In Situ Structural Studies of Anabaena Sensory Rhodopsin in the E. coli Membrane

Abstract

Magic-angle spinning nuclear magnetic resonance is well suited for the study of membrane proteins in the nativelike lipid environment. However, the natural cellular membrane is invariably more complex than the proteoliposomes most often used for solid-state NMR (SSNMR) studies, and differences may affect the structure and dynamics of the proteins under examination. In this work we use SSNMR and other biochemical and biophysical methods to probe the structure of a seven-transmembrane helical photoreceptor, Anabaena sensory rhodopsin (ASR), prepared in the Escherichia coli inner membrane, and compare it to that in a bilayer formed by DMPC/DMPA lipids. We find that ASR is organized into trimers in both environments but forms two-dimensional crystal lattices of different symmetries. It favors hexagonal packing in liposomes, but may form a square lattice in the E. coli membrane. To examine possible changes in structure site-specifically, we perform two- and three-dimensional SSNMR experiments and analyze the differences in chemical shifts and peak intensities. Overall, this analysis reveals that the structure of ASR is largely conserved in the inner membrane of E. coli, with many of the important structural features of rhodopsins previously observed in ASR in proteoliposomes being preserved. Small, site-specific perturbations in protein structure that occur as a result of the membrane changes indicate that the protein can subtly adapt to its environment without large structural rearrangement.

Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR

“…43,45 Moreover, hexagonal lattices formed by ASR and BR have similar lattice parameters (66.4 Å for ASR and 62.7 Å for BR). 64,67 Although the relative axial orientation of the trimers and the intertrimer interfaces are unknown for ASR, a high similarity to BR is expected due to comparable steric intertrimer constraints. Based on the analysis of the BR template, one can anticipate that C148 would make close intertrimer contacts to helices E and F, and the biradical can therefore induce a strong quenching in the neighboring trimer.…”

Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

“…While there are only a limited number of resolved peaks, a line width of 0.5-1 ppm appears representative for the entire spectrum. This line width is thought to be typical for solid-state NMR spectra of well-ordered membrane proteins in lipidic environment (Ward et al 2015;Brown and Ladizhansky 2015).…”

Isotope Labeling of Eukaryotic Membrane Proteins in Yeast for Solid-State NMR

“…Lipid compositions should be chosen in such a way that the reconstituted protein is stable and retains its native structure and function at high protein-to-lipid ratios needed to achieve good signal-to-noise ratios of ssNMR signals, and yields high spectral resolution. In principle, the higher protein-to-lipid ratios can lead to both better spectral resolution and higher signal amplitudes of ssNMR, which in some cases can be related to formation of 2D crystals, e.g., in the case of Anabaena sensory rhodopsin (Ward, Wang, et al, 2015). On the other hand, for some proteins, such as green proteorhodopsin, having somewhat lower protein-to-lipid ratios not leading to 2D crystallization produces more homogeneous samples with better spectral resolution (Shi et al, 2009;Yang et al, 2011).…”