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Identifying Anisotropic Constraints in Multiply Labeled Bacteriorhodopsin by ¹⁵N MAOSS NMR: A General Approach to Structural Studies of Membrane Proteins

A. James Mason ^* , Stephan L. Grage ^*, Suzana K. Straus ^*, Clemens Glaubitz  and Anthony Watts ^*

^* Oxford University Biomembrane Structure Unit, Department of Biochemistry, Oxford OX1 3QU, United Kingdom; and Centre for Biomolecular Magnetic Resonance and Institut für Biophysikalische Chemie, J. W. Goethe Universität, D-60439 Frankfurt, Germany

Correspondence: Address reprint requests to Anthony Watts, Oxford University Biomembrane Structure Unit, Dept. of Biochemistry, South Parks Rd., Oxford OX1 3QU, UK. Tel: +44-1865-275268; Fax: +44-1865-275234; E-mail: awatts{at}bioch.ox.ac.uk.

Structural models of membrane proteins can be refined with sets of multiple orientation constraints derived from structural NMR studies of specifically labeled amino acids. The magic angle oriented sample spinning (MAOSS) NMR approach was used to determine a set of orientational constraints in bacteriorhodopsin (bR) in the purple membrane (PM). This method combines the benefits of magic angle spinning (MAS), i.e., improved sensitivity and resolution, with the ability to measure the orientation of anisotropic interactions, which provide important structural information. The nine methionine residues in bacteriorhodopsin were isotopically ¹⁵N labeled and spectra simplified by deuterium exchange before cross-polarization magic angle spinning (CPMAS) experiments. The orientation of the principal axes of the ¹⁵N chemical shift anisotropy (CSA) tensors was determined with respect to the membrane normal for five of six residual resonances by analysis of relative spinning sideband intensities. The applicability of this approach to large proteins embedded in a membrane environment is discussed in light of these results.

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