Sequential conformation change and activation of chicken liver dihydrofolate reductase in low concentration of guanidine hydrochloride

Science in China. Series C, Life Sciences
Y X FanC Zou

Abstract

The conformation changes of dihydrofolate reductase (DHFR) from chicken liver in guanidine hydrochloride were monitored by protein intrinsic fluorescence, hydrophobic fluorescence probe TNS and limited proteolysis by proteinase K. The kinetics of the enzyme denaturation were also studied and compared with its activity changes. It was indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene (TNS) that a subtle conformational change of the enzyme in dilute GuHCl parallels GuHCl-induced activation. At GuHCl concentration higher than 0. 75 mol/L, the conformational change can be detected by increased susceptibility of the enzyme to proteinase K, but no significant gross conformational change of the enzyme molecule is observed by intrinsic fluorescence up to a GuHCl concentration of 1.2 mol/L. The results suggest that the denaturation of DHFR by GuHCl does not follow strictly the two-state model. The enzyme seems to open up sequentially with increasing concentrations of denaturants, mainly at the active site first, until to a critical concentration of the denaturant the overall conformation change of the molecule may occur cooperatively. Activation of DHFR in low GuHCl concentration is due to the subtle conformational cha...Continue Reading

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