FOLDING AND FUNCTIONAL COMPLEMENTATION OF ENGINEERED FRAGMENTS FROM YEAST PHOSPHOGLYCERATE KINASE

A set of protein fragments was produced by site-directed mutagenesis f ollowed by chemical cleavage of phosphoglycerate kinase according to a previously described method [Pecorari et al, (1993) Protein Eng. 6, 3 13-325]. The cleavage positions were chosen in order to correspond to limits between structural subdomains. These isolated fragments were st udied by circular dichroism, folding transitions, and cross-linking an alyses. It appears that fragments corresponding to globular subdomains in the protein can recover the expected helix content. However, the c ooperativity classically observed in the folding transitions of natura l proteins is only-observed for fragments larger than a domain. Previo us studies have shown that the isolated C-terminal domain is an autono mous folding unit which displays a single cooperative transition [Miss iakas et al. (1990) Biochemistry 29, 8683-8689]. The results presented here show that the presence in a fragment of a sequence overpassing t hat of the C-terminal domain modifies its folding process. Reassociati on experiments suggest that the efficiency of the complementation proc ess is not related to the folding autonomy of the isolated fragments.