How to Prepare Membrane Proteins for Solid- State NMR: A Case Study on the a-Helical Integral Membrane Protein Diacylglycerol Kinase from E. coli Mark Lorch, [a] Salem Fahem, [b] Christoph Kaiser, [a] Ingrid Weber, [a] A. James Mason, [a, c] James U. Bowie, [b] and Clemens Glaubitz* [a] Introduction The determination of membrane-protein structures is of undisputed importance. Despite the fact that genes coding for membrane proteins are estimated to represent ~ 30% of genomes, [6] these proteins make up a tiny proportion of the elucidated struc- tures (~ 0.5%). [7,8] X-ray crystallography is by far the most successful technique for determining 3D struc- tures of membrane proteins. However, the produc- tion of diffracting crystals of membrane proteins often involves decades of accumulated effort. Of the 148 available high-resolution structures of multitopic membrane proteins, only three have been elucidated by NMR and five by electron diffraction; the rest have been derived from X-ray-crystallographic stud- ies. [8] Solid-state NMR (SSNMR) has already shown that it is capa- ble of yielding structural information on insoluble peptides and proteins. The possibility of a complete de novo structure determination purely based on MAS-NMR recoupling tech- niques was first demonstrated for small insoluble peptides. [9,10] The techniques have been extended to soluble proteins that have been studied in the solid-state. [11] A great advantage that SSNMR has over other means of determining structures for membrane proteins is that it can be used to study proteins in a plethora of states. Diffraction methods are limited to study- ing 2D or 3D crystals and solubilised proteins must be used in solution NMR (with detergent or organic solvents) in poor membrane-mimetic environments. SSNMR can utilise all of the afore-mentioned states (frozen in the case of detergent and or- ganic-solvent-solubilised proteins) along with proteins that are reconstituted in lipid bilayers or even aggregated or as fibrils (Figure 1). The ability to study proteins embedded within a native-like lipid environment offers further advantages since it is possible to apply an activity assay directly to the sample being measured. This can prove that the protein of interest is in a native conformation. Furthermore, lipid reconstituted sam- Figure1. Methods of membrane protein sample preparation and the high resolution structural techniques that can be used to study them. Detergent-solubilised samples must be frozen if they are to be studied with SSNMR. Several studies have demonstrated that it is viable to use micro- crystalline preparations of water-soluble proteins as samples in solid-state NMR experiments. [1–5] Here, we investigate whether this approach holds any potential for studying water-insoluble systems, namely membrane proteins. For this case study, we have prepared proteoliposomes and small crystals of the a-helical membrane-protein diacylglycerol kinase (DGK). Preparations were characterised by 13 C- and 15 N-cross-polarization magic-angle spinning (CPMAS) NMR. It was found that crystalline samples produce better-resolved spectra than proteoliposomes. This makes them more suitable for structural NMR experiments. How- ever, reconstitution is the method of choice for biophysical stud- ies by solid-state NMR. In addition, we discuss the identification of lipids bound to membrane-protein crystals by 31 P-MAS NMR. [a] Dr. M. Lorch, C. Kaiser, I. Weber, Dr. A. J. Mason, Prof. C. Glaubitz Centre for Biomolecular Magnetic Resonance and Institut für Biophysikalische Chemie, J. W. Goethe Universität Marie-Curie-Straße 9, 60439 Frankfurt (Germany) Fax: (+ 49) 69798-29929 E-mail: glaubitz@em.uni-frankfurt.de [b] Dr. S. Fahem, Prof. J. U. Bowie MBI, University of California at Los Angeles 611 Charles E. Young Drive E., Los Angeles California 90095-1570 (USA) [c] Dr. A. J. Mason Current address: Institut ISIS, UniversitØ Louis Pasteur 8 rue Gaspard Monge, 67000 Strasbourg (France) ChemBioChem 2005, 6, 1693 – 1700 DOI: 10.1002/cbic.200500054 # 2005 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim 1693