The Angles between the C₁−, C₅−, and C₉−Methyl Bonds of the Retinylidene Chromophore and the Membrane Normal Increase in the M Intermediate of Bacteriorhodopsin:  Direct Determination with Solid-State ²H NMR

Abstract

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.

Dynamics and orientation of N + (CD 3 ) 3 -bromoacetylcholine bound to its binding site on the nicotinic acetylcholine receptor

“…These simulations suggest that although the overall lineshape shows a significant dependence the inner and outer wings of the 0°lineshape and the overall width of the 90°spectrum show very little dependence. Such weak correlation between the inner and outer wings of the deuterium spectra of uniaxially aligned samples and the mosaic spread has previously been reported on similar studies of the bacterial proton pump bacteriorhodopsin (44,45). Both of these observations would indicate that the estimate of the angle formed between the quaternary ammonium group and the membrane normal is not significantly compromised by the simulations used.…”

“…The deuterium spectra of the same sample before orientation acquired at a similar temperature indicated that significant motions were still present on the intermediate time scale even at these low temperatures (Ϫ120°C). Alternative simulations are available to calculate the lineshapes of uniaxially oriented samples (44,45); however, these also assume that intermediate motions do not dominate the lineshape. Lineshape simulations that incorporate such dynamic processes have been developed (46).…”

Coupling Between the Retinal Thermal Isomerization and the Glu194 Residue of Bacteriorhodopsin†

“…The retinal orientation in the LA state is essentially described by the roll and tilt angles of the nearly planar all‐trans chromophore (33). Actually, the retinal isomerization from all‐trans to 13‐cis in the DA state, as well as in the M state, keeps the same planar 6‐s trans chain configuration of the chromophore with a decrease in the inclination of the C5‐to‐N axis and a movement of the C5‐to‐C13 axis out of the membrane plane by 4–5° (34). These findings strongly point out that the structural changes should occur mostly at the end of the retinal molecule, near to the Schiff base linkage.…”

Determination of Membrane Peptide Orientation by 1H-Detected 2H NMR Spectroscopy

“…Moreover, the large quadrupolar couplings of N-D and C-D groups (ca. 200-300 kHz) make them sensitive probes of segmental orientation as well as segmental motion (20) (21).…”