Probing the folding and unfolding of wild-type and mutant forms of bacteriorhodopsin in micellar solutions: Evaluation of reversible unfolding conditions

Wild-type and mutant forms of bacteriorhodopsin (sbR) from Halobacterium sa linarium, produced by Escherichia coli overexpression of a synthetic gene, were reversibly unfolded in 1,2-dimristoyl-sn-glycero-3-phosphocholine (DMP C), 3-[(3-cholamidopropyl)dimethylamino]-2-hydroxyl-1-propane (CHAPSO), and sodium dodecyl sulfate (SDS) mixed micelles, To study the effect on protei n stability by substitutions on the hydrophobic surface with polar residues , the unfolding behavior of a G113Q, G116Q mutant [sbR(Q2)] was compared to the wild-type sbR [sbR(WT)], sbR(Q2) was more sensitive to SDS-induced unf olding than sbR(WT) under equilibrium conditions, and kinetic experiments s howed that sbR(Q2) was more sensitive to acid-induced denaturation and ther mal unfolding than sbR(WT). Since the mutations in sbR(Q2) were on the dete rgent-embedded hydrophobic surface of sbR, protein destabilization by these mutations supports the concept that the membrane-embedded segments are imp ortant for the stability of sbR. Our experiments provide the basis for stud ying the thermodynamic stability of sbR by evaluating reversible folding an d unfolding conditions in DMPC/CHAPSO/SDS mixed micelles.