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 2H NMR spectroscopy. Bacteriorhodopsin was regenerated with all-trans-retinal stereospecifically deuterated in one of the geminal methyl groups on C1 of the cyclohexene ring. A highly oriented sample, which is needed to obtain individual bond orientations from 2H NMR, was prepared by forming hydrated lamellar films of purple membranes on glass slides. A Monte Carlo method was developed to accurately simulate the 2H 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 T2e 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 C1-(1R)-1-CD3 bond and the purple membrane normal was determined with high accuracy from the simultaneous analysis of a series of 2H 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 C5-CD3 bond, to calculate the possible orientations of the cyclohexene ring in the membrane. The solutions obtained from 2H 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.

The problem of simulating the spectral line shapes of aligned immobile samples arises in solid-state NMR of various biological systems, including integral membrane proteins and peptides, receptor-bound ligands, and macroscopically oriented DNA fibers. An important issue with regard to the extraction of structural information is the correct treatment of the distribution of local symmetry axes relative to the average alignment axis (mosaic spread). Previous formulations have not considered explicitly the three-dimensional uniaxial character of the local axis disorder. Rather, the mosaic spread has been treated simply by convoluting the theoretical line shape function with an effectively two-dimensional distribution of the local symmetry axes. Here a closed-form line shape expression is derived for an axially symmetric distribution of bond orientations, which includes the uniaxial distribution of the local symmetry axis about the average alignment axis. As an illustration, the influences of the bond orientation and the degree of mosaic spread on deuterium (2H) NMR line shapes are investigated. The closed-form solution in terms of elliptic integrals gives virtually identical results to those of an alternative numerical Monte Carlo line shape simulation method. The derived line shape function yields the correct powder-type limit, and has been tested by simulating a tilt series of 2H NMR spectra of purple membranes containing bacteriorhodopsin with a specifically deuterated 1R methyl group in the retinal ring. The probability distribution for the bond orientations derived herein can be of potential interest for solid-state NMR spectroscopy of aligned biomolecules involving dipolar, quadrupolar, and chemical shift interactions, such as integral membrane proteins and peptides.

Structural restraints from residual tensorial couplings in high resolution NMR are usually incorporated into molecular structure calculation programs by an energy penalty function which depends on the knowledge of the alignment tensor. Here, we show that the alignment tensor enters in linear form into such a function. Therefore, the explicit appearance of the alignment tensor can be eliminated from the penalty function. This avoids the necessity of a determination of magnitude and rhombicity of the alignment tensor in the absence of structural information. The price for this procedure is a slightly shallower energy landscape. Simulations in the vicinity of the energy minimum for the backbone of human ubiquitin show that the reduction in curvature is on the order of a few percent.

The orientations of three methyl bonds of the retinylidene chromophore of bacteriorhodopsin were investigated in the M photointermediate using deuterium solid-state NMR ((2)H NMR). In this key intermediate, the chromophore has a 13-cis, 15-anti conformation and a deprotonated Schiff base. Purple membranes containing wild-type or mutant D96A bacteriorhodopsin were regenerated with retinals specifically deuterated in the methyl groups of either carbon C(1) or C(5) of the beta-ionone ring or carbon C(9) of the polyene chain. Oriented hydrated films were formed by drying concentrated suspensions on glass plates at 86% relative humidity. The lifetime of the M state was increased in the wild-type samples by applying a guanidine hydrochloride solution at pH 9.5 and in the D96A sample by raising the pH. (2)H NMR experiments were performed on the dark-adapted ground state (a 2:1 mixture of 13-cis, 15-syn and all-trans, 15-anti chromophores), the cryotrapped light-adapted state (all-trans, 15-anti), and the cryotrapped M intermediate (13-cis, 15-anti) at -50 degrees C. Bacteriorhodopsin was first completely converted to M under steady illumination of the hydrated films at +5 degrees C and then rapidly cooled to -50 degrees C in the dark. From a tilt series of the oriented sample in the magnetic field and an analysis of the (2)H NMR line shapes, the angles between the individual C-CD(3) bonds and the membrane normal could be determined even in the presence of a substantial degree of orientational disorder. While only minor differences were detected between dark- and light-adapted states, all three angles increase in the M state. This is consistent with an upward movement of the C(5)-C(13) part of the polyene chain toward the cytoplasmic surface or with increased torsional strain. The C(9)-CD(3) bond shows the largest orientational change of 7 degrees in M. This reorientation of the chromophore in the binding pocket provides direct structural support for previous suggestions (based on spectroscopic evidence) for a steric interaction in M between the C(9)-methyl group and Trp 182 in helix F.