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Indication of possible post-translational formation of disulphide bonds in the beta-sheet domain of human lysozyme.

ABSTRACT: Lysozyme has two distinct folding domains, and in most molecules the alpha-helical domain folds more quickly than the beta-sheet domain in vitro [Radford, Dobson and Evans (1992) Nature (London) 358, 302-307]. In order to investigate the relationship between the formation of disulphide bonds and protein folding in vivo, we carried out cysteine scanning mutagenesis to shift positions of the disulphide bonds in both the alpha-helical and beta-sheet domains of human lysozyme. Of the constructed mutants (nine in the beta-sheet domain and 13 in the alpha-helical domain), the mutant L79CC81A, in which Leu-79 and Cys-81 in the beta-sheet domain were replaced by Cys and Ala respectively, was secreted by yeast. The rest of the mutants were retained in the insoluble fraction of the cell, probably because of a failure of folding. The distance between the two alpha-carbons at positions 79 and 95 in the wild-type protein is too far to form a disulphide bond, but analysis of the primary structure revealed that the major part of L79CC81A was secreted with a non-native disulphide bond Cys79-Cys95 and two free cysteine residues at positions 65 and 77 in the beta-sheet domain. These results suggest that the beta-sheet domain of human lysozyme can tolerate the shift of locations of disulphide bonds, and the non-native folding of mutated polypeptide chains in in vivo folding. The free residues Cys-65 and Cys-77 formed a disulphide bond in vitro by air oxidation, yielding two isomers. On the basis of our previous results and present study it is suggested that the formation of Cys6-Cys128 is the first step of the in vivo correct folding of human lysozyme, and disulphide bonds in the beta-sheet domain are post-translationally formed in vivo.

SUBMITTER: Kanaya E

PROVIDER: S-EPMC1134233 | biostudies-other | 1993 Jun

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Disulphide bridges are well known to play key roles in stability, folding and functions of proteins. Introduction or deletion of disulphides by site-directed mutagenesis have produced varying effects on stability and folding depending upon the protein and location of disulphide in the 3-D structure. Given the lack of complete understanding it is worthwhile to learn from an analysis of extent of conservation of disulphides in homologous proteins. We have also addressed the question of what structural interactions replaces a disulphide in a homologue in another homologue. RESULTS: Using a dataset involving 34,752 pairwise comparisons of homologous protein domains corresponding to 300 protein domain families of known 3-D structures, we provide a comprehensive analysis of extent of conservation of disulphide bridges and their structural features. We report that only 54% of all the disulphide bonds compared between the homologous pairs are conserved, even if, a small fraction of the non-conserved disulphides do include cytoplasmic proteins. Also, only about one fourth of the distinct disulphides are conserved in all the members in protein families. We note that while conservation of disulphide is common in many families, disulphide bond mutations are quite prevalent. Interestingly, we note that there is no clear relationship between sequence identity between two homologous proteins and disulphide bond conservation. Our analysis on structural features at the sites where cysteines forming disulphide in one homologue are replaced by non-Cys residues show that the elimination of a disulphide in a homologue need not always result in stabilizing interactions between equivalent residues. CONCLUSION: We observe that in the homologous proteins, disulphide bonds are conserved only to a modest extent. Very interestingly, we note that extent of conservation of disulphide in homologous proteins is unrelated to the overall sequence identity between homologues. The non-conserved disulphides are often associated with variable structural features that were recruited to be associated with differentiation or specialisation of protein function.

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Indication of possible post-translational formation of disulphide bonds in the beta-sheet domain of human lysozyme.

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