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Strategies for acquisition of resonance assignment spectra of highly dynamic membrane proteins: a GPCR case study

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Abstract

In protein nuclear magnetic resonance (NMR), chemical shift assignment provides a wealth of information. However, acquisition of high-quality solid-state NMR spectra depends on protein-specific dynamics. For membrane proteins, bilayer heterogeneity further complicates this observation. Since the efficiency of cross-polarization transfer is strongly entwined with protein dynamics, optimal temperatures for spectral sensitivity and resolution will depend not only on inherent protein dynamics, but temperature-dependent phase properties of the bilayer environment. We acquired 1-, 2-, and 3D homo- and heteronuclear experiments of the chemokine receptor CCR3 in a 7:3 phosphatidylcholine:cholesterol lipid environment. 1D direct polarization, cross polarization (CP), and T2’ experiments indicate sample temperatures below − 25 °C facilitate higher CP enhancement and longer-lived transverse relaxation times. T1rho experiments indicate intermediate timescales are minimized below a sample temperature of − 20 °C. 2D DCP NCA experiments indicated optimal CP efficiency and resolution at a sample temperature of − 30 °C, corroborated by linewidth analysis in 3D NCACX at − 30 °C compared to − 5 °C. This optimal temperature is concluded to be directly related the lipid phase transition, measured to be between − 20 and 15 °C based on rINEPT signal of all-trans and trans-gauche lipid acyl conformations. Our results have critical implications in acquisition of SSNMR membrane protein assignment spectra, as we hypothesize that different lipid compositions with different phase transition properties influence protein dynamics and therefore the optimal acquisition temperature.

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Acknowledgements

We would like to thank NMRbox: National Center for Biomolecular NMR Data Processing and Analysis, a Biomedical Technology Research Resource (BTRR) for use of computational resources in this work, which is supported by NIH grant P41GM111135 (NIGMS).

Funding

This work was funded by NIH Grant R35GM124979 (Maximizing Investigators’ Research Award [MIRA] R35) awarded to Benjamin J. Wylie.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79415, USA

    Evan J. van Aalst, Jun Jang, Ty C. Halligan & Benjamin J. Wylie

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  1. Evan J. van Aalst
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Contributions

Conceptualization, EJvA and BJW; methodology, EJvA and BJW; software, EJvA, JJ, TCH, and BJW; validation, EJvA and BJW; formal analysis, EJvA, JJ, TCH, and BJW; investigation, EJvA and BJW; resources, BJW; data curation, EJvA, JJ, TCH, and BJW; writing—original draft preparation, EJvA; writing—review and editing, EJvA, JJ, TCH and BJW; visualization, EJvA and BJW; supervision, BJW; project administration, BJW; funding acquisition, BJW. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Benjamin J. Wylie.

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van Aalst, E.J., Jang, J., Halligan, T.C. et al. Strategies for acquisition of resonance assignment spectra of highly dynamic membrane proteins: a GPCR case study. J Biomol NMR 77, 191–202 (2023). https://doi.org/10.1007/s10858-023-00421-8

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