Name: purification and reconstitution of trpv1 for spectroscopic analysis
Uploaded: 2018-07-03T13:30:00
Description: Watch this Scientific Journal Video about Purification and Reconstitution of TRPV1 for Spectroscopic Analysis at JoVE.com

We are very grateful to Dr. H. Mchaourab for providing access to the EPR and DEER spectrometers and Dr. T. Rosenbaum for providing the full-length cysteine-less TRPV1 plasmid.

1. TRPV1 Mutagenesis
Note: A minimal TRPV1 construct for spectroscopic analysis26 was built from the full-length cysteine-less channel TRPV127 using the polymerase chain reaction (PCR) method (Figure 1). This cysteine-less minimal TRPV1 construct (referred to as eTRPV1 hereafter) consists of residues 110 - 603 and 627 - 764. eTRPV1 was cloned in pMO (a pcDNA3.1-based vector) for functional analysis and in a recombinant...

Current technologies for expression and purification of mammalian membrane proteins have made it possible to obtain sufficient amounts of protein for spectroscopic studies14,15,16,42. Here, we have adapted these technologies to express, purify, reconstitute, and perform spectroscopic analyses in TRPV1.
Among the critical steps in the protocol, below are the ones that...

With recent advances in single-particle cryo-electron microscopy (cryo-EM), mammalian ion channel structures have been obtained at an extraordinary rate. Particularly, structural studies of polymodal ion channels, such as the transient receptor potential vanilloid 1 (TRPV1), have provided further understanding of its activation mechanisms1,2,3,4,5. However, dynamic information about ion channels embedded in a membrane environment is required to understand their polymodal gating and drug-binding mechanisms.
Electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) spectroscopies have provided some of the most definitive mechanistic models for ion channels6,7,8,9,10,11,12,13. These approaches have been mainly restricted to the examination of prokaryotic and archeal ion channels that yield a large amount of detergent-purified proteins when overexpressed in bacteria. With the development of eukaryotic membrane proteins production in insect and mammalian cells for functional and structural characterization14,15,16, it is now possible to obtain biochemical amounts of detergent-purified proteins for spectroscopic studies.
The EPR and DEER signals arise from a&#160;paramagneticspin label (SL) (i.e., methanethiosulfonate) attached to a single-cysteine residue in the protein. The spin-labels report three types of structural information: motion, accessibilities, and distances. This information allows determining whether residues are buried within the protein or are exposed to the membrane or aqueous environment in the apo and ligand-bound states13,17,18,19. In the context of a high-resolution structure (when available), the EPR and DEER data provide a collection of constraints for deriving dynamic models in their native environment while monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Moreover, flexible regions that might be difficult to determine by X-ray crystallography or cryo-EM could be obtained by using these environmental data sets to assign secondary structures as well as location within the protein20. Cryo-EM structures obtained in lipid nanodiscs provided valuable information about the gating of ion channels3,21,22,23,24,25; however, spectroscopic approaches could provide dynamic information from conformational states (e.g., thermal changes) that might be difficult to determine using cryo-EM.
Many difficulties must be overcome to implement EPR and DEER, including lack of protein function when removing all cysteine residues (especially abundant in mammalian channels), low protein yield, protein instability during purification and after spin labeling, and protein aggregation in detergent or liposomes. Here, we have designed protocols to overcome these critical barriers and have obtained DEER and EPR spectra information for a mammalian sensory receptor. The purpose here is to describe methodologies for the expression, purification, labeling, and reconstitution of a functional minimal cysteine-less rat TRPV1 (eTRPV1) construct for spectroscopic analyses. This methodology is appropriate for those membrane proteins that keep their function despite the removal of cysteine residues or that contain cysteine forming disulfide-bonds. This collection of protocols could be adapted for the spectroscopic analysis of other mammalian ion channels.

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</table>

purification and reconstitution of trpv1 for spectroscopic analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

Functional Characterization of the Minimal Cysteine-less TRPV1 Construct (eTRPV1) and Single-cysteine Mutants
The first step toward spectroscopic studies is to engineer and characterize cysteine-less protein constructs (Figure 2A) that are functional and yield biochemical amounts of proteins. eTRPV1 is functional as determined by Ca2+ imaging and TEVC (Figure 2B...

Watch this Scientific Journal Video about Purification and Reconstitution of TRPV1 for Spectroscopic Analysis at JoVE.com

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

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