Studies on pyrylretinal analogues of bacteriorhodopsin

The retinal analogues 3-methyl-5-(1-pyryl)-2E,4E-pentadienal (1) and 3,7-di methyl-9-(1 -pyryl) -2E,JE,6E,8E-non-atetraenal (2), which contain the tetr a aromatic pyryl system, have been synthesized and characterized in order t o examine the effect of the extended ring system on the binding capabilitie s and the function of bacteriorhodopsin (bR), The two bR mutants, E194Q and E204Q, known to have distinct proton-pumping patterns, were also examined so that the effect of the bulky ring system on the proton-pumping mechanism could be studied. Both retinals formed pigments with all three bacterioops ins, and these pigments were found to have absorption maxima in the range 4 98-516 nm, All the analogue pigments showed activity as proton pumps, The p igment formed from wild-type apoprotein bR with 1 (with the shortened polye ne side chain) showed an M intermediate at 400 nm and exhibited fast proton release followed by proton uptake. Extending the polyene side chain to the length identical with retinal, analogue 2 with wild-type apoprotein gave a pigment that shows M and O intermediates at 435 mm and 650 nm, respectivel y, This pigment shows both fast and slow proton release at pH 7, suggesting that the pK(a) of the proton release group (in the M-state) is higher in t his pigment compared to native bR, Hydrogen azide ions were found to accele rate the rise and decay of the O intermediate at neutral pH in pyryl 2 pigm ent. The pigments formed between 2 and E194Q and E204Q showed proton-pumpin g behavior similar to pigments formed with the native retinal, suggesting t hat the size of the chromophore ring does not alter the protein conformatio n at these sites.