title: Tracking individual membrane proteins and their biochemistry: The power of direct observation.
abstract: The advent of single molecule fluorescence microscopy has allowed experimental molecular biophysics and biochemistry to transcend traditional ensemble measurements, where the behavior of individual proteins could not be precisely sampled. The recent explosion in popularity of new super-resolution and super-localization techniques coupled with technical advances in optical designs and fast highly sensitive cameras with single photon sensitivity and millisecond time resolution have made it possible to track key motions, reactions, and interactions of individual proteins with high temporal resolution and spatial resolution well beyond the diffraction limit. Within the purview of membrane proteins and ligand gated ion channels (LGICs), these outstanding advances in single molecule microscopy allow for the direct observation of discrete biochemical states and their fluctuation dynamics. Such observations are fundamentally important for understanding molecular-level mechanisms governing these systems. Examples reviewed here include the effects of allostery on the stoichiometry of ligand binding in the presence of fluorescent ligands; the observation of subdomain partitioning of membrane proteins due to microenvironment effects; and the use of single particle tracking experiments to elucidate characteristics of membrane protein diffusion and the direct measurement of thermodynamic properties, which govern the free energy landscape of protein dimerization. The review of such characteristic topics represents a snapshot of efforts to push the boundaries of fluorescence microscopy of membrane proteins to the absolute limit. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'.
authors.affiliation: Department of Chemistry, Washington State University, PO Box 644630, Pullman, WA, 99164-4630, United States.
authors.forename: Adam O
authors.initials: AO
authors.lastname: Barden
authors.affiliation: Department of Chemistry, Washington State University, PO Box 644630, Pullman, WA, 99164-4630, United States.
authors.forename: Adam S
authors.initials: AS
authors.lastname: Goler
authors.affiliation: Department of Chemistry, Washington State University, PO Box 644630, Pullman, WA, 99164-4630, United States.
authors.forename: Sara C
authors.initials: SC
authors.lastname: Humphreys
authors.affiliation: Department of Chemistry, Washington State University, PO Box 644630, Pullman, WA, 99164-4630, United States.
authors.forename: Samaneh
authors.initials: S
authors.lastname: Tabatabaei
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012, Bern, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Lochner
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012, Bern, Switzerland.
authors.forename: Marc-David
authors.initials: MD
authors.lastname: Ruepp
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012, Bern, Switzerland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Jack
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012, Bern, Switzerland.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Simonin
authors.affiliation: Pharmacology Department, Cambridge University, Tennis Court Road, Cambridge, CB2 1PD, United Kingdom.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thompson
authors.affiliation: Department of Chemistry, Washington State University, PO Box 644630, Pullman, WA, 99164-4630, United States.
authors.forename: Jeffrey P
authors.initials: JP
authors.lastname: Jones
authors.affiliation: Department of Chemistry, Washington State University, PO Box 644630, Pullman, WA, 99164-4630, United States. Electronic address: brozik@wsu.edu.
authors.forename: James A
authors.initials: JA
authors.lastname: Brozik
mesh_terms: D000818:Animals; D001671:Biochemistry; D001703:Biophysics; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D058446:Ligand-Gated Ion Channels / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D021381:Protein Transport / Q000502:physiology; D013269:Stochastic Processes
chemical_list: D005456:Fluorescent Dyes; D058446:Ligand-Gated Ion Channels; D008565:Membrane Proteins
keywords: 5HT3; Fluorescence; GPCR G-protein coupled receptor; LGIC; Ligand-gated ion channel; Molecular machine; P2X1; Protein tracking; Single molecule fluorescence imaging; Stochastic