Structure and function in bacteriorhodopsin: The effect of the interhelical loops on the protein folding kinetics

The loops connecting the seven transmembrane helices of bacteriorhodopsin h ave each been replaced in turn by structureless linkers of Gly-Gly-Ser repe at sequences, and the effect on the protein folding kinetics has been deter mined. An SDS-denatured state of each loop mutant bacterio-opsin was folded in L-alpha -1,2-dihexanoylphosphatidylcholine/L-alpha -1,2-dimyristoylphos phatidylcholine micelles, containing retinal, to give functional bacteriorh odopsin. Stopped-now mixing was used to initiate the folding reaction, givi ng a time resolution of milliseconds, and changes in protein fluorescence w ere used to monitor folding. All loop mutant proteins folded according to t he same reaction scheme as wild-type protein. The folding kinetics of the A B, BC and DE loop mutants were the same as wild-type protein, despite the b lue-shifted chromophore band of the BC loop mutant bR state. A partially fo lded apoprotein intermediate state of the AB loop mutant did however appear to decay in the absence of retinal. The most significant effects on the fo lding kinetics were seen for mutant protein with structureless linkers in p lace of the CD, EF and FG loops. The rate-limiting apoprotein folding step of the CD loop mutant was about ten times slower than wild-type, whilst tha t of the EF loop mutant was almost four times slower than wild-type. Wild-t ype behaviour was observed for the other folding and retinal binding events of the CD and EF loop mutant proteins. These effects of the CD and EF loop mutations on apoprotein folding correlate with the fact that these two loo p mutants also have the least stable, partially folded apoprotein intermedi ate of all the loop mutants, and are the most affected by a decrease in lip id lateral pressure. Ln contrast, the FG loop mutant exhibited wild-type ap oprotein folding, but altered covalent binding of retinal and final folding to bacteriorhodopsin. This correlates with the fact that the FG loop mutan t bacteriorhodopsin is the most susceptible to denaturation by SDS of all t he loop mutants, but its partially folded apoprotein intermediate is more s table than that of the CD and EF mutants. Thus the CD and EF loops may cont ribute to the transition state for the rate-limiting apoprotein folding ste p and the FG loop to that for final folding and covalent binding of retinal . (C) 2001 Academic Press.