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Resonance assignments of a membrane protein in phospholipid bilayers by combining multiple strategies of oriented sample solid-state NMR

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Abstract

Oriented sample solid-state NMR spectroscopy can be used to determine the three-dimensional structures of membrane proteins in magnetically or mechanically aligned lipid bilayers. The bottleneck for applying this technique to larger and more challenging proteins is making resonance assignments, which is conventionally accomplished through the preparation of multiple selectively isotopically labeled samples and performing an analysis of residues in regular secondary structure based on Polarity Index Slant Angle (PISA) Wheels and Dipolar Waves. Here we report the complete resonance assignment of the full-length mercury transporter, MerF, an 81-residue protein, which is challenging because of overlapping PISA Wheel patterns from its two trans-membrane helices, by using a combination of solid-state NMR techniques that improve the spectral resolution and provide correlations between residues and resonances. These techniques include experiments that take advantage of the improved resolution of the MSHOT4-Pi4/Pi pulse sequence; the transfer of resonance assignments through frequency alignment of heteronuclear dipolar couplings, or through dipolar coupling correlated isotropic chemical shift analysis; 15N/15N dilute spin exchange experiments; and the use of the proton-evolved local field experiment with isotropic shift analysis to assign the irregular terminal and loop regions of the protein, which is the major “blind spot” of the PISA Wheel/Dipolar Wave method.

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Acknowledgments

We thank Chris Grant and Albert Wu for assistance with instrumentation. The research was supported by Grants RO1GM099986 and P41EB002031 from the National Institutes of Health. It utilized the Biomedical Technology Resource for NMR Molecular Imaging of Proteins at the University of California, San Diego.

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  1. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093-0307, USA

    George J. Lu & Stanley J. Opella

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  1. George J. Lu
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  2. Stanley J. Opella
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Correspondence to Stanley J. Opella.

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Lu, G.J., Opella, S.J. Resonance assignments of a membrane protein in phospholipid bilayers by combining multiple strategies of oriented sample solid-state NMR. J Biomol NMR 58, 69–81 (2014). https://doi.org/10.1007/s10858-013-9806-y

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