Topographic studies of spin-labeled bacteriorhodopsin. Evidence for buried carboxyl residues and immobilization of the COOH-terminal tail.
Chemical modification and electron spin resonance techniques were used to study the topography of carboxyl residues in purple membranes. The results showed that buried carboxyl groups are located in hydrophobic protein domains at least 16 A from the membrane surface, and that the carboxyl-terminal tail is partially immobilized. Carboxyl groups on bacteriorhodopsin in purple membranes were covalently spin-labeled with 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl using N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline as a highly specific coupling agent. Spin-labeled bacteriorhodopsin preparations containing an average of 2.1 +/- 0.5 spins/molecule retained photocycling and proton-pumping functions. Accessibility to the paramagnetic broadening agents, Fe(CN)3-6 and Ni2+, revealed a highly mobile surface group quenched at low concentrations of these agents, and a buried, immobilized group whose ESR signal remained at high quencher concentration. Treatment with denaturing agents greatly increased the mobility and quenching of these buried residues. A series of stearic acid spin labels bound to purple membranes was used to define the depth of paramagnetic interactions. Fe(CN)3-6 interactions were limited to surfaces whereas Ni2+ and Cu2+ effects extended into hydrophobic domains. A double modification procedure, which first blocked surface groups, selectively spin-labeled only buried carboxyl group(s) having a strongly immobilized signal. ESR analysis of the isolated carboxyl-terminal tail after trypsin treatment showed it had increased mobility, indicating that it is moderately immobilized in the native structure. These data provide evidence consistent with several models of bacteriorhodopsin tertiary structure which place carboxyls within hydrophobic domains of the protein.
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins; D002338:Carotenoids; D004578:Electron Spin Resonance Spectroscopy
D002338:Carotenoids; D001436:Bacteriorhodopsins