Single‐Molecule FRET of Membrane Transport Proteins

Abstract

Uncovering the structure and function of biomolecules is a fundamental goal in structural biology. Membrane-embedded transport proteins are ubiquitous in all kingdoms of life. Despite structural flexibility, their mechanisms are typically studied by ensemble biochemical methods or by static high-resolution structures, which complicate a detailed understanding of their dynamics. Here, we review the recent progress of single molecule Förster Resonance Energy Transfer (smFRET) in determining mechanisms and timescales of substrate transport across membranes. These studies do not only demonstrate the versatility and suitability of state-of-the-art smFRET tools for studying membrane transport proteins but they also highlight the importance of membrane mimicking environments in preserving the function of these proteins. The current achievements advance our understanding of transport mechanisms and have the potential to facilitate future progress in drug design.

Structural snapshots of human PepT1 and PepT2 reveal mechanistic insights into substrate and drug transport across epithelial membranes

“…Based on this finding, the substrate promiscuity observed for POTs can be explained and potentially be used for modification approaches for more efficient uptake in the future. (50,51) in agreement with the deposited POT structures. Multiple stabilizing interactions, mainly salt-bridges between the N-and C-terminal bundle, need to be broken and formed during a transport cycle (27,52).…”

“…Nevertheless, all structures of bacterial homologues (47 Protein Databank (PDB) entries representing ten different bacterial POTs) have been captured in the inward-open or inward-open partially occluded state, limiting our understanding of the conformational changes during transport. To date, it is unclear why both human transporters display different conformational states under the measured conditions, since a recent single molecule FRET study on the bacterial POT DtpA confirmed the inward-open state as the lowest energy state in detergent solution ( 50, 51 ) in agreement with the deposited POT structures. Multiple stabilizing interactions, mainly salt-bridges between the N- and C-terminal bundle, need to be broken and formed during a transport cycle ( 27, 52 ).…”

An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET

“…Time-resolved FRET experiments complement single-molecule FRET and DEER spectroscopy experiments. Single-molecule FRET also measures the proportion of time the protein dwells in different states and, in addition, provides information on the transition rates between states, not currently possible with fluorescent lifetime measurements ( Mazal and Haran, 2019 ; Quast and Margeat, 2019 ; Metskas and Rhoades, 2020 ; Feng et al, 2021 ; Bartels et al, 2021 ). Single-molecule measurements, however, are slow owing to technical limitations that generally require integration of photons for ≥10 ms.…”

Lipid Membrane Mimetics in Functional and Structural Studies of Integral Membrane Proteins

“…It captures conformational motions within one protein molecule/complex in real time, although measurements on many molecules/complexes are needed to average the effect of modulating protein conformation by, for example, ligand binding. smFRET has been used in multiple studies on detergent-residing IMPs to monitor their conformational responses to ligands, changes in pH, or other stimuli [137][138][139].…”

“…Single-molecule fluorescence spectroscopy and microscopy have also been used to study conformations of IMPs in liposomes. This technique was used to successfully assess the dimerization of fluorescently labeled IMPs [277,278] and the conformational dynamics of membrane transporters in real time [137,279] The concept of amphipols-amphipathic polymers that can solubilize and stabilize IMPs in their native state without the need for detergent-emerged in 1994. Amphipols' mechanism was validated in a study of four IMPs: bacteriorhodopsin, a bacterial photosynthetic reaction center, cytochrome b6f, and matrix porin [280].…”