Revisiting the folding kinetics of bacteriorhodopsin.

The elucidation of the physical principles that govern the folding and stability of membrane proteins is one of the greatest challenges in protein science. Several insights into the folding of α-helical membrane proteins have come from the investigation of the conformational equilibrium of H. halobium bacteriorhodopsin (bR) in mixed micelles using SDS as a denaturant. In an effort to confirm that folded bR and SDS-denatured bR reach the same conformational equilibrium, we found that bR folding is significantly slower than has been previously known. Interrogation of the effect of the experimental variables on folding kinetics reveals that the rate of folding is dependent not only on the mole fraction of SDS but also on the molar concentrations of mixed micelle components, a variable that was not controlled in the previous study of bR folding kinetics. Moreover, when the molar concentrations of mixed micelle components are fixed at the concentrations commonly employed for bR equilibrium studies, conformational relaxation in the transition zone is slower than hydrolysis of the retinal Schiff base. As a result, the conformational equilibrium between folded bR and SDS-denatured bR cannot be achieved under the conventional condition. Our finding suggests that the molar concentrations of mixed micelle components are important experimental variables in the investigation of the kinetics and thermodynamics of bR folding and should be accounted for to ensure the accurate assessment of the conformational equilibrium of bR without the interference of retinal hydrolysis.