Incomplete refolding of a fragment of the N-terminal domain of pig muscle 3-phosphoglycerate kinase that lacks a subdomain. Comparison with refolding of the complementary C-terminal fragment.
Refolding of pig muscle 3-phosphoglycerate kinase (PGK) from a mixture of its complementary proteolytic fragments that did not correspond to the individual domains resulted in a high degree of reactivation [Vas, M., Sinev, M.A., Kotova, N. & Semisotnov, G.V. (1990) Eur. J. Biochem. 189, 575--579]. An independent refolding of the 27.7 kDa C-terminal proteolytic fragment (which encompasses the whole C domain) has been noted, but the refolding ability of the 16.8-kDa N-terminal proteolytic fragment, which lacks a single subdomain from the N domain, remained to be seen. Here the refolding processes of the isolated fragments are compared. Within the first few seconds of initiation of refolding, pulse-proteolysis experiments show the formation of a structure with moderate protease resistance for both fragments. This structure, however, remains unchanged upon further incubation of the N-terminal fragment, whereas refolding of the C-terminal fragment continues as detected by a further increase in proteolytic resistance. The non-native character of the folding intermediate of the N fragment is indicated by the elevated fluorescence intensity of the bound hydrophobic probe 8-anilino-1-naphtalene sulphonate. Its CD spectrum shows the formation of secondary structure distinct from the native one. The noncooperative phase-transition observed in microcalorimetry indicates the absence of a rigid tertiary structure, in contrast with the refolded C-terminal fragment for which a cooperative transition is seen. Size-exclusion chromatography supported the globular character of the intermediate, and showed its propensity to form dimers. No binding of the substrate, 3-phosphoglycerate (3-PGri), to the isolated N-terminal fragment, could be detected but the presence of the complementary C-terminal fragment led to restoration of the substrate binding ability of the N domain. Thus, refolding of the isolated N-terminal fragment yields a highly flexible, globular, potentially productive intermediate with non-native secondary structure and highly exposed hydrophobic clusters, which favour dimerization.
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary.
A N
AN
Szilágyi
N V
NV
Kotova
G V
GV
Semisotnov
M
M
Vas
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D009132:Muscles / Q000201:enzymology*; D010735:Phosphoglycerate Kinase / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013552:Swine
D010735:Phosphoglycerate Kinase