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

The orientations of three methyl bonds of the retinylidene chromophore of bacteriorhodopsin were investigated in the M photointermediate using deuterium solid-state NMR (²H 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 C₁ or C₅ of the β-ionone ring or carbon C₉ 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. ²H 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 °C. Bacteriorhodopsin was first completely converted to M under steady illumination of the hydrated films at +5 °C and then rapidly cooled to -50 °C in the dark. From a tilt series of the oriented sample in the magnetic field and an analysis of the ²H NMR line shapes, the angles between the individual C-CD₃ 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 C₅-C₁₃ part of the polyene chain toward the cytoplasmic surface or with increased torsional strain. The C₉-CD₃ bond shows the largest orientational change of 7°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 C₉-methyl group and Trp 182 in helix F.