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Abstract

We present a solid-state NMR study of metarhodopsin-1, the pre-discharge intermediate of the photochemical signal transduction cascade of rhodopsin, which is the 41 kDa integral membrane protein that triggers phototransduction in vertebrate rod cells. The H-C10-C11-H torsional angles of the retinylidene chromophore in bovine rhodopsin and metarhodopsin-I were determined simultaneously in the photo-activated membrane-bound state, using double-quantum heteronuclear local field spectroscopy. The torsional angles were estimated to be [phi] = 160+/-10 degrees for rhodopsin and phi = 180+/-25 degrees for metarhodopsin-I. The result is consistent with current models of the photo-induced conformational transitions in the chromophore, in which the 11-Z retinal ground state is twisted, while the later photointermediates have a planar all-E conformation.

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

“…43 A conformational change could be detected in a trapped photointermediate. 44 The same experiment has also been demonstrated on carbohydrates. 77, 78 The experiment may be extended to allow the determination of 15 N-13 C-13 C-15 N torsional angles in peptides and proteins.…”

“…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.…”

Determination of Solid-State NMR Structures of Proteins by Means of Three-Dimensional 15N−13C−13C Dipolar Correlation Spectroscopy and Chemical Shift Analysis

“…FIGURE 2: Schematic representation of R-helix, 3 10 -helix, antiparallel -sheet, and parallel -sheet secondary structure motives. The numbering of the residues (i and j) corresponds to the numbering in Table 1. experiments were performed on a DMX-400 spectrometer (Bruker, Karlsruhe, Germany) operating at a field of 9.…”

“…In the past years, solid-state NMR 1 has become a promising method for obtaining structural information about these systems, via the measurement of accurate distances (1-7), φ and backbone torsion angles (8)(9)(10), and chemical shift anisotropy (11,12). In these studies, samples labeled only in the positions of interest were investigated.…”

“…This can be accomplished by employing a reduced labeling scheme, in which chemically bonded carbons are not simultaneously labeled and hence the number of strong dipolar couplings between connected nuclei is reduced. For proteins expressed in bacterial systems, this can be achieved by using [2- 13 C]glycerol or [1,[3][4][5][6][7][8][9][10][11][12][13] C]glycerol as the only carbon source in the media (15)(16)(17). In combination with this labeling pattern, long-range 13 C- 13 C distance restraints may be collected by using a broad-band recoupling method like the proton-driven spindiffusion (PDSD) mixing scheme (18).…”