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 i ts complementary proteolytic fragments that did not correspond to the indiv idual 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 (whi ch encompasses the whole C domain) has been noted, but the refolding abilit y of the 16.8-kDa N-terminal proteolytic fragment, which lacks a single sub domain from the N domain, remained to be seen. Here the refolding processes of the isolated fragments are compared. Within the first few seconds of in itiation of refolding, pulse-proteolysis experiments show the formation of a structure with moderate protease resistance for both fragments. This stru cture, however, remains unchanged upon further incubation of the N-terminal fragment, whereas refolding of the C-terminal fragment continues as detect ed by a further increase in proteolytic resistance. The non-native characte r of the folding intermediate of the N fragment is indicated by the elevate d fluorescence intensity of the bound hydrophobic probe 8-anilino-1-naphtal ene 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 tra nsition is seen. Size-exclusion chromatography supported the globular chara cter of the intermediate, and showed its propensity to form dimers. No bind ing of the substrate, 3-phosphoglycerate (3-PGri), to the isolated N-termin al fragment, could be detected but the presence of the complementary C-term inal 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 se condary structure and highly exposed hydrophobic clusters, which favour dim erization.