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Fluorine-19 labeling of the tryptophan residues in the G protein-coupled receptor NK1R using the 5-fluoroindole precursor in Pichia pastoris expression

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Abstract

In NMR spectroscopy of biomolecular systems, the use of fluorine-19 probes benefits from a clean background and high sensitivity. Therefore, 19F-labeling procedures are of wide-spread interest. Here, we use 5-fluoroindole as a precursor for cost-effective residue-specific introduction of 5-fluorotryptophan (5F-Trp) into G protein-coupled receptors (GPCRs) expressed in Pichia pastoris. The method was successfully implemented with the neurokinin 1 receptor (NK1R). The 19F-NMR spectra of 5F-Trp-labeled NK1R showed one well-separated high field-shifted resonance, which was assigned by mutational studies to the “toggle switch tryptophan”. Residue-selective labeling thus enables site-specific investigations of this functionally important residue. The method described here is inexpensive, requires minimal genetic manipulation and can be expected to be applicable for yeast expression of GPCRs at large.

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Acknowledgements

D.L. acknowledges funding from the National Natural Science Foundation of China (No. 31971153). K.W. is the Cecil H. and Ida M. Green Professor of Structural Biology at Scripps Research. We acknowledge the use of the NMR Core Facilities of the iHuman Institute and help with MS data collection by Dr. Jiakang Chen from the Shanghai Institute for Advanced Immunochemical Studies at ShanghaiTech University.

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Authors and Affiliations

  1. iHuman Institute, ShanghaiTech University, Shanghai, 201210, China

    Benxun Pan, Canyong Guo, Dongsheng Liu & Kurt Wüthrich

  2. School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China

    Benxun Pan, Canyong Guo & Kurt Wüthrich

  3. Department of Integrative Structural and Computational Biology, Scripps Research, La Jolla, CA, 92037, USA

    Kurt Wüthrich

  4. Institute of Molecular Biology and Biophysics, ETH Zürich, Otto-Stern-Weg 5, Zürich, 8093, Switzerland

    Kurt Wüthrich

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  1. Benxun Pan
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Contributions

K.W. and D.L. supervised the project. B.P. carried out protein production and recorded NMR spectra. B.P., C.G., D.L. and K.W. analyzed the data and prepared the manuscript.

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Correspondence to Kurt Wüthrich.

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Pan, B., Guo, C., Liu, D. et al. Fluorine-19 labeling of the tryptophan residues in the G protein-coupled receptor NK1R using the 5-fluoroindole precursor in Pichia pastoris expression. J Biomol NMR (2024). https://doi.org/10.1007/s10858-024-00439-6

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