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 compounds, as well as in the membrane protein bacteriorhodopsin (bR), regenerated 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. These data provide supporting evidence that the conformation of the retinal chromophore in bR is 6-s-trans. The 6-s-cis conformer is characterized 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 activation energy barrier of 5 +/- 1 kJ/mol. In bR, it is possible to obtain an approximate activation energy of 9 kJ/mol. This data is inconsistent with a 6-s-cis Conformer but is consistent with the existence of a 6-s-trans-retinal Schiff base in bR with some interaction with the protein matrix. These results suggest that methyl rotor motions can be used to probe the van der Waals contact between a ligand and a protein binding pocket. The 6-s-trans conformer of the [16,17-(CD3)(2)] retinal in frozen hexane exhibits a major kinetic component with an activation energy barrier of 14 +/- 2 kJ/mol. For the [16,17-(CD3)(2)] retinal in bR, line shapes indicate an activation energy of 13 +/- 2 kJ/mol within error of that for the 6-s-trans model compound. Our results illustrate the use of deuterium NMR techniques to probe local group motions in a relatively large membrane protein like bR and they illustrate a novel solution to a structural problem by measuring molecular dynamics of pertinent functional groups.