The angles between the C-1-, C-5-, and C-9-methyl bonds of the retinylidene chromophore and the membrane normal increase in the M intermediate of bacteriorhodopsin: Direct determination with solid-state H-2 NMR

The orientations of three methyl bonds of the retinylidene chromophore of b acteriorhodopsin were investigated in the M photointermediate using deuteri um solid-state NMR (H-2 NMR), In this key intermediate, the chromophore has a 13-cis, 15-anti conformation and a deprotonated Schiff base. Purple memb ranes containing wild-type or mutant D96A bacteriorhodopsin were regenerate d with retinals specifically deuterated in the methyl groups of either carb on C1 or Cs of the beta-ionone ring or carbon Cs of the polyene chain. Orie nted 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 p erformed 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) a t -50 degrees C. Bacteriorhodopsin was first completely converted to M unde r steady illumination of the hydrated films at +5 OC and then rapidly coole d to -50 OC in the dark. From a tilt series of the oriented sample in the m agnetic 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 eve n 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 upwa rd movement of the C-5-C-13 part of the polyene chain toward the cytoplasmi c surface or with increased torsional strain. The C-9-CD3 bond shows the la rgest orientational change of 7 degrees in M. This reorientation of the chr omophore in the binding pocket provides direct structural support for previ ous suggestions (based on spectroscopic evidence) for a steric interaction in M between the Cs-methyl group and Trp 182 in helix F.