title: Time-shared experiments for efficient assignment of triple-selectively labeled proteins.
abstract: Combinatorial triple-selective labeling facilitates the NMR assignment process for proteins that are subject to signal overlap and insufficient signal-to-noise in standard triple-resonance experiments. Aiming at maximum amino-acid type and sequence-specific information, the method represents a trade-off between the number of selectively labeled samples that have to be prepared and the number of spectra to be recorded per sample. In order to address the demand of long measurement times, we here propose pulse sequences in which individual phase-shifted transients are stored separately and recombined later to produce several 2D HN(CX) type spectra that are usually acquired sequentially. Sign encoding by the phases of (13)C 90° pulses allows to either select or discriminate against (13)C' or (13)C(α) spins coupled to (15)N. As a result, (1)H-(15)N correlation maps of the various isotopomeric species present in triple-selectively labeled proteins are deconvoluted which in turn reduces problems due to spectral overlap. The new methods are demonstrated with four different membrane proteins with rotational correlation times ranging from 18 to 52 ns.
authors.affiliation: Institute of Biophysical Chemistry & Center for Biomolecular Magnetic Resonance, Goethe University, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Löhr
authors.affiliation: Institute of Biophysical Chemistry & Center for Biomolecular Magnetic Resonance, Goethe University, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany.
authors.forename: Aisha
authors.initials: A
authors.lastname: Laguerre
authors.affiliation: Institute of Biochemistry, Goethe University, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Bock
authors.affiliation: Institute of Biophysical Chemistry & Center for Biomolecular Magnetic Resonance, Goethe University, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany.
authors.forename: Sina
authors.initials: S
authors.lastname: Reckel
authors.affiliation: Vertex Pharmaceuticals Inc., Cambridge, MA 02139, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Connolly
authors.affiliation: Vertex Pharmaceuticals Inc., Cambridge, MA 02139, USA.
authors.forename: Norzehan
authors.initials: N
authors.lastname: Abdul-Manan
authors.affiliation: Institute of Biochemistry, Goethe University, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany.
authors.forename: Franz
authors.initials: F
authors.lastname: Tumulka
authors.affiliation: Institute of Biochemistry, Goethe University, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany.
authors.forename: Rupert
authors.initials: R
authors.lastname: Abele
authors.affiliation: Vertex Pharmaceuticals Inc., Cambridge, MA 02139, USA.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Moore
authors.affiliation: Institute of Biophysical Chemistry & Center for Biomolecular Magnetic Resonance, Goethe University, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany. Electronic address: vdoetsch@em.uni-frankfurt.de.
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012815:Signal Processing, Computer-Assisted; D059629:Signal-To-Noise Ratio; D013113:Spin Labels
chemical_list: D008565:Membrane Proteins; D013113:Spin Labels
keywords: BEST-TROSY; Cell-free expression; Deuteration; Hadamard decoding; Isotope labeling; Membrane proteins; Triple-resonance NMR