Incomplete refolding of a fragment of the N‐terminal domain of pig muscle 3‐phosphoglycerate kinase that lacks a subdomain

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‐ P Gri), 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.