Direct Determination of a Molecular Torsional Angle in the Membrane Protein Rhodopsin by Solid-State NMR

Abstract

A solid-state NMR method (double-quantum heteronuclear local field NMR) is applied to a 13C2 labeled sample of the 41 kD integral membrane protein rhodopsin. The technique operates under magic-angle-spinning conditions, with good sensitivity and resolution, and allows a direct determination of molecular torsional angles, without estimating intemuclear distances. In rhodopsin, we determine the H-C1CHC11-H torsional angle of the retinylidene chromophore to be 160 dt 10°, indicating a significant deviation from the planar 10-11-s-trans conformation. Double-quantum heteronuclear local field NMR is shown to be a feasible method for the accurate determination of local molecular conformation in large molecular systems which are unsuitable for crystallography.i

Computational Studies of the Primary Phototransduction Event in Visual Rhodopsin

“…[33][34][35] In contrast, however, resonance Raman data 25,36,37 and recent double-quantum solid-state NMR experiments 32 converge toward single-bond character for C14-C15, suggesting that the polaronic charge defect might hop over the vicinity of the Schiff base, settling in the middle of the polyene chain next to C12. 32 An objective of recent DFT QM/MM studies has been to resolve such a contradictory picture by providing a detailed first principles description of the electronic density of the chromophore as influenced by the protein environment.…”

1 H and 13 C MAS NMR evidence for pronounced ligand–protein interactions involving the ionone ring of the retinylidene chromophore in rhodopsin

“…The 11-cis-retinal was obtained by a standard illumination and HPLC purification procedure (9). All subsequent manipulations with the purified 11-cis isomer and rhodopsin were performed in the dark or under dim red light conditions with Ͼ 620 nm.…”

“…Solid-state 13 C magic angle spinning (MAS) NMR spectroscopy has been used in the past to resolve essential details of the spatial and electronic structure of the chromophore. These studies have focused on the retinylidene chain by using 13 C-labeled retinals and have assigned the chemical shifts for the polyene carbon atoms (6)(7)(8)(9)(10)(11)(12).…”

Symmetry‐Based Pulse Sequences in Magic‐Angle Spinning Solid‐State NMR

“…The evolution of the 13 C 2 double-quantum coherences in the presence of local fields from the neighboring protons allowed determination of a H-C-C-H torsional angle in the retinylidene chromophore. 43 A conformational change could be detected in a trapped photointermediate. 44 The same experiment has also been demonstrated on carbohydrates.…”

“…These methods were applied to a variety of problems, including the measurement of molecular torsional angles in a membrane protein. 43,44 As well as being practically successful, the theory behind C7 was very powerful, and could be used for a variety of problems. A further extension of the symmetry theory led to a pallette of symmetries suitable for γ -encoded doublequantum recoupling, including methods which are applicable at high spinning frequencies.…”