Kinetic evidence for an obligatory intermediate in the folding of the membrane protein bacteriorhodopsin.
A photodiode array in conjunction with a rapid stopped-flow mixing method, with a millisecond time resolution, is used here to study the refolding of the membrane protein bacteriorhodopsin from an apoprotein state with a native-like secondary structure in mixed phospholipid/detergent micelles. Refolding to the native state is initiated by the rapid mixing of all-trans-retinal and the apoprotein bacterioopsin in mixed micelles. A lag phase of several seconds is observed in the appearance of the native state, as monitored by the increase in absorbance of the native chromophore. This observation demonstrates unequivocally that an intermediate is obligatory in the formation of bacteriorhodopsin. It is further shown that this intermediate is spectroscopically distinct from free retinal (absorbance maximum approximately 380 nm) and bacteriorhodopsin (absorbance maximum approximately 560 nm) and absorbs maximally at 430 nm. Evidence for the decay of the 430 nm intermediate into bacteriorhodopsin via three distinct parallel pathways is also provided. Taken together, these findings are used to describe a model in which distinct populations of the apoprotein in mixed micelles appear to fold along separate pathways via their corresponding intermediates into the native state. How the results of this study provide new insights into the mechanisms of protein folding is discussed.
Farooq
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Department of Biochemistry, Imperial College of Science, Technology & Medicine, London, UK. amjad@inka.mssm.edu
D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism; D019617:Halobacterium salinarum; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008823:Micelles; D008956:Models, Chemical; D017510:Protein Folding; D017433:Protein Structure, Secondary; D018717:Purple Membrane / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry
D001059:Apoproteins; D008565:Membrane Proteins; D008823:Micelles; D001436:Bacteriorhodopsins