Purification and reconstitution of TRPV1 for spectroscopic analysis

Francisco J. Sierra-Valdez, Richard A. Stein, Phanindra Velissety, Valeria Vasquez, Julio Cordero-Morales

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Article numbere57796
JournalJournal of Visualized Experiments
Volume2018
Issue number137
DOIs
StatePublished - Jul 3 2018

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Spectroscopic analysis
Electron resonance
Ion Channels
Purification
Electrons
Paramagnetic resonance
Electron Spin Resonance Spectroscopy
Ions
Electron microscopy
Cryoelectron Microscopy
Transient Receptor Potential Channels
Proteins
X ray crystallography
Detergents
Binding sites
Dynamic analysis
Conformations
Membrane Proteins
Positive ions
Chemical activation

All Science Journal Classification (ASJC) codes

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Cite this

Purification and reconstitution of TRPV1 for spectroscopic analysis. / Sierra-Valdez, Francisco J.; Stein, Richard A.; Velissety, Phanindra; Vasquez, Valeria; Cordero-Morales, Julio.

In: Journal of Visualized Experiments, Vol. 2018, No. 137, e57796, 03.07.2018.

Research output: Contribution to journalArticle

Sierra-Valdez, Francisco J. ; Stein, Richard A. ; Velissety, Phanindra ; Vasquez, Valeria ; Cordero-Morales, Julio. / Purification and reconstitution of TRPV1 for spectroscopic analysis. In: Journal of Visualized Experiments. 2018 ; Vol. 2018, No. 137.
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