Circumnavigating Misfolding Traps in the Energy Landscape through Protein Engineering:Suppression of Molten Globule and Aggregation in Carbonic Anhydrase

摘要

The native state of the enzyme human carbonic anhydrase (HCA II) has been stabilized by the introduction of a disulfide bond,the oxidized A23C/L203C mutant.This stabilized protein variant undergoes an apparent two-state unfolding process with suppression of the otherwise stable equilibrium,molten-globule intermediate,which is normally very prone to aggregation.Stopped-flow measurements also showed that lower amounts of the transiently occurring molten globule were formed during refolding.This led to a markedly lowered tendency for aggregation during equilibrium denaturing conditions and,more importantly,to significantly higher reactivation yields upon refolding of the fully denatured protein.Thus,a general strategy to circumvent aggregation during the refolding of proteins could be to stabilize the native state of a protein at the expense of partially folded intermediates,thereby shifting the unfolding behavior from a three-state process to a two-state one.