Intermediates in the assembly of bacteriorhodopsin investigated by time-resolved absorption spectroscopy.
The in vitro folding and assembly kinetics of bacteriorhodopsin have been studied by absorption spectroscopy. Folding is initiated by rapid stopped-flow mixing of denatured apoprotein (bacterio-opsin) in SDS micelles and mixed dimyristoylglycerophosphocholine/Chaps micelles containing retinal. The apparent mixing rate of the two types of micelles has been determined by time-resolving the changes in light scattering by 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 constant of similar magnitude to the latter has previously been assigned to a protein-folding event on the basis of protein fluorescence studies [Booth, P. J., Farooq, A. & Flitsch, S. L. (1996) Biochemistry 35, 5902-5909]. However the results presented here show that this rate constant may be associated with a rearrangement of the mixed detergent/lipid micelles. When the changes in the retinal absorption band are time-resolved during assembly of bacteriorhodopsin, a retinal-protein intermediate, with an absorption maximum of about 430 nm, has been identified. This absorption maximum lies between that of unbound retinal (at about 380 nm) and the native chromophore (at about 560 nm). A comparison of fluorescence and absorption data, together with previous evidence [Booth, 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 that the covalent Schiff-base link to retinal is not formed in the 430-nm-absorbing intermediate.
Booth
P J
PJ
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK. p.j.booth@ic.ac.uk
Farooq
A
A
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism; D002793:Cholic Acids; D003902:Detergents; D007700:Kinetics; D008027:Light; D008823:Micelles; D017510:Protein Folding; D012172:Retinaldehyde / Q000378:metabolism; D012542:Scattering, Radiation; D012967:Sodium Dodecyl Sulfate; D013050:Spectrometry, Fluorescence; D013054:Spectrophotometry, Atomic; D013997:Time Factors
D002793:Cholic Acids; D003902:Detergents; D008823:Micelles; D012967:Sodium Dodecyl Sulfate; D001436:Bacteriorhodopsins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D012172:Retinaldehyde