The Role of the Positively Charged Amino Acids in Bacteriorhodopsin

This study has used chemical modification techniques to investigate the role of the only two types of positively charged amino acids, lysines and arginines, in the membrane-bound protein, bacteriorhodopsin (bR). The results of these chemical modifications have significance both for the structure of bR and for the molecular mechanism of light-activated photocycling and proton pumping. The implications for the secondary structure of bR are: LYS 40 is totally exposed to the aqueous phase, while the other five reactive lysines are partially buried in the hydrophobic domain; LYS 30, LYS 129 and LYS 159 are buried by only one or two residues within the hydrophobic domain; all but two arginines are totally exposed to the aqueous phase; and at least three ionic bridges form between arginines and carboxyl groups. These results were used to construct a new model of the secondary structure of bR. The implication for the tertiary structure of bR is that Model A (126) is the preferred model of fitting the bR sequence into the helices seen by electron diffraction. The implication for the quaternary structure is that one structural role of retinal is to increase the intermolecular distances between bR molecules in a trimer. This focuses attention on the bR trimer as the primary structural unit. Regarding the molecular mechanism of photocycling and proton pumping, two specific arginines were responsible for the slowdown of the photocycle. This result was obtained by a quantitative analysis of the inhibition of M decay as a function of fraction of arginines modified by 2,3-butanedione. Possible roles of the two important arginines, including controlling the conformational changes of the protein by ionic linkages to carboxyl groups and participating directly in proton pathways, are discussed. Bifunctional imidoester modification of lysines revealed that a conformational change of the bR protein is needed for photocycling, since cross-linking lysines slowed the photocycle. 2,3-Butanedione modification of arginines supported this result, since this modification caused a marked slowdown of the photocycle and also protein conformational changes as evidenced by tryptophan fluorescence and circular dichroism spectroscopy.