STRUCTURE AND FUNCTIONAL COMPLEMENTATION OF ENGINEERED FRAGMENTS FROMYEAST PHOSPHOGLYCERATE KINASE

Previous studies have shown that, although the isolated structural dom ains of yeast phosphoglycerate kinase recover a quasi-native structure in vitro as well as in vivo, they do not reassociate nor generate a f unctional enzyme. The aim of this work was first to study the folding of complementary fragments different from structural domains and secon d to determine the requirements for their reassociation and functional complementation. The method used for producing rigorously dermed frag ments consists of the introduction of a unique cysteinyl residue in th e protein followed by a specific cleavage by 5'5'-dithiobis(2-nitroben zoate)/potassium cyanide at this residue. Two pairs of complementary f ragments were thus obtained, 1-96/97-415 and 1-248/249-415. The struct ure and stabilities of the different fragments were studied. The short fragments, i.e. 1-96 and 249-415 were found to contain some secondary structure, but to have a low stability. Each large fragment has a hig h structural content and a stability close to that of the correspondin g domain. In contrast to that observed with the isolated domains, a we ak but significant complementation was observed for the two pairs of f ragments; the pair of fragments 1-248/249-415 recovered 8% of the acti vity of the native enzyme upon complementation. An independent refoldi ng of the complementary fragments before reassociation decreased the y ield of complementation for the pair of fragments 1-96/97-415, but did not affect the complementation for the other pair (1-248/249-415). Fr om the present data and previous work on the isolated domains, it appe ars that the correct folding of the isolated fragments is not a prereq uisite for their complementation.