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Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly 15N labeled integral membrane proteins in magnetically aligned lipid bilayers

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Abstract

Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring 1H-15N dipolar couplings (DC) and 15N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal (rotational) angles of the protein domains can be directly derived from analytical expression of DC and CSA values, or, alternatively, obtained by refining protein structures using these values as harmonic restraints in simulated annealing calculations. The Achilles’ heel of this approach is the lack of suitable experiments for sequential assignment of the amide resonances. In this Article, we present a new pulse sequence that integrates proton driven spin diffusion (PDSD) with sensitivity-enhanced PISEMA in a 3D experiment ([1H,15N]-SE-PISEMA-PDSD). The incorporation of 2D 15N/15N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the 15N resonances. The feasibility of this approach is demonstrated for the membrane protein sarcolipin reconstituted in magnetically aligned lipid bicelles. Taken with low electric field probe technology, this approach will propel the determination of sequential assignment as well as structure and topology of larger integral membrane proteins in aligned lipid bilayers.

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Abbreviations

SLN:

Sarcolipin

SERCA:

sarcoplasmic reticulum Ca2+-ATPase

DMPC:

1,2-dimyristoyl-sn-glycero-3-phosphocholine

D6PC:

1,2-dihexanoyl-sn-glycero-3-phosphocholine

POPC:

1-palmitoyl,2-oleyl-sn-glycero-3-phosphocholine

SE-PISEMA:

Sensitivity enhanced polarization inversion spin exchange at magic angle

PISA:

Polar index slant angle

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Acknowledgments

The authors would like to thank Kim Ha and Raffaello Verardi for helping with protein purification and sample preparation. This work was supported by NIH to G.V. (GM 64742).

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Author notes

  1. Nathaniel J. Traaseth

    Present address: Chemistry Department, New York University, New York, NY, 10003, USA

Authors and Affiliations

  1. Department of Chemistry, University of Minnesota, Minneapolis, MN, 55455-0431, USA

    Kaustubh R. Mote & Gianluigi Veglia

  2. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 207 Pleasant St. S. E, Minneapolis, MN, 55455, USA

    T. Gopinath, Nathaniel J. Traaseth & Gianluigi Veglia

  3. National High Magnetic Field Laboratory, Tallahassee, FL, 32310, USA

    Jason Kitchen, Peter L. Gor’kov & William W. Brey

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  1. Kaustubh R. Mote
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Correspondence to Gianluigi Veglia.

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Mote, K.R., Gopinath, T., Traaseth, N.J. et al. Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly 15N labeled integral membrane proteins in magnetically aligned lipid bilayers. J Biomol NMR 51, 339–346 (2011). https://doi.org/10.1007/s10858-011-9571-8

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