Structure and functional complementation of engineered fragments from yeast phosphoglycerate kinase.
Previous studies have shown that, although the isolated structural domains of yeast phosphoglycerate kinase recover a quasi-native structure in vitro as well as in vivo, they do not reassociate nor generate a functional enzyme. The aim of this work was first to study the folding of complementary fragments different from structural domains and second to determine the requirements for their reassociation and functional complementation. The method used for producing rigorously defined fragments consists of the introduction of a unique cysteinyl residue in the protein followed by a specific cleavage by 5'5'-dithiobis(2-nitrobenzoate)/potassium cyanide at this residue. Two pairs of complementary fragments were thus obtained, 1-96/97-415 and 1-248/249-415. The structure 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 high structural content and a stability close to that of the corresponding domain. In contrast to that observed with the isolated domains, a weak but significant complementation was observed for the two pairs of fragments; the pair of fragments 1-248/249-415 recovered 8% of the activity of the native enzyme upon complementation. An independent refolding of the complementary fragments before reassociation decreased the yield 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). From the present data and previous work on the isolated domains, it appears that the correct folding of the isolated fragments is not a prerequisite for their complementation.
Pecorari
F
F
Laboratoire d'Enzymologie physicochimique et moléculaire, Université de Paris-Sud, Orsay, France.
Minard
P
P
Desmadril
M
M
Yon
J M
JM
D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D005818:Genetic Engineering; D005976:Glutaral; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D015003:Yeasts / Q000201:enzymology*
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D010735:Phosphoglycerate Kinase; D005976:Glutaral