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The GFP thermal shift assay for screening ligand and lipid interactions to solute carrier transporters

Nature Protocols volume 16, pages 5357–5376 (2021)Cite this article

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Abstract

Solute carrier (SLC) transporters represent the second-largest fraction of the membrane proteome after G-protein-coupled receptors, but have been underutilized as drug targets and the function of many members of this family is still unknown. They are technically challenging to work with as they are difficult to express and highly dynamic, making them unstable in detergent solution. Many SLCs lack known inhibitors that could be utilized for stabilization. Furthermore, as they bind their physiological substrates with high micromolar to low millimolar affinities, binding and transport assays have proven to be particularly challenging to implement. Previously, we reported a GFP-based method for the overexpression and purification of membrane proteins in Saccharomyces cerevisiae. Here, we extend this expression platform with the GFP thermal shift (GFP-TS) assay, which is a simplified version of fluorescence-detection size-exclusion chromatography that combines the sample versatility of fluorescence-detection size-exclusion chromatography with the high-throughput capability of dye-based thermal shift assays. We demonstrate how GFP-TS can be used for detecting specific ligand interactions of SLC transporter fusions and measuring their affinities in crude detergent-solubilized membranes. We further show how GFP-TS can be employed on purified SLC transporter fusions to screen for specific lipid–protein interactions, which is an important complement to native mass spectrometry approaches that cannot cope easily with crude lipid-mixture preparations. This protocol is simple to perform and can be followed by researchers with a basic background in protein chemistry. Starting with an SLC transporter construct that can be expressed and purified from S. cerevisiae in a well-folded state, this protocol extension can be completed in ~4–5 d.

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Fig. 1: Flowchart of the GFP-TS assay.
Fig. 2: Development and validation of the GFP-TS assay.
Fig. 3: Optimization of the GFP-TS assay.
Fig. 4: GFP-TS assay for functional characterization of hCST and deorphanization of a plant homolog.
Fig. 5: GFP-TS assay for ligand interaction studies to human SLC35A1 and a plant homolog.
Fig. 6: Monitoring lipid–protein interactions with FSEC-TS and GFP-TS.

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All data generated or analyzed during this study are included in this published article.

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Acknowledgements

We thank M. Claesson and other present and former lab members for their input into the described protocol. This work was funded by grants from the Knut and Alice Wallenberg Foundation (KAW), the Novo Nordisk Foundation (no. 34188) and a European Research Council (ERC) Consolidator Grant EXCHANGE (grant no. ERC-CoG-820187) to D.D.

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Author notes

  1. These authors contributed equally: Yurie Chatzikyriakidou, Do-Hwan Ahn.

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

    Yurie Chatzikyriakidou, Do-Hwan Ahn, Emmanuel Nji & David Drew

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  1. Yurie Chatzikyriakidou
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  3. Emmanuel Nji
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Contributions

The supporting experiments additional to those previously described in ref. 14 were carried out by Y.C. and D.A. The manuscript was prepared by all authors.

Corresponding author

Correspondence to David Drew.

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The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Eric R. Geertsma, Dianfan Li and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

Related links

Key references using this protocol

Nji, E. et al. Nat. Commun. 9, 4253 (2018): https://doi.org/10.1038/s41467-018-06702-3

Nji, E. et al. Nat. Struct. Mol. Biol. 26, 415–423 (2019): https://doi.org/10.1038/s41594-019-0225-y

Winkelmann, I. et al. EMBO J. 39, 4541–4559 (2020): https://doi.org/10.15252/embj.2020105908

This protocol is an extension to: Nat. Protoc. 3, 784–798, (2008): https://doi.org/10.1038/nprot.2008.44

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Chatzikyriakidou, Y., Ahn, DH., Nji, E. et al. The GFP thermal shift assay for screening ligand and lipid interactions to solute carrier transporters. Nat Protoc 16, 5357–5376 (2021). https://doi.org/10.1038/s41596-021-00619-w

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