INDICATION OF POSSIBLE POSTTRANSLATIONAL FORMATION OF DISULFIDE BONDSIN THE BETA-SHEET DOMAIN OF HUMAN LYSOZYME

Lysozyme has two distinct folding domains, and in most molecules the a lpha-helical domain folds more quickly than the beta-sheet domain in v itro [Radford, Dobson and Evans (1992) Nature (London) 358, 302-3071. In order to investigate the relationship between the formation of disu lphide bonds and protein folding in vivo, we carried out cysteine scan ning mutagenesis to shift positions of the disulphide bonds in both th e alpha-helical and beta-sheet domains of human lysozyme. Of the const ructed mutants (nine in the beta-sheet domain and 13 in the alpha-heli cal domain). the mutant L79CC81A, in which Leu-79 and Cys-81 in the be ta-sheet domain were replaced by Cys and Ala respectively, was secrete d by yeast. The rest of the mutants were retained in the insoluble fra ction of the cell, probably because of a failure of folding. The dista nce between the two a-carbons at positions 79 and 95 in the wild-type protein is too far to form a disulphide bond, hut analysis of the prim ary structure revealed that the major part of L79CC81A 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 sugges t that the beta-sheet domain of human lysozyme can tolerate the shift of locations of disulphide bonds. and the non-native folding of mutate d polypeptide chains in in vivo folding. The free residues Cys-65 and Cys-77 formed a disulphide bond in vitro by air oxidation, yielding tw o isomers. On the basis of our previous results and present study it i s suggested that the formation of Cys6-Cys128 is the first step of the in vivo correct folding of human lysozyme, and disulphide bonds in th e beta-sheet domain are post-translationally formed in