DEUTERIUM SOLID-STATE NUCLEAR-MAGNETIC-RESONANCE STUDIES OF METHYL-GROUP DYNAMICS IN BACTERIORHODOPSIN AND RETINAL MODEL COMPOUNDS - EVIDENCE FOR A 6-S-TRANS CHROMOPHORE IN THE PROTEIN

Solid-state deuterium NMR spectroscopy is used to examine the dynamic behavior of 18-CD3 methyl groups in microcrystalline 6-s-cis-retinoic acid (triclinic) and 6-s-trans-retinoic acid (monoclinic) model compou nds, as well as in the membrane protein bacteriorhodopsin (bR), regene rated with CD3-labeled retinal. Temperature dependent quadrupolar echo line shapes and T-1 anisotropy measurements were used to characterize activation energies for 3-fold hopping motion of the methyl groups. T hese data provide supporting evidence that the conformation of the ret inal chromophore in bR is 6-s-trans. The 6-s-cis conformer is characte rized by strong eclipsing interactions between the 8-C proton and the 18-C methyl group protons; the 18-CD3 group shows an activation energy barrier for methyl 3-fold hopping of 14.5 +/- 1 kJ/mol. In contrast, the 18-CD3 group in the 6-s-trans isomer shows a considerably lower ac tivation energy barrier of 5 +/- 1 kJ/mol. In bR, it is possible to ob tain an approximate activation energy of 9 kJ/mol. This data is incons istent with a 6-s-cis Conformer but is consistent with the existence o f a 6-s-trans-retinal Schiff base in bR with some interaction with the protein matrix. These results suggest that methyl rotor motions can b e used to probe the van der Waals contact between a ligand and a prote in binding pocket. The 6-s-trans conformer of the [16,17-(CD3)(2)] ret inal in frozen hexane exhibits a major kinetic component with an activ ation energy barrier of 14 +/- 2 kJ/mol. For the [16,17-(CD3)(2)] reti nal in bR, line shapes indicate an activation energy of 13 +/- 2 kJ/mo l within error of that for the 6-s-trans model compound. Our results i llustrate the use of deuterium NMR techniques to probe local group mot ions in a relatively large membrane protein like bR and they illustrat e a novel solution to a structural problem by measuring molecular dyna mics of pertinent functional groups.