EVIDENCE THAT BILAYER BENDING RIGIDITY AFFECTS MEMBRANE-PROTEIN FOLDING

The regeneration kinetics of the integral membrane protein bacteriorho dopsin have been investigated in a lipid-based refolding system, Previ ous studies on bacteriorhodopsin regeneration have involved detergent- based systems, and in particular mixed dimyristoylphosphatidylcholine (DMPC)/CHAPS micelles, Here, we show that the short chain lipid dihexa noylphosphatidylcholine (DHPC) can be substituted for the detergent CH APS and that bacteriorhodopsin can be regenerated to high yield in mix ed DMPC/DHPC micelles, Bacteriorhodopsin refolding kinetics are measur ed in the mixed DMPC/DHPC micelles. Rapid, stopped flow mixing is empl oyed to initiate refolding of denatured bacterioopsin in SDS micelles with mixed DMPC/DHPC micelles and time-resolved fluorescence spectrosc opy to follow changes in protein fluorescence during folding. Essentia lly identical refolding kinetics are observed for mixed DMPC/CHAPS and mixed DMPC/DHPC micelles. Only one second-order retinal/apoprotein re action is identified, in which retinal binds to a partially folded apo protein intermediate, and the free energy of this retinal binding reac tion is found to be the same in both types of mixed micelles, Formatio n of the partially folded apoproptein intermediate is a rate-limiting step in protein folding and appears to be biexponential, Both apparent rate constants are found to be dependent on the relative proportion o f DMPC present in the mixed DMPC/DHPC micelles as well as on the pH of the aqueous phase. Increasing the DMPC concentration should increase the bending rigidity of the amphiphilic bilayer, and this is found to slow the rate of formation of the partially folded apoprotein intermed iate. Increasing the mole fraction of DMPC from 0.3 to 0.6 slows the t wo apparent rate constants associated with formation of this intermedi ate from 0.29 and 0.031 to O.11 and 0.013 s(-1), respectively. Formati on of the intermediate also slows with increasing pH, from 0.11 and 0. 013 s(-1) at pH 6 to 0.033 and 0.0053 s(-1) at pH 8, Since this pH cha nge has no known effect on the phase behavior of lecithins, this is mo re likely to represent a direct effect on the protein itself. Thus, it appears to be possible to control the rate-limiting process in bacter ioopsin folding through both bilayer bending rigidity and pH.