A previous study performed using steady state fluorescence has revealed the existence of residual structures surrounding the two @entity2340 residues in an unfolded form of @entity375 @entity21590 [Garcia, P., et al. (1995) Biochemistry 34, 397-404]. In this paper, we present a more detailed characterization of these residual structures, through the study of two single @entity2340 -containing mutants of yPGK, @entity69424 and @entity69425 . Denaturation experiments have first been performed at low temperatures to assess the nature of the interactions stabilizing these residual structures. On the other hand, the compactness and dynamics of the protein matrix were probed using @entity2340 fluorescence quenching by @entity3323 at various denaturant concentrations. Taking into consideration the changes in sample viscosity induced by addition of @entity2097 made feasible the use of this technique during the denaturation process. These different approaches have shown that the residual structures around @entity2340 308 are mainly stabilized by hydrophobic interactions and are more compact and less fluctuant than the ones surrounding @entity2340 333. Native and denatured yPGK have also been studied by time-resolved fluorescence spectroscopy. In the native protein, @entity2340 buried in the core, W333, is mainly associated with a lifetime close to 0.1 ns, whereas @entity2340 that is partially accessible to the solvent, W308, has a lifetime close to 0. 5 ns. The time-resolved @entity2340 fluorescence emission of wild-type yPGK can be accounted for quantitatively by the summed emissions of its two single @entity2340 mutants. The significance of minor long lifetime components is discussed for the two @entity2340 residues. This new assignment of fluorescent decay times has allowed for the detection of a folding intermediate in which the environment of @entity2340 333 is modified for denaturant concentrations lower than those for @entity2340 308.

Steady state and time-resolved fluorescence study of residual structures in an unfolded form of @entity375 XXXX .

@entity21590 :: ('850370(Tax:4932)', 'Gene') :: ['phosphoglycerate kinase'] @entity2340 :: ('CHEBI:27897', 'Chemical') :: ['tryptophan'] @entity3323 :: ('MESH:D020106', 'Chemical') :: ['acrylamide'] @entity2097 :: ('MESH:D019791', 'Chemical') :: ['guanidinium chloride'] @entity69425 :: ('p|SUB|W|308|Y', 'ProteinMutation') :: ['W308Y'] @entity375 :: ('4932', 'Species') :: ['yeast'] @entity69424 :: ('p|SUB|W|333|F', 'ProteinMutation') :: ['W333F']
@entity21590 :: (850370(Tax:4932),Gene) :: ['phosphoglycerate kinase']
