Chromophore Orientation In Bacteriorhodopsin Determined From The Angular Dependence Of Deuterium Nuclear Magnetic Resonance Spectra Of Oriented Purple Membranes
BIOCHEMISTRY(1998)
Abstract
The orientation of prosthetic groups in membrane proteins is of considerable importance in understanding their functional role in energy conversion, signal transduction, and ion transport. In this work, the orientation of the retinylidene chromophore of bacteriorhodopsin (bR) was investigated using H-2 NMR spectroscopy. Bacteriorhodopsin was regenerated with all-trans-retinal stereospecifically deuterated in one of the geminal methyl groups on C-1 of the cyclohexene ring. A highly oriented sample, which is needed to obtain individual bond orientations from H-2 NMR, was prepared by forming hydrated lamellar films of purple membranes on glass slides. A Monte Carlo method was developed to accurately 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 independent measurements of the intrinsic line width (1.6 kHz from T-2e experiments) and the effective quadrupolar coupling constant (38.8-39.8 kHz from analysis of the line shape of a powder-type sample). The angle between the C-1-(1R)-1-CD3 bond and the purple membrane normal was determined with high accuracy from the simultaneous analysis of a series of H-2 NMR spectra recorded at different inclinations of the uniaxially oriented 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 the previously determined angle of the C-5-CD3 bond, to calculate the possible orientations of the cyclohexene ring in the membrane. The solutions obtained from H-2 NMR were then combined with additional constraints from linear dichroism and electron cryomicroscopy to obtain the allowed orientations of retinal in the noncentrosymmetric membrane structure. The combined data indicate that the methyl groups on the polyene chain point toward the cytoplasmic side of the membrane and the N-H bond of the Schiff base to the extracellular side, i.e., toward the side of proton release in the pump pathway.
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Key words
membrane protein,ion transport,nmr spectroscopy,monte carlo method,electron cryomicroscopy,energy conversion,nuclear magnetic resonance,signal transduction,magnetic field,coupling constant,linear dichroism
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