Abstract Obtaining NMR assignments for slowly tumbling molecules such as detergent-solubilized membrane proteins is often compromised by low sensitivity as well as spectral overlap. Both problems can be addressed by amino-acid specific isotope labeling in conjunction with 15Nâ??1H correlation experiments. In this work an extended combinatorial selective in vitro labeling scheme is proposed that seeks to reduce the number of samples required for assignment. Including three different species of amino acids in each sample, 15N, 1-13C, and fully 13C/15N labeled, permits identification of more amino acid types and sequential pairs than would be possible with previously published combinatorial methods. The new protocol involves recording of up to five 2D triple-resonance experiments to distinguish the various isotopomeric dipeptide species. The pattern of backbone NH cross peaks in this series of spectra adds a new dimension to the combinatorial grid, which otherwise mostly relies on comparison of [15N, 1H]â??HSQC and possibly 2D HN(CO) spectra of samples with different labeled amino acid compositions. Application to two α-helical membrane proteins shows that using no more than three samples information can be accumulated such that backbone assignments can be completed solely based on 3D HNCA/HN(CO)CA experiments. Alternatively, in the case of severe signal overlap in certain regions of the standard suite of triple-resonance spectra acquired on uniformly labeled protein, or missing signals due to a lack of efficiency of 3D experiments, the remaining gaps can be filled.
Content Type Journal Article
Category Article
Pages 1-14
DOI 10.1007/s10858-012-9601-1
Authors
Frank Löhr, Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany
Sina Reckel, Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany
Mikhail Karbyshev, Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany
Peter J. Connolly, Vertex Pharmaceuticals Inc., Cambridge, MA 02139, USA
Norzehan Abdul-Manan, Vertex Pharmaceuticals Inc., Cambridge, MA 02139, USA
Frank Bernhard, Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany
Jonathan M. Moore, Vertex Pharmaceuticals Inc., Cambridge, MA 02139, USA
Volker Dötsch, Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany
Frequency-selective heteronuclear dephasing and selective carbonyl labeling to deconvolute crowded spectra of membrane proteins by magic angle spinning NMR.
Frequency-selective heteronuclear dephasing and selective carbonyl labeling to deconvolute crowded spectra of membrane proteins by magic angle spinning NMR.
Frequency-selective heteronuclear dephasing and selective carbonyl labeling to deconvolute crowded spectra of membrane proteins by magic angle spinning NMR.
J Magn Reson. 2011 Mar 17;
Authors: Traaseth NJ, Veglia G
We present a new method that combines carbonyl-selective labeling with frequency-selective heteronuclear recoupling to resolve the spectral overlap of magic angle spinning (MAS) NMR...
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04-13-2011 11:57 PM
Frequency-Selective Heteronuclear Dephasing and Selective Carbonyl Labeling to Deconvolute Crowded Spectra of Membrane Proteins By Magic Angle Spinning NMR
Frequency-Selective Heteronuclear Dephasing and Selective Carbonyl Labeling to Deconvolute Crowded Spectra of Membrane Proteins By Magic Angle Spinning NMR
Publication year: 2011
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 17 March 2011</br>
Nathaniel J., Traaseth , Gianluigi, Veglia</br>
We present a new method that combines carbonyl-selective labeling with frequency-selective heteronuclear recoupling to resolve the spectral overlap of magic angle spinning (MAS) NMR spectra of membrane proteins in fluid lipid membranes with broad lines and...
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03-18-2011 06:43 AM
Pseudo-4D triple resonance experiments to resolve HN overlap in the backbone assignment of unfolded proteins
Pseudo-4D triple resonance experiments to resolve HN overlap in the backbone assignment of unfolded proteins
Abstract The solution NMR resonance assignment of the protein backbone is most commonly carried out using triple resonance experiments that involve 15N and 1HN resonances. The assignment becomes problematic when there is resonance overlap of 15Nâ??1HN cross peaks. For such residues, one cannot unambiguously link the â??leftâ?? side of the NH root to the â??rightâ?? side, and the residues associated with such overlapping HN resonances remain often unassigned. Here we present a...
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12-31-2010 08:38 PM
Optimization of amino acid type-specific (13)C and (15)N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm.
Optimization of amino acid type-specific (13)C and (15)N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm.
Optimization of amino acid type-specific (13)C and (15)N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm.
J Biomol NMR. 2010 Dec 18;
Authors: Hefke F, Bagaria A, Reckel S, Ullrich SJ, Dötsch V, Glaubitz C, Güntert P
We present a computational method for finding optimal labeling patterns for the backbone...
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12-21-2010 01:00 PM
Optimization of amino acid type-specific 13C and 15N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm
Optimization of amino acid type-specific 13C and 15N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm
Abstract We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273â??6279 (1982)), types of amino acids are labeled with 13C or/and 15N such that cross peaks between 13CO(i â?? 1) and 15NH(i) result only for pairs...
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[NMR paper] A combinatorial selective labeling method for the assignment of backbone amide NMR re
A combinatorial selective labeling method for the assignment of backbone amide NMR resonances.
Related Articles A combinatorial selective labeling method for the assignment of backbone amide NMR resonances.
J Am Chem Soc. 2004 Apr 28;126(16):5020-1
Authors: Parker MJ, Aulton-Jones M, Hounslow AM, Craven CJ
A combinatorial selective labeling (CSL) method is presented for the assignment of backbone amide NMR resonances, which has a particular application in the identification of protein-ligand interaction sites. The method builds on the dual...
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[NMR paper] Selective and extensive 13C labeling of a membrane protein for solid-state NMR invest
Selective and extensive 13C labeling of a membrane protein for solid-state NMR investigations.
Related Articles Selective and extensive 13C labeling of a membrane protein for solid-state NMR investigations.
J Biomol NMR. 1999 May;14(1):71-4
Authors: Hong M, Jakes K
The selective and extensive 13C labeling of mostly hydrophobic amino acid residues in a 25 kDa membrane protein, the colicin Ia channel domain, is reported. The novel 13C labeling approach takes advantage of the amino acid biosynthetic pathways in bacteria and suppresses the...
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[NMR paper] Solid-state NMR triple-resonance backbone assignments in a protein.
Solid-state NMR triple-resonance backbone assignments in a protein.
Related Articles Solid-state NMR triple-resonance backbone assignments in a protein.
J Biomol NMR. 1999 Apr;13(4):337-42
Authors: Tan WM, Gu Z, Zeri AC, Opella SJ
Triple-resonance solid-state NMR spectroscopy is demonstrated to sequentially assign the 13C' and 15N amide backbone resonances of adjacent residues in an oriented protein sample. The observed 13C' chemical shift frequency provides an orientational constraint complementary to those measured from the 1H and 15N amide...
Combinatorial triple-selective labeling as a tool to assist membrane protein backbone resonance assignment
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