CONFORMATIONAL FLEXIBILITY IN A STAPHYLOCOCCAL NUCLEASE MUTANT K45C FROM TIME-RESOLVED RESONANCE ENERGY-TRANSFER MEASUREMENTS

Thermal fluctuations exist in native proteins and other macromolecules in solution. Some may play a role in ligand or receptor binding, cont rol rates of enzymatic catalysis, or define a range of conformations a segment can adopt in solution. We apply the method of time-resolved r esonance energy transfer to study the conformational flexibility of a staphylococcal nuclease mutant, K45C, where lysine 45 located at a fle xible loop is replaced by a cysteine. We labeled the thiol group with DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) and used the TNB group cova lently attached to the protein as an energy acceptor from a single try ptophan at residue 140 as the donor. Conformational flexibility occurr ing on the time scale of nanoseconds oi longer is dispersed as an appa rent distance distribution in-time-resolved resonance energy transfer measurements. Below room temperature the apparent distance distributio n was fitted with a symmetric Lorentzian model with a full width at ha lf maximum height of about 6 Angstrom, indicating substantial degrees df heterogeneity between residues 45 and 140. At room or higher temper ature where the protein is in its native state, the apparent distance distribution is asymmetric, indicating the presence of static disorder s. Segments in the protein that contribute to the static disorder can be converted to mobile ones with the addition of denaturing guanidiniu m chloride.