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

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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 15N–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]–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.

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Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (DO545/7-1 and SFB 807), NIH (U54 GM094608) and the Cluster of Excellence Frankfurt (Macromolecular Complexes).

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Authors and Affiliations

  1. Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe University, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany

    Frank Löhr, Sina Reckel, Mikhail Karbyshev, Frank Bernhard & Volker Dötsch

  2. Vertex Pharmaceuticals Inc., Cambridge, MA, 02139, USA

    Peter J. Connolly, Norzehan Abdul-Manan & Jonathan M. Moore

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  1. Frank Löhr
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  2. Sina Reckel
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  3. Mikhail Karbyshev
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Correspondence to Volker Dötsch.

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Löhr, F., Reckel, S., Karbyshev, M. et al. Combinatorial triple-selective labeling as a tool to assist membrane protein backbone resonance assignment. J Biomol NMR 52, 197–210 (2012). https://doi.org/10.1007/s10858-012-9601-1

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