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.

Picturing the Membrane‐assisted Choreography of Cytochrome P450 with Lipid Nanodiscs

“…[91][92] Highresolution determination of molecular structure using diffraction methods requires high quality 3D crystals that are hard to obtain for membrane proteins, although significant breakthrough have been made by lipid-cubic phase crystallography and cryo-EM techniques. Model membrane systems -planar supported bilayers [95][96] and giant-unilamellar vesicles (GUVs), [97] among others -have aided our understanding on the lipid organization of proteins in cell membranes. Model membrane systems -planar supported bilayers [95][96] and giant-unilamellar vesicles (GUVs), [97] among others -have aided our understanding on the lipid organization of proteins in cell membranes.…”

“…[93][94] On the other hand, fluorescence-based techniques allow to visualize protein-lipid interaction in model membranes and cells. [98] The advent of super-resolution imaging has impacted the microscopy field by overcoming the diffraction limit, [96] and so solving structure that are separated by~200 nm. [98] The advent of super-resolution imaging has impacted the microscopy field by overcoming the diffraction limit, [96] and so solving structure that are separated by~200 nm.…”

Single-Molecule Kinetics in Living Cells

“…Diffusion is also generally slower in the membrane, which means that it is relatively easy to get good signals. Thus, membrane-bound single-molecule kinetics is in itself a big field that has been reviewed elsewhere (6,7).…”

High performance computing for three-dimensional agent-based molecular models

“…New advances in super-resolution and super-localisation techniques have allowed experimental molecular biophysics and biochemistry to go beyond ensemble measurements and obtain data at the single molecule level [4,15]. Such experiments are able to track down key motions, reactions, and interactions of individual molecules with high temporal and spatial resolution.…”