   7.2.2.8 | guanidine-HCl |
the incubation of the enzyme reconstituted in phospholipid/detergent mixed micelles with high concentrations of guanidinium hydrochloride at 25°C induces a reversible decrease in fluorescence quantum yield, far-UV ellipticity, and loss of ATPase and phosphatase activities. Refolding of the enzyme from this unfolded state leads to recovery of full biological activity and all the structural features of the native enzyme. The unfolding shows typical characteristics of a two-state process. Most of the secondary and tertiary structures are disrupted. The fraction of Trp fluorescence accessible to soluble quenchers shifts from 0.52 in the native state to 0.96 in the unfolded state, with a significant spectral redshift. Hydrophobic patches in the enzyme, mainly located in the transmembrane region, are disrupted as indicated by 1-anilino-naphtalene-8-sulfonate fluorescence. Nevertheless, the unfolded state has a small but detectable amount of residual structure, which might play a key role in both CopA folding and adaptation for working at high temperatures |
725711 |
