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

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.

Domain interactions direct misfolding and amyloid formation of yeast phosphoglycerate kinase

“…The folding pathways of a multidomain enzyme can strongly depend on the relative stability of the domains and the strength of the interdomain contacts 26–28. Due to its two‐domain structure, PGK has been used successfully as a simple model for the investigation of domain interactions 29–34…”

Protein Stability, Folding and Misfolding in Human PGK1 Deficiency

“…PGK enzymes have been studied extensively as a model system for the folding/unfolding of two-domains proteins, including bacterial, yeast and mammalian PGKs [13,14,15,16,17,18,19,20]. Despite their size and structural complexity, the unfolding of PGK enzymes is often described well by relatively simple models.…”