Structure and functional complementation of engineered fragments from yeast phosphoglycerate kinase |
| |
Authors: | Pecorari Frederic; Minard Philippe; Desmadril Michel; Yon Jeannine M |
| |
Affiliation: | Laboratoire dEnzymologie physicochimique et moléculaire, Unité de Recherche Associée du Centre National de la Recherche Scientifique Université de Paris-Sud 91405F, Orsay, France |
| |
Abstract: | Previous studies have shown that, although the isolated structuraldomains of yeast phosphoglycerate kinase recover a quasi-nativestructure in vitro as well as in vivo, they do not reassociatenor generate a functional enzyme. The aim of this work was firstto study the folding of complementary fragments different fromstructural domains and second to determine the requirementsfor their reassociation and functional complementation. Themethod used for producing rigorously defined fragments consistsof the introduction of a unique cysteinyl residue in the proteinfollowed by a specific cleavage by 5'5'-dithiobis(2-nitrobenzoate)/potassiumcyanide at this residue. Two pairs of complementary fragmentswere thus obtained, 196/97415 and 1248/249415.The structure and stabilities of the different fragments werestudied. The short fragments, i.e. 196 and 249415were found to contain some secondary structure, but to havea low stability. Each large fragment has a high structural contentand a stability close to that of the corresponding domain. Incontrast to that observed with the isolated domains, a weakbut significant complementation was observed for the two pairsof fragments; the pair of fragments 1248/249415recovered 8% of the activity of the native enzyme upon complementation.An independent refolding of the complementary fragments beforereassociation decreased the yield of complementation for thepair of fragments 196/97415, but did not affectthe complementation for the other pair (1248/249415).From the present data and previous work on the isolated domains,it appears that the correct folding of the isolated fragmentsis not a prerequisite for their complementation. |
| |
Keywords: | complementation/ folding/ fragments/ phosphoglycerate kinase/ site-directed mutagenesis |
本文献已被 Oxford 等数据库收录! |
|