Hiroshi Taniuchi

Implication of the structure and stability of disulfide intermediates of lysozyme on the mechanism of renaturation

SummaryThe conformational properties and stability of reduced hen egg lysozyme and those of the disulfide intermediates species formed during renaturation of reduced lysozyme have been reviewed. This information and that related with RNase A and other proteins are interpreted to outline a possible mechanism of renaturation of the reduced protein.

A study of core domains, and the core domain-domain interaction of cytochrome c fragment complex

To gain insight into the folding mechanism of the cytochrome c complex, we prepared a complete set of homologous and hybrid two-fragment ferric complexes of four different types and related complexes from horse, tuna, yeast iso-l, and Candida cytochromes c. The complexes were characterized for structural properties. Apparent equilibrium constants of the complexes were determined to calculate delta G0 for binding. The results have allowed us to assign four core domains of the complex. A core domain is a structural region containing a hydrophobic core and the surrounding shell which folds and unfolds as a unit. Core domain 1 folds by itself and consists essentially of the right channel structure, found by R. E. Dickerson and colleagues, and a part of the heme. Core domains 2, 3, and 4, respectively, are assigned based on the cores located on the left (the Fe-S bond) and right sides and at the bottom of heme. Evidence of the core domain-domain interaction to stabilize the Fe-S bond, combined with the kinetic studies by G. R. Parr and H. Taniuchi, has led to a model of two alternative folding orders of the core domains for the horse type I complex: domain 1----3----2----4 or 1----2----3----4. Furthermore, delta G0 variation between the complexes has shown non-additive behavior, indicating the existence of a residue-residue interaction between the heme- and apofragments in the complex. Evidence suggests that this interaction in most cases occurs within or through the core groups of the ordered interface between the heme- and the apo-fragments formed by folding of core domains 1, 2, and 3. Evidence also suggests that such core group interaction manifests itself in the interaction to stabilize the Fe-S bond and may be manifested in the core domain-domain interaction.

A kinetic study of the folding of nuclease B, a possible precursor of staphylococcal nuclease A

Nuclease B, which contains an additional flexible amino acid sequence of 19 amino acid residues bound to the NH2-terminus of nuclease A, an extracellular nuclease of Staphylococcus aureus, has been investigated in order to determine the influence of the extra residues on the refolding of the nuclease A portion from the acid denaturated state by monitoring the change in tryptophan fluorescence using a stopped-flow technique. It was found that the kinetic parameters of this refolding is similar within experimental error for nuclease A and nuclease B for the entire course (up to 40 s) studied. Therefore, the extra residues do not appear to have any detectable effect on the dynamic events involved in the refolding process. Thus, the folding of the nuclease A portion of nuclease B appears to be thermodynamically and kinetically independent of the 19 residues at the amino-terminus.

A study of hydrogen exchange of monoclonal antibodies : specificity of the antigen-binding induced conformational stabilization

Amide-hydrogen exchange of three anti-yeast iso-1-cytochrome-c IgG monoclonal antibodies and the Fab, prepared from one of them, were studied by infrared spectrophotometry in the presence and absence of the deuterated immunogen and evolutionarily related species (the deuterated immunogen contained a population of a dimer. Each subunit of the dimer appeared to bind to the antibodies in a manner similar to the monomer). The number of hydrogens of the antibodies whose exchange was suppressed on binding to the immunogen was found to exceed that estimated for the residues shielded by the immunogen. Analysis of the data suggests that such suppression of hydrogen exchange occurs mainly for the Fab domains, but not for the Fc. One of the antibodies showed two distinct classes of amide-hydrogens. Class-1 hydrogens (approx. 36/site) exchange faster than class 2 (approx. 37/site). The exchange of class-1 hydrogens was suppressed by binding to the immunogen, but not to the evolutionarily related species. The exchange of class-2 hydrogens was suppressed by binding to the evolutionarily related species, as well as to the immunogen. Thus, the suppression of exchange of class-1 hydrogens appears to occur by some kind of conformational stabilization, the mechanism of which differentiates between the deuterated immunogen and the evolutionarily related species. Evidence suggests that the trans-interactions of the Fab domains may modulate the hydrogen exchange. If it is assumed that the antigen-binding strengthens the trans-interactions in such a way that the exchange of the slower exchanging hydrogens is suppressed, this could explain the suppression of exchange of class-2 hydrogens.

Functional correlation in amino acid residue mutations of yeast iso-2-cytochrome c that is consistent with the prediction of the concomitantly variable codon theory in cytochrome c evolution.

