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

Abstract

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.

Correlation between conformational stability of the ternary enzyme–substrate complex and domain closure of 3‐phosphoglycerate kinase

“…The best approach by which to obtain direct information about the extent of domain–domain cooperativity, is to carry out unfolding–refolding experiments, as noninteracting structural domains generally correspond to separate folding units. Both unfolding [36–50] and refolding [36–38,46–62] experiments using denaturants have been carried out extensively with PGK, but mainly in the absence of substrates, with the intact enzyme [36,37,39,41,43,44,46,48], its engineered mutants [39–41,46,55,62] and its various molecular fragments [40,50–55,57–63]. These studies show that the two domains exhibit slightly different stabilities and unfolding/refolding of the two domains probably occurs in a sequential order within the PGK molecule.…”

“…The best approach by which to obtain direct information about the extent of domain-domain cooperativity, is to carry out unfolding-refolding experiments, as noninteracting structural domains generally correspond to separate folding units. Both unfolding [36][37][38][39][40][41][42][43][44][45][46][47][48][49][50] and refolding [36-38, [46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62] experiments using denaturants have been carried out extensively with PGK, but mainly in the absence of substrates, with the intact enzyme [36,37,39,41,43,44,46,48], its engineered mutants [39][40][41]46,55,62] and its various molecular fragments [40,[50][51][52]…”

Role of loops in the folding and stability of yeast phosphoglycerate kinase

“…1[25]). PGK from various sources has been studied by several independent groups as a folding model representative of multidomain proteins [26–31].…”