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.
Highlights
SmFRET is becoming increasingly popular in the investigation of membrane transporter dynamics (Table 1), complementing well-established methods such as Nuclear Magnetic Resonance (NMR),[28,29,30] Small-Angle X-ray Scattering (SAXS)[31,32,33] and Electron Paramagnetic Resonance (EPR).[34,35,36]
To understand transport mechanisms on a molecular basis, data interpretation of single molecule Förster Resonance Energy Transfer (smFRET) experiments can highly benefit from available high-resolution structures, combined with MD simulations, mass spectrometry, SAXS and NMR studies
The power of combining smFRET studies with NMR spectroscopy has already been demonstrated on model systems such as EmrE and members of the glutamate transporter family.[174,181,200]
Summary
For the last two decades,[1] smFRET techniques have been extensively used to study the properties of molecular machines,[2] intrinsically disordered proteins (IDPs),[3,4,5,6,7,8] protein folding processes,[9,10,11,12,13,14,15,16] protein-ligand[17,18,19] and protein-nucleic acid interactions,[20,21,22,23] as well as other structure-function relationships and dynamic processes.[24,25,26] SmFRET is a powerful and versatile tool to gain molecular and mechanistic insights because of its high spatial resolution (2– 10 nm) combined with a wide range of accessible timescales (ns-minutes).[1,10,27] smFRET is becoming increasingly popular in the investigation of membrane transporter dynamics (Table 1), complementing well-established methods such as Nuclear Magnetic Resonance (NMR),[28,29,30] Small-Angle X-ray Scattering (SAXS)[31,32,33] and Electron Paramagnetic Resonance (EPR).[34,35,36]. Membrane proteins play a key role in cell metabolism: they are encoded by roughly 30 % of the human genome[59,60] and account for an estimated 60 % of drug targets,[61] a full understanding of the interplay between conformational dynamics and their function is largely elusive. Among these proteins, transporters constitute a large class of integral membrane proteins. Proposed transport models are “rocker switch” for Sugar-Will-Eventually-beExported-Transporters (SWEETs) or “clamp and switch” for Major-Facilitator-Superfamily (MFS) transporters, “rocking bundle” for the Neurotransmitter-Sodium-Symporters (NSS) family and the “elevator” mechanism for members of the ExcitatoryAmino-Acid-Transporters (EAAT) family.[67,70,71,72,73,74] For primary active
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