Introduction of internal cysteines as conformational probes in yeast phosphoglycerate kinase

Several mutants of yeast phosphoglycerate kinase, each containingonly one internal cysteine residue, were constructed from asingle mutant devoid of cysteine. These cysteines were introducedas local conformational probes in selected buried positions.The enzyme activity, conformational characteristics and stabilityindicated that the mutations introduced only small perturbationsin the molecule. The folding–unfolding process mediatedby guanidine hydrochloride under equilibrium conditions wasstudied by following the variations in ellipticity and the reactivityof the cysteine residue towards 5,5'-dithiobis(nitrobenzoate).The process was found to be reversible except for mutant C97A,V49C,suggesting that this region located in helix I might be crucialin determining an intermediate on the folding pathway. The transitionsobtained by the two signals did not coincide, indicating thatthe local structures, in several parts inside the molecule,are more sensitive to the denaturant than the overall conformation.