NMR analysis of staphylococcal nuclease thermal quench refolding kinetics

Thermally unfolded staphylococcal nuclease has been rapidly quenched to temperatures near 0 °C and the refolding behavior examined using an NMR kinetic experiment. Unfolded protein, exhibiting random coil chemical shifts, persists following the quench and refolds in two distinct kinetic phases. A protein folding intermediate with a trans Lys 116‐Pro 117 peptide bond is transiently overpopulated and relaxes to the predominantly cis native cis‐trans equilibrium. The rate of trans → cis isomerization in the nativelike nuclease intermediate is approximately 100‐fold faster than that observed in a Lys‐Pro model peptide. The activation enthalpy of 20 kcal/mol observed for the nuclease Lys 116‐Pro 117 peptide bond is comparable to that observed for other X‐Pro isomerizations.