THERMODYNAMICS OF THE UNFOLDING AND SPECTROSCOPIC PROPERTIES OF THE V66W MUTANT OF STAPHYLOCOCCAL NUCLEASE AND ITS 1-136-FRAGMENT

Spectroscopic studies have been performed to characterize the solution structure of the V66W mutant of Staphylococcal nuclease and the corre sponding 1-136 fragment, referred to as V66W'. Whereas wild-type nucle ase has a single tryptophan residue at position 140, the V66W mutant h as a second tryptophan residue at position 66, which is the only such residue in V66W'. Steady-state and time-resolved fluorescence studies show Trp-66 in V66W' to have a blue emission, a relatively large fluor escence quantum yield, a long lifetime, a significant degree of protec tion from solute quenchers, and to depolarize with a relatively long r otational correlation time. These results characterize Trp-66 in V66W' as being a buried residue, which indicates that this fragment retains some global structure. Circular dichroism (CD) data are consistent wi th the fragment having lost most of the a-helical content of the wild type, while retaining beta-sheet structure. The CD spectrum in the aro matic region also suggests that Trp-66 in the fragment experiences an asymmetric environment, which is not identical to that in the full len gth mutant, V66W. In addition, optical detection of triplet state magn etic resonance (ODMR) spectroscopy can clearly resolve the tryptophan residues and demonstrates differences between the local environment of Trp-66 in V66W and in V66W', as well as small differences in the Trp- 140 environment in wild type and in V66W. Guanidine-HCl induced and th ermally induced unfolding studies were performed by simultaneously acq uiring CD and fluorescence data as a function of the perturbation and then performing a global analysis of such multiple data sets in terms of two-state and three-state unfolding models. Whereas data for wild-t ype nuclease and the V66W' fragment are well characterized by a two-st ate unfolding model, data for the V66W mutant are better characterized by a three-state process. That is, both the denaturant- and temperatu re-induced unfolding of V66W involves the significant population of an equilibrium unfolding intermediate. Our global analyses yield thermod ynamic parameters for the unfolding transitions, and we show that the data for V66W can be described by a constrained three-state model in w hich the transition of the intermediate to the fully unfolded state is fixed to have the same thermodynamic parameters that describe the unf olding of the V66W' fragment.