COMPACT THERMALLY-DENATURED STATE OF A STAPHYLOCOCCAL NUCLEASE MUTANTFROM RESONANCE ENERGY-TRANSFER MEASUREMENTS

Thermal denaturation of a staphylococcal nuclease mutant K78C, where l ysine 78 is replaced by cysteine. was studied by circular dichroism (C D) and resonance energy transfer. CD spectra suggest that residual str uctures remain in the denatured state. Steady-state energy transfer fr om intrinsic tyrosines to a single and intrinsic tryptophan was measur ed at different temperatures. In the thermally-denatured state of K78C . there is still a substantial degree of energy transfer from tyrosine (s) to tryptophan. indicating residual structures in the denatured sta te. The cysteine residue in mutant K78C was labeled with a cysteine sp ecific probe IAEDANS. Fluorescence decays of the tryptophan were measu red to estimate distance distributions between Trp 140 and IAEDANS at position 78. Measurements were done as a function of temperature from 4-degrees-C (native) to 65-degrees-C (denatured) both with and without Ca2+ and inhibitor pdTp. Below 30-degrees-C, the apparent distance di stribution of both the ligand-free nuclease and the enzyme with bound pdTp can be adequately described by a Gaussian model. Above 40-degrees -C. where the ligand-free nuclease but not the ternary complex begins to denature, two different populations are required to fit the data bo th with and without pdTp. One population has a compact structure and t he other has an expanded structure. As temperature rises, the populati on of the expanded structure increases. At the highest temperature. th e non-native compact structure is still the major form (60 to 70%). Th e overall thermally-denatured states of staphylococcal nuclease mutant K78C in the absence and presence of ligands are thus compact and hete rogeneous.