Solution-State NMR Spectroscopy of Membrane Proteins in Detergent Micelles: Structure of the Klebsiella pneumoniae Outer
Structure determination of integral membrane proteins is one of the most important challenges of structural biology. Over the last 7 years, solution-state NMR spectroscopy has become an increasingly useful approach for 3D structure determination and dynam
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1. Introduction Solution-state NMR spectroscopy is becoming an increasingly useful approach for structural and dynamical analysis of large molecular complexes, such as membrane proteins (MPs) solubilized in detergent micelles (1). Such complexes with large apparent molecular weight (i.e. typically above 50 kDa) exhibit slow molecular tumbling, resulting in fast transverse relaxation mechanisms, leading to increased linewidth and reduced sensitivity compared to spectra of smaller protein routinely solved by solution-state NMR
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_17, © Springer Science+Business Media, LLC 2010
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spectroscopy. To minimize the deleterious effects of nuclear spin relaxation on solution NMR spectra, the use of the so-called transverse relaxation-optimized spectroscopy (TROSY) (2) at very high field combined with a high level of protein deuteration (>80% 2H) provide large gains in both sensitivity and resolution (3). Using this approach, complete or nearly complete backbone resonance assignments of integral membrane proteins (up to 280 residues) were obtained, leading to the determination of several b-barrel global folds over the last decade (4–8). To circumvent the limited amount of long range 1H–1H NOEs in perdeuterated proteins, the development of efficient tools for obtaining additional conformational restraints such as restraints obtained from residual dipolar couplings (9, 10), paramagnetic relaxation enhancements experiments (11), or selective methyl protonated otherwise perdeuterated samples (12, 13), were reported for the structure refinement of membrane proteins (14–18). These approaches are particularly useful in the case of helical membrane proteins, which exhibit only few long-range NOEs, which are crucial for the global fold determination (15, 19, 20). This chapter highlights the techniques and experimental protocols used for the investigation of the molecular structure of the 216-residue transmembrane domain of the Outer Membrane Protein A from Klebsiella pneumoniae (KpOmpA) in detergent micelles by liquid-state NMR spectroscopy (21). Among the complete set of state-of-the-art NMR methods for backbone assignment of large protein, we performed a 3D 13C-TOCSY-(15N,1H)-TROSY (22) for aliphatic carbon side chain assignment, a 4D 15N,15N separated NOESY (23) for unambiguous assignment of HN–HN NOEs, and selective methyl protonation approaches for the measurement of distance constraints involving methyl groups. Because structure determination of membrane protein placed strong constraints on the expression system efficiency (with the requirement of tens of milligram amount of pure MPs labelled with three low abundant isotopes) and on protein stability (in the presence of highly concentrated detergent molecules), the overexpression of KpOmpA in E. coli inclusion bodies with different 2 H,13C,15N isotope labelling patterns, its in vitro refolding in
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