognized by T cells.

We prepared a lysozyme mutant (Q41S/R61S) introducing Asn-type glycosylation signal sites by yeast expression system. On purification by cation exchange column at pH 7, three fractions were obtained. Peptide mapping and mass-spectrometry showed the fractions were the derivatives glycosylated at both Asn39 and Asn59, at only Asn39, and not glycosylated. It was revealed that the processing of Asn-linked oligosaccharide at Asn39 and Asn59 occurred independently in yeast cells. The denaturation temperatures of these derivatives by differential scanning calorimetry were 76.0, 68.8, and 67.5 degrees C at pH 3, respectively. The stabilization of glycosylated lysozyme depends on the degree of glycosylation. We concluded that stabilized proteins can be constructed by glycosylation at proper sites. Thermodynamic stabilization by the artificial double glycosylations on a protein has not yet been reported.

We examined the effects of osmolytes, sucrose and trehalose, on the deterioration of hen lysozyme as a model protein. Sucrose and trehalose depressed the aggregation of lysozyme molecules caused by heating at 100 degrees C at pH 6. Since lysozyme was fully denatured under these conditions, the effects of sucrose and trehalose on the denatured state of lysozyme were investigated using reduced S-alkylated lysozyme, a model of denatured hen lysozyme. From analyses of circular dichroism spectra and fluorescence spectra, sucrose and trehalose were found to induce alpha-helical conformations and some tertiary structures around tryptophan residues in the reduced S-alkylated lysozyme. Moreover, these compounds also depressed chemical reactions such as deamidation and racemization, which often cause the deterioration of proteins, on the reduced S-alkylated lysozyme. Therefore, the data suggest that sucrose and trehalose have a propensity to depress such deterioration as the aggregation of protein molecules or chemical reactions in proteins by inducing some tertiary structures (including alpha-helical structures) in the polypeptide chain.

X-ray structure determination of proteins by using the multiple-wavelength anomalous dispersion method targeting selenomethionine is now widely employed. Isoleucine was examined for the second choice of the substitution of methionine next to leucine. We performed a systematic mutational study of the substitutions of methionine for isoleucine. All mutated lysozymes were less stable than the wild-type by about 1 kcal/mol and it is suggested that this instability was caused by the change in residual hydrophobicity from isoleucine to methionine. The X-ray structures of all mutant lysozymes were very similar to that of the wild-type. In addition, both the accessible surface areas and the conformation of the side chain of methionine in all mutant lysozymes were similar to those of the side chain at the respective isoleucine in the wild-type. Therefore, it is suggested that the mutation from isoleucine to methionine in a protein can be considered as a "safe" substitution.

Stabilization of a protein using cavity-filling strategy has hardly been successful because of unfavorable van der Waals contacts. We succeeded in stabilizing lysozymes by cavity-filling mutations. The mutations were checked by a simple energy minimization in advance. It was shown clearly that the sum of free energy change caused by the hydrophobicity and the cavity size was correlated very well with protein stability. We also considered the aromatic–aromatic interaction. It is reconfirmed that the cavity-filling mutation in a hydrophobic core is a very useful method to stabilize a protein when the mutation candidate is selected carefully.