INTERMEDIATES IN THE ASSEMBLY OF BACTERIORHODOPSIN INVESTIGATED BY TIME-RESOLVED ABSORPTION-SPECTROSCOPY

The in vitro folding and assembly kinetics of bacteriorhodopsin have b een studied by absorption spectroscopy. Folding is initiated by rapid stopped-flow mixing of denatured apoprotein (bacterio-opsin) in SDS mi celles and mixed dimyristoylglycerophosphocholine/Chaps micelles conta ining retinal. The apparent mixing rate of the two types of micelles h as been determined by time-resolving the changes in light scattering b y the micelles, Micelle mixing appears to occur in two stages: a fast phase with an apparent rate constant of about 420 s(-1), and a second phase with an apparent rate constant of about 10 s(-1). A rate constan t of similar magnitude to the latter has previously been assigned to a protein-folding event on the basis of protein fluorescence studies [B ooth, P. J., Farooq, A. & Flitsch, S. L. (1996) Biochemistry 35, 5902- 5909]. However the results presented here shout that this rate constan t may be associated with a rearrangement of the mixed detergent/lipid micelles. When the changes in the retinal absorption band are time-res olved during assembly of bacteriorhodopsin, a retinal-protein intermed iate, with an absorption maximum of about 430 nm, has been identified. This absorption maximum lies between that of unbound retinal (at abou t 380 nm) and the native chromophore (at about 560 nm). A comparison o f fluorescence and absorption data, together with previous evidence [B ooth, P. J., Flitsch, S. L., Stern, L. J., Greenhalgh, D. A., Kim, P. S., & Khorana, H. G. (1995) Nat. Struct. Biol. 2, 139-143], suggests t hat the covalent Schiff-base link to retinal is not formed in the 430- nm-absorbing intermediate.