Conformation of a Seven‐Helical Transmembrane Photosensor in the Lipid Environment

Abstract

Solid-state NMR spectroscopy (SSNMR) has emerged as one of the main tools for structural and dynamic investigation of membrane proteins in their native-like lipid environment, and has already provided a wealth of information on a number of biologically and medically important systems. [1][2][3][4][5][6][7][8] Applications to large helical proteins are underway and promise to add to our understanding of membrane biology. [9][10][11][12][13] Herein we present a magic-angle spinning [14] (MAS) SSNMR study of a seven-helical membrane photoreceptor, sensory rhodopsin from Anabaena sp. PCC 7120 (ASR). [15] We report the assignment of backbone and side-chain signals of the protein, analysis of its secondary structure, and analysis of the environment of many polar residues. We use sitespecific H/D exchange measurements to determine the wateraccessible surface of the protein and its topology within the lipid bilayer. Although the secondary structure of ASR derived from our data is overall consistent with that previously determined by X-ray crystallography, [16] we have identified a number of important differences and additions, which allowed us to build a refined structural model.We employed 3D chemical shift correlation spectroscopy performed on a single lipid-reconstituted uniformly 13 C, 15 Nlabeled sample. From a structural perspective, ASR shares its seven-helical architecture with G-protein-coupled receptors. Our studies demonstrate that a similar methodology can in principle be applied to this class of proteins without the need for crystallization and/or detergent solubilization. Importantly, the structural information obtained from SSNMR pertains to a protein in the lipid environment, closely related to its native state. ASR reconstituted in lipids gives wellresolved spectra with high signal-to-noise ratios, with typical carbon and nitrogen line widths of 0.5 ppm (Figure 1 and Figure S1 in the Supporting Information). The protein is functional and stable in this environment. [17] Spectroscopic assignments were obtained from five 3D chemical shift correlation experiments acquired on a single sample: CONCA, two NCACX experiments with dipoleassisted rotamer resonance (DARR) [18] mixing times of 20 ms and 50 ms, and two NCOCX experiments with DARR mixing times of 50 ms and 100 ms. While the CONCA spectrum provides nearly complete backbone resolution and establishes inter-residue correlations between CO[i], N[i+1], and CA-[i+1] atoms, the NCACX and NCOCX experiments allow to record chemical shifts of the side-chain carbon atoms for identification of the amino acid type. Shorter mixing time experiments provide mostly one-and two-bond correlations, for example, N[i+1]-CO[i]-CA[i] and N[i]-CA[i]-CO[i]/CB[i] in NCOCX and NCACX experiments, while longer mixing times establish shifts of the entire carbon side chain, and provide additional inter-residue correlations for assignment validation.The three types of spectra can be coanalyzed to construct a sequential backbone walk (Figure 2 and Figure S2). Back- Figure 1. 2D DARR 1...

Novel expression and characterization of a light driven proton pump archaerhodopsin 4 in a Halobacterium salinarum strain

“…6C is a 13 C-15 N DCP correlation experiment on [ 13 C 9 , 15 N]-Leu labeled native AR4 (black) and recombinant AR4 (red). The chemical shifts of the backbone 13 CO, 13 CA and 15 N for the recombinant AR4 are all in the ranges typical for a well formed α-helical structure, and in a good agreement with chemical shifts of Leu residues in BR and other retinal proteins by both solution NMR and solid-state NMR [63][64][65][66]. Spinning sidebands from the CO peaks in all the 13 C-13 C correlation experiments of AR4 clearly indicate that AR4 has a relative rigid transmembrane domain than that of recombinant AR4 which may attribute to the effect of bacterioruberin on the native AR4, as shown by the projections for both t 1 and t 2 dimensions to the 2D spectra in Fig.…”

Properties of the DREAM scheme and its optimization for application to proteins

“…The resonance assignment of proteins with more than 200 residues is now feasible using uniformly labeled samples, with the 33 kDa C-terminal domain of the yeast prion Ure2p (285 residues) presently being the largest system with extensive de novo assignments in the solid state . Other large systems with extensive resonance assignments include HET-s(1-227) (Schuetz et al 2010) and DsbA (223 residues) (Sperling et al 2010;Shi et al 2010). An important limitation is the need to acquire data showing a sufficiently high signal-to-noise ratio of the relevant cross peaks for each of the 3D spectra required for the assignment process (Schuetz et al 2010;Habenstein et al 2011).…”

Recent Solid-State NMR Studies of Membrane-Bound Peptides and Proteins

“…Well-resolved correlated peaks with high signal-to-noise ratios (S/N) and narrow line widths of 0.5 ppm were observed in 2D DARR 13 C-13 C correlated spectra of uniformly labeled ASR (Fig. 10) [185], indicating that the ASR sample was stable and characterized by excellent structural homogeneity in the membranes. 15 N CP-MAS analysis indicated an all-trans configuration for retinal formed all-trans configuration.…”

“…Although the X-ray structure of ASR [186] lacks the BC loop region, there are two short beta strands. Around 90% of the backbone and amino acid side chain could be assigned in experiments involving sparsely labeled ASR [171,185,187]. The water-accessible surface and transmembrane domain of the protein as well as light-induced conformational changes were characterized through H/D exchange experiments.…”