EXPRESS: Single-Molecule Fluorescence Techniques for Membrane Protein Dynamics Analysis

Fluorescence-based single molecule techniques, mainly including fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence resonance energy transfer (smFRET), are able to analyze the conformational dynamics and diversity of biological macromolecules. They have been applied to analysis of the dynamics of membrane proteins, such as membrane receptors and membrane transport proteins, due to their superior ability in resolving spatio-temporal heterogeneity and the demand of trace amounts of analytes. In this review, we first introduced the basic principle involved in FCS and smFRET. Then we summarized the labelling and immobilization strategies of membrane protein molecules, the confocal-based and TIRF-based instrumental configuration, and the data processing methods. The applications to membrane protein dynamics analysis are described in detail with the focus on how to select suitable fluorophores, labelling sites, experimental setup and analysis methods. In the last part, the remaining challenges to be addressed and further development in this field are also briefly discussed.

Tracking Membrane Protein Dynamics in Real Time

Membrane proteins govern critical cellular processes and are central to human health and associated disease. Understanding of membrane protein function is obscured by the vast ranges of structural dynamics—both in the spatial and time regime—displayed in the protein and surrounding membrane. The membrane lipids have emerged as allosteric modulators of membrane protein function, which further adds to the complexity. In this review, we discuss several examples of membrane dependency. A particular focus is on how molecular dynamics (MD) simulation have aided to map membrane protein dynamics and how enhanced sampling methods can enable observing the otherwise inaccessible biological time scale. Also, time-resolved X-ray scattering in solution is highlighted as a powerful tool to track membrane protein dynamics, in particular when combined with MD simulation to identify transient intermediate states. Finally, we discuss future directions of how to further develop this promising approach to determine structural dynamics of both the protein and the surrounding lipids. Graphic Abstract

FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins

Fluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these issues, we present here a robust computer program, called FluoSim, which is an interactive simulator of membrane protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. FluoSim integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the localization and intensity of thousands of independent molecules in 2D cellular geometries, providing simulated data directly comparable to actual experiments. FluoSim was thoroughly validated against experimental data obtained on the canonical neurexin-neuroligin adhesion complex at cell–cell contacts. This unified software allows one to model and predict membrane protein dynamics and localization at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments.