Hz. Zhang et al., ORIENTATION OF THE RETINAL 9-METHYL GROUP IN BACTERIORHODOPSIN AS STUDIED BY PHOTOAFFINITY-LABELING, Journal of the American Chemical Society, 116(15), 1994, pp. 6823-6831
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.