CHROMOPHORE ORIENTATION IN BACTERIORHODOPSIN DETERMINED FROM THE ANGULAR-DEPENDENCE OF DEUTERIUM NUCLEAR-MAGNETIC-RESONANCE SPECTRA OF ORIENTED PURPLE MEMBRANES
S. Moltke et al., CHROMOPHORE ORIENTATION IN BACTERIORHODOPSIN DETERMINED FROM THE ANGULAR-DEPENDENCE OF DEUTERIUM NUCLEAR-MAGNETIC-RESONANCE SPECTRA OF ORIENTED PURPLE MEMBRANES, Biochemistry, 37(34), 1998, pp. 11821-11835
The orientation of prosthetic groups in membrane proteins is of consid
erable importance in understanding their functional role in energy con
version, signal transduction, and ion transport. In this work, the ori
entation of the retinylidene chromophore of bacteriorhodopsin (bR) was
investigated using H-2 NMR spectroscopy. Bacteriorhodopsin was regene
rated with all-trans-retinal stereospecifically deuterated in one of t
he geminal methyl groups on C-1 of the cyclohexene ring. A highly orie
nted sample, which is needed to obtain individual bond orientations fr
om H-2 NMR, was prepared by forming hydrated lamellar films of purple
membranes on glass slides. A Monte Carlo method was developed to accur
ately simulate the H-2 NMR line shape due to the distribution of bond
angles and the orientational disorder of the membranes. The number of
free parameters in the line shape simulation was reduced by independen
t measurements of the intrinsic line width (1.6 kHz from T-2e experime
nts) and the effective quadrupolar coupling constant (38.8-39.8 kHz fr
om analysis of the line shape of a powder-type sample). The angle betw
een the C-1-(1R)-1-CD3 bond and the purple membrane normal was determi
ned with high accuracy from the simultaneous analysis of a series of H
-2 NMR spectra recorded at different inclinations of the uniaxially or
iented sample in the magnetic field at 20 and -50 degrees C. The value
of 68.7 +/- 2.0 degrees in dark-adapted bR was used, together with th
e previously determined angle of the C-5-CD3 bond, to calculate the po
ssible orientations of the cyclohexene ring in the membrane. The solut
ions obtained from H-2 NMR were then combined with additional constrai
nts from linear dichroism and electron cryomicroscopy to obtain the al
lowed orientations of retinal in the noncentrosymmetric membrane struc
ture. The combined data indicate that the methyl groups on the polyene
chain point toward the cytoplasmic side of the membrane and the N-H b
ond of the Schiff base to the extracellular side, i.e., toward the sid
e of proton release in the pump pathway.