<title>Time-resolved fluorescence studies of the thermal and guanidine-induced unfolding of nuclease A and its unstable mutant</title>

Abstract

The thermal and guanidine(DOT)HCl induced unfolding of Staphylococcal nuclease A, and its hybrid mutant, NCASG28, have been investigated by time-resolved studies of the fluorescence of the single tryptophan residue, Trp-140. The NCASG28 mutant has a much lower thermodynamic stability than the wild type. The fluorescence decay of both proteins is found to change significantly as temperature and guanidine concentration is increased. These changes appear to reflect unfolding transitions in the proteins. When analyzed in terms of a bi- exponential decay law, the amplitude ((alpha) ) of the long fluorescence decay time ((tau) ) decreases with unfolding; likewise the (alpha) for the short (tau) increases with unfolding. A global analysis of the temperature and guanidine dependence data sets was performed. The recovered pre-exponential values were then analyzed in terms of a two-state unfolding transition. The time-resolved data are consistent with such a two-state model and analysis yielded values of thermodynamic parameters, including the enthalpy change for the thermal transition and the `m' value for the guanidine-induced unfolding transition.© (1992) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.