Unfolding-refolding transition of a hinge bending enzyme: horse muscle phosphoglycerate kinase induced by guanidine hydrochloride

Abstract

The unfolding-refolding transition of horse muscle phosphoglycerate kinase induced by guanidine hydrochloride was studied under equilibrium conditions using four different signals: fluorescence intensity at 336 nm, UV difference absorbance at 286 and 292 nm, ellipticity at 220 nm, and enzyme activity. From the following arguments, we found that the process deviates from a two-state model and intermediates are significantly populated even at equilibrium: (1) the noncoincidence of the transition curves and (2) the asymmetry of the transition curve obtained from CD measurements. From these different data and the thermodynamic analysis, it was suggested that the two domains of the horse muscle phosphoglycerate kinase refold independently of one another with different equilibrium constants, the most favorable constant referring to the folding of the C-terminal domain which contains all tryptophans.

Alteration Around the Active Site of Rhodanese during Urea-induced Denaturation and Its Implications for Folding

“…Importantly, in CMR, cysteine 247 loses its ability to form disulfide linkages with other cysteine residues during refolding. As expected, it shows excellent reversibility during refolding from urea, in the absence of any assistance (8). These observations, along with the position of the highly reactive active site cysteine at the cleft formed by two globular domains, make rhodanese a good model system to study the conformational change around the active site during folding of the protein.…”

“…Multiphasic transitions are observed for "hinge-bending" type proteins such as T4 lysozyme (25) and phosphoglycerate kinase (8). Rhodanese, whose two globular domains are linked by a connecting region consisting of a single strand of polypeptide chain also shows at least a two-step unfolding transition.…”

A comparison of the reactivity and stability of wild type and His388 Gln mutant phosphoglycerate kinase from yeast

“…Other GdnHCl equilibrium denaturation studies have provided some evidence that the domains of yeast 1371 and horse muscle PGK [38] may unfold and refold independently. Denaturation transitions followed by fluorescence gave higher c, values and larger values of AGrlzo than those from other spectral properties or activity measurements.…”

“…A ratio of these parameters greater than unity is considered evidence for a significant population of a folding intermediate [40] and it has been suggested that in PGK this species arises through the independent unfolding of one of its domains. In the light of the fluorescence transitions observed for PGK [37,38] it would seem likely that the N domain would partially or completely unfold before the more stable C domain. Differentiating the polynomial described by Adams et al [37] using the same fractional exposure of residues as these authors to correct AG, we obtain the values of mca, listed in Table 2.…”

Quasi‐irreversibility in the unfolding‐refolding transition of phosphoglycerate kinase induced by guanidine hydrochloride

“…The structural properties of the inactive species were studied; all the fl structures were recovered, but about 29% of the helical structures remained unfolded, and two SH groups were buried. Simulated kinetics were compared with the experimental results and were used to extend the minimum folding scheme previously proposed from equilibrium and kinetic studies [Betton et al (1984) Biochemistry 23,6654-6661 ; Betton et al (1985) Biochemistry 24,4570 -45771. The intermediates trapped under these conditions were structured but devoid of catalytic activity.…”