KINETIC EVIDENCE FOR AN OBLIGATORY INTERMEDIATE IN THE FOLDING OF THEMEMBRANE-PROTEIN BACTERIORHODOPSIN

A photodiode array in conjunction with a rapid stopped-flow mixing met hod, with a millisecond time resolution, is used here to study the ref olding of the membrane protein bacteriorhodopsin from an apoprotein st ate with a native-like secondary structure in mixed phospholipid/deter gent 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 appearanc e of the native state, as monitored by the increase in absorbance of t he native chromophore. This observation demonstrates unequivocally tha t an intermediate is obligatory in the formation of bacteriorhodopsin. It is further shown that this intermediate is spectroscopically disti nct from free retinal (absorbance maximum similar to 380 nm) and bacte riorhodopsin (absorbance maximum similar to 560 nm) and absorbs maxima lly at 430 nm. Evidence for the decay of the 430 nm intermediate into bacteriorhodopsin via three distinct parallel pathways is also provide d. Taken together, these findings are used to describe a model in whic h distinct populations of the apoprotein in mixed micelles appear to f old 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.