Seven-helical membrane proteins represent a challenge for structural biology. Here, we report the first NMR structure determination of a detergent-solubilized seven-helical transmembrane (7TM) protein, the phototaxis receptor sensory rhodopsin II (pSRII) from Natronomonas pharaonis, as a proof of principle. The overall quality of the structure ensemble is extremely good (backbone root mean squared deviation of 0.48 Å) and agrees well with previously determined X-ray structures. Furthermore, measurements in more native-like small phospholipid bicelles indicate that the protein structure is the same as in detergent micelles, suggesting that environment specific effects are minimal when using mild detergents. We use our case study as a platform to discuss the feasibility of similar solution NMR studies for other 7TM proteins including members of the family of G protein-coupled receptors (GPCRs).
Solution-state NMR spectroscopy is rapidly winning importance in the study of the structure and dynamics of integral membrane proteins (IMPs). The majority of investigations concentrate on the more amenable b-barrel-type proteins. [1,2] The larger group of a-helical IMPs is more challenging to study and only a few structures of proteins with up to three helices have been determined by solution-state NMR spectroscopy, including several oligomeric structures. [3][4][5][6][7][8][9][10][11][12] We have characterized a multispan seven-helical membrane protein receptor, sensory rhodopsin pSRII from Natronomonas pharaonis, using high-resolution solution NMR methods. We report the near-complete backbone assignment (> 98 %) of the 241-residue chain, secondary structure determination, and analysis of the backbone dynamics. Structurally, pSRII can be considered as a G protein-coupled receptor (GPCR) analogue, with the same seven-helical transmembrane architecture. The structure of pSRII has previously been solved by X-ray diffraction [13,14] and more recently this protein was also studied by solid-state NMR spectroscopy. [15] In the latter study, problems with signal overlap were partly alleviated through reverse labeling of the abundant amino acids, allowing assignment of 40 % of the primary sequence.In contrast, our approach is based on solution-state NMR spectroscopy and employs uniform 13 C, 15 N-labeling in combination with a high level of side-chain deuteration (> 95 %) to improve resolution and sensitivity. The choice of solubilizing detergent is crucial for NMR studies in solution: the native structural integrity of the protein must be preserved in the protein-detergent complex and the increase in molecular weight upon complex formation should be minimal, [16] so that adequate sensitivity is maintained to allow application of transverse relaxation-optimized spectroscopy (TROSY). [17]
The biochemical processes of living cells involve a numerous series of reactions that work with exceptional specificity and efficiency. The tight control of this intricate reaction network stems from the architecture of the proteins that drive the chemical reactions and mediate protein-protein interactions. Indeed, the structure of these proteins will determine both their function and interaction partners. A detailed understanding of the proximity and orientation of pivotal functional groups can reveal the molecular mechanistic basis for the activity of a protein. Together with X-ray crystallography and electron microscopy, NMR spectroscopy plays an important role in solving three-dimensional structures of proteins at atomic resolution. In the challenging field of membrane proteins, retinal-binding proteins are often employed as model systems and prototypes to develop biophysical techniques for the study of structural and functional mechanistic aspects. The recent determination of two 3D structures of seven-helical trans-membrane retinal proteins by solution-state NMR spectroscopy highlights the potential of solution NMR techniques in contributing to our understanding of membrane proteins. This review summarizes the multiple strategies available for expression of isotopically labeled membrane proteins. Different environments for mimicking lipid bilayers will be presented, along with the most important NMR methods and labeling schemes used to generate high-quality NMR spectra. The article concludes with an overview of types of conformational restraints used for generation of high-resolution structures of membrane proteins. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.
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