STRUCTURE OF THE INTERHELICAL LOOPS AND CARBOXYL-TERMINUS OF BACTERIORHODOPSIN BY X-RAY-DIFFRACTION USING SITE-DIRECTED HEAVY-ATOM LABELING

The positions of single amino acids in the interhelical loop regions a nd the C-terminal tail of bacteriorhodopsin (bR) were investigated by X-ray diffraction using site-directed heavy-atom labeling. Since wild- type bR does not contain any cysteines, appropriate cysteine mutants w ere produced with a unique sulfhydryl group at specific positions. The se sites were then labeled with mercury using the sulfhydryl specific reagent p-chloromercuribenzoate (p-CMB). The cysteine mutants D96A/V10 1C, V130C, A160C, and G231C were derivatized with labeling stoichiomet ries of 0.93 +/- 5%, 0.85 +/- 5%, 0.79 +/- 7%, and 0.77 +/- 8%, respec tively (Hg per bR). No incorporation was observed with wild-type bR un der the same conditions. All mutants and heavy-atom derivatives were f ully active as judged by the kinetics of the photocycle and of the pro ton release and uptake. Moreover, the unit cell dimensions of the two- dimensional P-3 lattice were unchanged by the mutations and the deriva tization. This allowed the position of the mercury atoms, projected on to the plane of the membrane, to be calculated from the intensity diff erences in the X-ray diffraction pattern between labeled and unlabeled samples using Fourier difference methods. The X-ray diffraction data were collected at room temperature from oriented purple membrane films at 100% relative humidity without the use of dehydrating solvents. Th ese native conditions of temperature, humidity, and solvent are expect ed to preserve the structure of the surface-exposed loops. Sharp maxim a corresponding to a single mercury atom were found in the difference density maps for D96A/V101C and V130C. Residues 101 and 130 are in the short loops connecting helices C/D and D/E, respectively. No localize d difference density was found for A160C and G231C. Residue 160 is in the longer loop connecting helices E and F, whereas residue 231 is in the C-terminal tail. Residues 160 and 231 are apparently in a more dis ordered and mobile part of the structure.