ORIENTATION OF THE RETINAL 9-METHYL GROUP IN BACTERIORHODOPSIN AS STUDIED BY PHOTOAFFINITY-LABELING

High-resolution electron cryomicroscopy has clarified the tertiary str ucture of the proton-pumping bacteriorhodopsin (BR) at a resolution of 3.5 Angstrom in the direction parallel to the membrane. However, due to the lower resolution in the perpendicular direction, it could not b e determined whether the retinal side-chain methyls at C-9 and C-13 we re directed toward the extracellular surface or the cytoplasmic side o f the membrane. The direction of the Me groups is of fundamental impor tance because it is directly related to the mechanisms of proton pumpi ng. Our past bioorganic studies with C-9 substituents containing cc-su lfate groups suggested that the methyls are directed to the exterior. In contrast, measurements of linear dichroism, neutron diffraction and solid-state deuterium NMR have concluded the opposite. In order to se cure further evidence, two tritiated analogs of retinal (5 and 6), eac h with a photolabile phenyl azide moiety appended through a 13-Angstro m spacer arm, one pointing in the same direction and the other pointin g opposite to the polyene methyl groups, were synthesized and incorpor ated into the apoprotein to reconstitute functional bacteriorhodopsin analogs. The cross-linked residues labeled by analog 5, in which the p hotoactive group is directed opposite to the methyl groups, were ident ified as Arg-175 and Asn-176 by Edman degradation of cyanogen bromide- cleaved peptides. These two amino acids are located on the cytoplasmic side of alpha-helix F, indicating that the 9-Me in this photolabile a nalog points toward the extracellular space. These studies also reveal ed that the chromophore plane is closely perpendicular to the plane of the membrane and that the beta-ionone ring of retinal is tilted towar d the extracellular space at a 27 degrees angle relative to the plane of the membrane. Photoaffinity studies with analog 6 did not give conc lusive results.