Circumnavigating misfolding traps in the energy landscape through protein engineering: suppression of molten globule and aggregation in carbonic anhydrase.
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.
Karlsson
Martin
M
IFM-Department of Chemistry, Linköping University, SE-581 83 Linköping, Sweden.
Mårtensson
Lars-Göran
LG
Olofsson
Patrik
P
Carlsson
Uno
U
D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding
D004220:Disulfides; D002256:Carbonic Anhydrases