Fitch and Markowitz theory of concomitantly variable codons (covarions) in evolution predicted the existence of functional correlation in amino acid residue mutations among present-day cytochromes c. Mutational analysis was carried out on yeast iso-2-cytochrome c, where hydrophobic core residues I20, M64, L85, and M98 and surface residue L9 were mutated, in selected combinations, to those found in mammalian and bird cytochromes c. The functionality assay is based upon the ability of yeast cells to grow in YPGE medium. Furthermore, experiments on the single M64L and M98L mutations as well as the double M64L/M98L mutation using NMR showed that the effects of these mutations are to perturb the structural integrity of the protein. We identified functional correlation in two cases of a pair of residue mutations, the I20 → V and M98 → L pair and the L9 → I and L85 → I pair. In both cases, only one of the two alternative, putative evolutionary pathways leads to a functional protein and the corresponding pairs of residue mutations are among those found in present-day cytochromes c. Since valine is predicted to be at position 20 in the ancestral form of cytochrome c, the present data provide an explanation for the ancient requirement of leucine rather than methionine in position 98. The present data provide further evidence for the role of those specific atom–atom interactions in directing a pathway in the evolutionary changes of the amino acid sequence that have taken place in cytochrome c, in accordance with Fitch and Markowitz.

A Study of the Influence of the Hydrophobic Core Residues of Yeast Iso-2-cytochrome c on Phosphate Binding: A Probe of the Hydrophobic Core-Surface Charge Interactions

To gain insight into the role of hydrophobic core-surface charge interactions in stabilizing cytochrome c, we investigated the influence of hydrophobic core residues on phosphate binding by mutating residues in yeast iso-2-cytochrome c to those corresponding to iso-1-cytochrome c in various combinations. Heat transition of ultraviolet CD was followed as a function of pH in the presence and absence of phosphate. Thermodynamic parameters were deduced. It was found that the I20V/V43A/M98L mutation in the hydrophobic core, whose locations are remote from the putative phosphate sites, modulates phosphate interactions. The modulation is pH dependent. The I20V/M98L and V43A mutation effects are nonadditive. The results lead to a model analogous to that of Tsao, Evans, and Wennerstrom, where a domain associated with the ordered hydrophobic core is sensitive to the fields generated by the surface charges. Such an explanation would be in accord with the observed difference in thermal stability between iso-2 and horse cytochromes c.

ANTIBODY IMMUNODIVERSITY : A STUDY ON THE MARKED SPECIFICITY DIFFERENCE BETWEEN TWO ANTI-YEAST ISO-1 CYTOCHROME C MONOCLONAL ANTIBODIES WHOSE EPITOPES ARE CLOSELY RELATED

Anti-yeast iso-1 cytochrome c (cyt. c) monoclonal antibodies 2-96-12 and 4-74-6 have closely related epitopes (antigenic determinants). However, while the specificity of 4-74-6 is stringent, 2-96-12 cross-reacts with many evolutionarily related cytochromes c. Such a marked difference in specificity of antibodies with overlapping epitopes may represent unique antibody immunodiversity. Thus, we constructed Fv fragment models consisting of the variable domains of the heavy and light chains of 2-96-12 and 4-74-6 and that of another anti-iso-1 cyt. c as a control to gain insight into the origin of this difference in specificity. Our models show that 4-74-6 and 2-96-12 contain five and two aromatic side chains, respectively, in or near the central area of the antigen-combining site. The side chains of Arg95H (heavy chain) in 2-96-12 and Arg91L (light chain) in 4-74-6 project toward the central area of the combining site in our model. Antigen docking to our Fv models, combined with previous immunological studies, suggests that iso-1 cyt. c Asp60 may interact with Arg95H in 2-96-12 and Arg91L in 4-74-6 and that both epitopes of 2-96-12 and 4-74-7 may include iso-1 cyt. c Leu58, Asp60, Asn62, and Asn63. The effect of the Arg95H to Lys mutation on the antigen binding is also in accord with our model. The difference in specificity may be partly explained by a greater degree of conformational flexibility in and around the central area of the combining site in 2-96-12 compared to 4-74-6 due to differences in aromatic side chain packing.

On the renaturation of reduced hen egg white lysozyme containing two blocked sulfhydryl groups.

Formation of native lysozyme from the reduced form involves many pathways in two processes: incorrect pairing of half-cystine residues by oxidation and rearrangement of disulfide (SS) bonds. The energy barrier against suflhydryl (SH)-disulfide interchange of the native or nativelike species thus formed causes accumulation of these species. For example, the enzymatically active isomers containing three presumably native SS bonds and one open SS bond may be thermodynamically favorable over the nonnative isomers and can be formed from reduced lysozyme or lysozyme containing scrambled SS bonds by nonobligatory and flexible pathways. As an extension of these observations formation of nativelike species from reduced lysozyme containing the average of two carboxymethyl (CM)-cysteine was investigated.

On the mechanism of renaturation of proteins containing disulfide bonds.

The renaturation of bovine pancreatic ribonuclease A and hen egg white lysozyme from their reduced forms involves two statistical processes, pairing of half-cystine residues by oxidation and rearrangement of disulfide bonds by enzyme or thiol catalyzed sulfhydryl-disulfide interchange. The stability against sulfhydryl-disulfide interchange of the native or nativelike conformation thus attained, which could be a form containing three native disulfide bonds and one open disulfide bond, causes the system to accumulate the renatured enzyme. Thus, the native-like conformation is associated with the lowest free energy only in the late phase of folding.