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. 2021:652:31-48.
doi: 10.1016/bs.mie.2021.02.006. Epub 2021 Mar 12.

TRPV3 expression and purification for structure determination by Cryo-EM

Arthur Neuberger  1 , Kirill D Nadezhdin  1 , Alexander I Sobolevsky  2
Affiliations

TRPV3 expression and purification for structure determination by Cryo-EM

Arthur Neuberger et al. Methods Enzymol. 2021.

Abstract

The transient receptor potential vanilloid-superfamily member 3 (TRPV3) channel is implicated in a variety of physiological processes, including temperature sensing, nociception and itch, maintenance of the skin barrier, wound healing, hair growth, and embryonic development. TRPV3 is also associated with various skin diseases, including Olmsted syndrome, atopic dermatitis, and rosacea. Studies of TRPV3 are of fundamental importance for structural pharmacology aimed at the design of drugs targeting this channel and for understanding the molecular basis of temperature sensing. Here we describe a detailed protocol for expression and purification of chemically pure and stable TRPV3 protein that is suitable for structural and functional characterization of this channel, in particular for cryo-EM sample preparation and high-resolution 3D reconstruction.

Keywords: Conformation; Cryo-EM; Expression; Gating; Protein; Purification; Structure; TRP channels; TRPV3.

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Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Figures

Fig. 1.
Fig. 1.
Phylogenetic tree of TRP channels. There is a human (Homo sapiens) gene for each of the shown TRP channels, except TRPC2, which is a pseudogene in human but not in rat (Rattus norvegicus), and TRPN that has not been identified in mammals but is present in fruit fly (Drosophila melanogaster).
Fig. 2.
Fig. 2.
FSEC profiles for TRPV3 orthologues from rabbit (Oryctolagus cuniculus), frog (Xenopus laevis), mouse (Mus musculus), and chicken (Gallus gallus), expressed in HEK-293S cells and extracted using 1% DDM. The samples were run on a Superose™ 6 10/300 GL SEC column followed by eGFP fluorescence.
Fig. 3.
Fig. 3.
Schematic of mTRPV3 expression. Created using BioRender.com.
Fig. 4.
Fig. 4.
Schematic of mTRPV3 purification. Created using BioRender.com.
Fig. 5.
Fig. 5.
SEC profiles for mTRPV3 in GDN and nanodiscs. (A), SEC profile for mTRPV3 purified in 0.01% GDN. (B), SEC profile for mTRPV3 reconstituted in MSP2N2 nanodiscs.
Fig. 6.
Fig. 6.
Schematic of mTRPV3 nanodisc reconstitution. Created using BioRender.com.
Fig. 7.
Fig. 7.
Example micrograph and 2D class averages for mTRPV3 reconstituted in MSP2N2 nanodiscs. Left panel shows an example cryo-EM micrograph collected on Titan Krios TEM operating at 300 kV, equipped with a post-column GIF Quantum energy filter and a Gatan K3 Summit DED camera, using a ~0.4 Å pixel size and a total dose of ~60 e−Å−2 (dose rate of ~16 e−pixel−1s−1 across 50 frames for 2.5 s total exposure time). Example particles of frozen-hydrated mTRPV3 reconstituted in MSP2N2 nanodiscs are circled in pink. Right panel shows 2D class averages calculated using cryoSPARC.
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References

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