CHROMOPHORE ORIENTATION IN BACTERIORHODOPSIN DETERMINED FROM THE ANGULAR-DEPENDENCE OF DEUTERIUM NUCLEAR-MAGNETIC-RESONANCE SPECTRA OF ORIENTED PURPLE MEMBRANES

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.