Available online at www.sciencedirect.com Recent contributions from solid-state NMR to the understanding of membrane protein structure and function Peter J Judge and Anthony Watts The plasma membrane functions as a semi-permeable barrier, defining the interior (or cytoplasm) of an individual cell. This highly dynamic and complex macromolecular assembly comprises predominantly lipids and proteins held together by entropic forces and provide the interface through which a cell interacts with its immediate environment. The extended sheet- like bilayer structure formed by the phospholipids is a highly adaptable platform whose structure and composition may be tuned to provide specialised functionality. Although a number of biophysical techniques including X-ray crystallography have been used to determine membrane protein structures, these methods are unable to replicate and accommodate the complexity and diversity of natural membranes. Solid state NMR (ssNMR) is a versatile method for structural biology and can be used to provide new insights into the structures of membrane components and their mutual interactions. The extensive variety of sample forms amenable for study by ssNMR, allows data to be collected from proteins in conditions that more faithfully resemble those of native environment, and therefore is much closer to a functional state. Address Biomembrane Structure Unit, Biochemistry Dept., University of Oxford, Oxford, OX1 3QU, UK Corresponding author: Watts, Anthony (anthony.watts@bioch.ox.ac.uk) Current Opinion in Chemical Biology 2011, 15:690–695 This review comes from a themed issue on Analytical Techniques Edited by Morgan Alexander and Ian Glimore Available online 19th August 2011 1367-5931/$ – see front matter Published by Elsevier Ltd. DOI 10.1016/j.cbpa.2011.07.021 Biophysical characterisation of membrane proteins Many biophysical studies of proteins begin with expres- sion in a suitable vector and isolation from other cellular components. Membrane protein purification is compli- cated by the presence of the lipid component of the membrane, which is frequently removed by detergent solubilisation in order to increase the concentration of the desired protein [1,2]. Many membrane proteins require specific lipids to be present in the membrane to be fully active [3  ] and lipid species such as sphingolipids and cholesterol, may form into enriched domains that facilitate short-term, local organisation of parts of a natural membrane [4,5]. Biophysical experiments which require detergent-solubilised and delipidated membrane protein samples, are unable to replicate the complexity of natural membranes and may occasionally produce misleading descriptions of little functional meaning. Around 200 unique membrane protein structures have been obtained by X-ray crystallography (http://blanco.- biomol.uci.edu/Membrane_Proteins_xtal.html) and most are crystallised from detergent-solubilised preparations from which much of the native lipid has been removed [6]; a notable exception is the 7-transmembrane helical photoreceptor, bacteriorhodopsin (bR), which is routinely crystallised directly from the membranes in which it occurs naturally with only minor purification steps. Although tight binding of lipids to proteins is often reported in structural models from X-ray crystallography studies, the electron density corresponding to non- protein molecules is typically ill-defined and the lipid or detergent species may not be unambiguously assign- able [7,8]. Crystallisation of membrane proteins remains a major hurdle for X-ray diffraction studies and many small bioactive peptides are elusive to crystallographic methods. Most X-ray diffraction data are acquired at cryo-temperatures at which the dynamic motions of pep- tides and proteins present under physiological conditions are suppressed and other biophysical approaches must be used to provide this detail. Solid state NMR Solid state NMR is a methodology commonly applied to a range of macromolecular (M r  100 kDa) complexes which can be regarded as solid or solid-like, including membranes and membrane proteins [9]. Samples of this type cannot be studied by solution state NMR methods, as the anisotropic interactions are not averaged by mol- ecular tumbling on the NMR time-scales of <ms, resulting in the broadening of individual resonances. Solid state NMR is uniquely able to exploit the intrinsic anisotropy of macromolecular assemblies, and is broadly able to provide orientation information, distance restraints and torsion constraints to provide structural detail at sub-A ˚ resolution. The same technique is also able to provide information about protein and lipid dynamics, allowing a more complete picture of the beha- viour of a transmembrane protein [10  ]. A key advantage of solid state NMR is the flexibility of sample compo- sitions and lipids may be included to mimic more closely the characteristics of the native membrane in which a given protein would reside. Current Opinion in Chemical Biology 2011, 15:690–695 www.sciencedirect.com