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Solution NMR studies of α-helical membrane proteins are often complicated by severe spectral crowding. In addition, hydrophobic environments like detergent micelles, isotropic bicelles or nanodiscs lead to considerably reduced molecular tumbling rates which translates into line-broadening and low sensitivity. Both difficulties can be addressed by selective isotope labeling methods. In this publication, we propose a combinatorial protocol that utilizes four different classes of labeled amino acids, in which the three backbone heteronuclei (amide nitrogen, α-carbon and carbonyl carbon) are enriched in N or C isotopes individually as well as simultaneously. This results in eight different combinations of dipeptides giving rise to cross peaks in H-N correlated spectra. Their differentiation is achieved by recording a series of HN-detected 2D triple-resonance spectra. The utility of this new scheme is demonstrated with a homodimeric 142-residue membrane protein in DHPC micelles. Restricting the number of selectively labeled samples to three allowed the identification of the amino-acid type for 77 % and provided sequential information for 47 % of its residues. This enabled us to complete the backbone resonance assignment of the uniformly labeled protein merely with the help of a 3D HNCA spectrum, which can be collected with reasonable sensitivity even for relatively large, non-deuterated proteins. [ABSTRACT FROM AUTHOR]

Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N(5)-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyl-tetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na(+) across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS., (Copyright © 2016. Published by Elsevier B.V.)