Effects of denaturants and substitutions of hydrophobic residues on backbone dynamics of denatured staphylococcal nuclease.
Analysis of residual dipolar couplings (RDCs) in the Delta131Delta fragment of staphylococcal nuclease has demonstrated that its ensemble-averaged structure is resistant to perturbations such as high concentrations of urea, low pH, and substitution of hydrophobic residues, suggesting that its residual structure is encoded by local side-chain/backbone interactions. In the present study, the effects of these same perturbations on the backbone dynamics of Delta131Delta were examined through (1)H-(15)N relaxation methods. Unlike the global structure reported by RDCs, the transverse relaxation rates R(2) were quite sensitive to denaturing conditions. At pH 5.2, Delta131Delta exhibits an uneven R(2) profile with several characteristic peaks involving hydrophobic chain segments. Protonation of carboxyl side chains by lowering the pH reduces the values of R(2) along the entire chain, yet these characteristic peaks remain. In contrast, high concentrations of urea or the substitution of 10 hydrophobic residues eliminates these peaks and reduces the R(2) values by a greater amount. The combination of low pH and high urea leads to further decreases in R(2). These denaturant-induced increases in backbone mobility are also reflected in decreases in (15)N NOEs and in relaxation interference parameters, with the former reporting an increase in fast motions and the latter a decrease in slow motions. Comparison between the changes in chain dynamics and the corresponding changes in Stokes radius and the patterns of RDCs suggests that regional variations in backbone dynamics in denatured nuclease arise primarily from local contacts between hydrophobic side chains and local interactions involving charged carboxyl groups.