Structure and function in bacteriorhodopsin: The role of the interhelical loops in the folding and stability of bacteriorhodopsin

Bacteriorhodospin functions as a light-driven proton pump in Halobacterium salinarium. The functional protein consists of an apoprotein, bacterioopsin , with seven transmembrane alpha helices together with a covalently bound a ll-trans retinal chromophore. In order to study the role of the interhelica l loop conformations in the structure and function of bacteriorhodopsin, we have constructed bacterioopsin genes where each loop is replaced, one at a time, by a peptide linker consisting of Gly-Gly-Ser-repeat sequences, whic h are believed to have flexible conformations. These mutant proteins have b een expressed in Escherichia coli, purified and reconstituted with all-tran s retinal in L-alpha -1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3- (3-cholamidopropyl)dimethylammonio-1-propane sulfonate (CHAPS)/SDS and L-al pha -1,2-dihexanoylphosphatidylcholine (DHPC)/DMPC/SDS micelles. Wild-type- like chromophore formation was observed in all the mutants containing singl e loop replacements. In the BC and FG mutants, an additional chromophore ba nd with an absorption band at about 480 nm was observed, which was in equil ibrium with the 550 nm, wild-type band. The position of the equilibrium dep ended on temperature, SDS and relative DMPC concentration. The proton pumpi ng activity of all of the mutants was comparable to that of wild-type bR ex cept for the BC and FG mutants, which had lower activity. All of the loop m utants were more sensitive to denaturation by SDS than the wild-type protei n, except the mutant where the DE loop was replaced. These results suggest that a specific conformation of all the loops of bR, except the DB loop, co ntributes to bR stability and is required for the correct folding and funct ion of the protein. An increase in the relative proportion of DHPC in DHPC/ DMPC micelles, which reduces the micelle rigidity and alters the micelle sh ape, resulted in lower folding yields of all loop mutants except the BC and DE: mutants. This effect of micelle rigidity on the bR folding yield corre lated with a loss in stability of a partially folded, seven-transmembrane a poprotein intermediate state in SDS/DMPC/CHAPS micelles. The folding yield and stability of the apoprotein intermediate state both decreased for the l oop mutants in the order WT similar to BC similar to DE > FG > AB > EF grea ter than or equal to CD, where the EF and CD loop mutants were the least st able. (C) 2001 Academic Press.