Single-Molecule Fluorescence Techniques for Membrane Protein Dynamics Analysis

Abstract

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.

Reduction of protein disulfide isomerase results in open conformations and stimulates dynamic exchange between structural ensembles

“…Atto550 (donor) and Atto647N (acceptor) were chosen because of their long fluorescence lifetime and excellent photostability, as these features are necessary to carry out multiparameter confocal smFRET studies. The R 0 for this FRET pair was determined to be 62.3 Å, which agrees with previous reports (20).…”

“…S8 ). Furthermore, addition of DTT caused a greater shift toward lower FRET for 57/401 and 88/401, which was anticipated because of the lower value of R 0 of the sulfoCy dyes (∼56 Å) ( 20 ). Thus, data with the sulfoCy dyes support the conclusions made with the Atto dyes and reveal that the high FRET population in 57/401 and 88/401 is dynamic too.…”

Principles of Resonance Energy Transfer

“…Conformational changes in the 10 µs to 10 s range can be best studied using diffusion‐based smFRET (Tomov et al., 2012). With the established and improved smFRET schemes, several problems have been studied, such as the structural dynamics of ion channels (Martinac, 2017), membrane proteins (Castell, Dijkman, Wiseman, & Goddard, 2018; Ward, Ye, Schuster, Wei, & Barrera, 2021; Yang, Xu, Wang, Chen, & Zhao, 2021), intrinsically disordered proteins (LeBlanc, Kulkarni, & Weninger, 2018; Metskas & Rhoades, 2020), DNA polymerases (Millar, 2022), and DNA nanostructures (Pal, 2022). The multicolor version of smFRET, in which more than one donor‐acceptor pair is used, can provide even more information about protein‐DNA interactions, chromatin remodeling and even protein translation (Feng et al., 2021).…”

A Flexible and Polymer‐Based Chemiresistive CO2 Gas Sensor at Room Temperature

“…Carbon dioxide (CO 2 ) detection plays a crucial role in different fields such as air‐quality monitoring, [ 1 ] fire detection, [ 2 ] green‐house gas monitoring, [ 3 ] health, [ 4 ] marine and environmental studies, [ 5 ] and food supply chain. [ 6 ] Conventionally, different methods including fluorescence, [ 7 ] gas chromatography, [ 8 ] infrared spectrometry, [ 9 ] photoacoustic spectroscopy, [ 10 ] and severinghaus type electrode [ 11 ] have been used for CO 2 measurement. Although traditional CO 2 detection methods are highly selective and sensitive, they usually suffer from relatively high cost, bulkiness, high power consumption, and vulnerability to electromagnetic interference.…”