Determination of Membrane Protein Structures Using Solution and Solid-State NMR
NMR is an essential tool to characterize the structure, dynamics, and interactions of biomolecules at an atomic level. Its application to membrane protein (MP) structure determination is challenging and currently an active and rapidly developing field. Ma
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1. Introduction Both solution and solid-state NMR methods have been used for a long time to gain insights into the structure, dynamics, and interactions of membrane peptides and proteins (MPs) as well as of its main partners within the membrane, i.e., lipids. But, it is only in the past two decades that NMR reveals its Jean-Jacques Lacapère (ed.), Membrane Protein Structure Determination: Methods and Protocols, Methods in Molecular Biology, vol. 654, DOI 10.1007/978-1-60761-762-4_14, © Springer Science+Business Media, LLC 2010
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abilities to determine MPs structures. This chapter focuses on the methodologies currently used for membrane structure determination as well as on the most recent advances in that field. The main advantage of NMR is the variety of environments that can be used: from native membrane environment to various membrane mimetic environments including planar bilayers, bicelles (1), micelles, and organic solvents, at different salt concentration, temperature, and pH values. NMR studies of MPs in their native membrane is only feasible if large amount of proteins (mg) can be purified which has been found for few MPs like bacteriorhodopsin. Among membrane mimetic environments, planar synthetic bilayer and bicelles more closely mimic the membrane environment. Detergent micelles provide good approximations of the interfacial and hydrophobic regions of membrane but do not account for the bidimensional geometry, local curvature, heterogeneity, and dynamics of biological membranes. Organic solvent mixtures are isotropic media that can mimic the hydrophobic region but cannot account for the lipid–water interface. They have been essentially used for the studies of transmembrane (TM) peptides. The main criterion that distinguishes solution versus solidstate NMR is the motional properties of the protein–lipid sample. As a matter of fact, large proteins or macromolecular assemblies (corresponding to MW greater than ~40 kDa) are challenging to study using solution NMR methods due to their slow overall rotational correlation time. Typically, assignment and NMR structure of monomeric membrane protein close to 30 kDa can be achieved. Eighty-five percent signal assignments of the tetrameric KcsA potassium channel in DPC micelles (apparent molecular weight in the range of 130 kDa) have been obtained. On contrast, the protein size is in theory not a limiting factor for solid-state NMR (ssNMR) but in practice, spectral resolution impaired studies of large MPs and specific ssNMR strategies including specific stable-isotope labelling of proteins are currently under development focusing on this limiting factor. Both solution and solid-state NMR methods are currently used for the structural studies of small membrane peptides but are still very challenging for MPs. The main factors responsible for these difficulties are (1) sample preparations including large amount of pure protein in an appropriate membrane mimetic model and (2) signal overlaps due to the amino acid sequences of MPs composed
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