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 labeling 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, labeling 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.
Keywords: FCS; Fluorescence correlation spectroscopy; dynamics analysis; membrane protein; single-molecule fluorescence resonance energy transfer; smFRET.