Combinatorial triple-selective labeling as a tool to assist membrane protein backbone resonance assignment
 

Combinatorial triple-selective labeling as a tool to assist membrane protein backbone resonance assignment


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

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738

Source: Journal of Biomolecular NMR