Using molecular dynamics to probe the structural basis for enhanced stability in thermal stable cytochromes P450.

High-temperature molecular dynamics (MD) has been used to assess if MD can be employed as a useful tool for probing the structural basis for enhanced stability in thermal stable cytochromes P450. CYP119, the most thermal stable P450 known, unfolds more slowly during 500 K MD simulations than P450s that melt at lower temperatures, P450cam and P450cin. A comparison of the 500 K MD trajectories shows that the Cys ligand loop, a critically important structural feature just under the heme, in both P450cin and P450cam completely unfolds while this region is quite stable in CYP119. In CYP119, this region is stabilized by tight nonpolar interactions involving Tyr26 and Leu308. The corresponding residues in P450cam are Gly and Thr, respectively. The in silico generated Y26A/L308A CYP119 double mutant is substantially less stable than wild-type CYP119, and the Cys ligand loop unfolds in a manner similar to that of P450cam. The MD thus has identified a potential "hot spot" important for stability. As an experimental test of the MD results, the Y26A/L308A double mutant was prepared, and thermal melting curves show that the double mutant exhibits a melting temperature (T(m)) 16 degrees C lower than that of wild-type CYP119. Control mutations that were predicted by MD not to destabilize the protein were also generated, and the experimental melting temperature was not significantly different from that of the wild-type enzyme. Therefore, high-temperature MD is a useful tool in predicting the structural underpinnings of thermal stability in P450s.