Abstract
Conformational dynamics underlie enzyme function, yet are generally inaccessible via traditional structural approaches. FRET has the potential to measure conformational dynamics in vitro and in intact cells, but technical barriers have thus far limited its accuracy, particularly in membrane proteins. Here, we combine amber codon suppression to introduce a donor fluorescent noncanonical amino acid with a new, biocompatible approach for labeling proteins with acceptor transition metals in a method called ACCuRET (Anap Cyclen-Cu2+ resonance energy transfer). We show that ACCuRET measures absolute distances and distance changes with high precision and accuracy using maltose binding protein as a benchmark. Using cell unroofing, we show that ACCuRET can accurately measure rearrangements of proteins in native membranes. Finally, we implement a computational method for correcting the measured distances for the distance distributions observed in proteins. ACCuRET thus provides a flexible, powerful method for measuring conformational dynamics in both soluble proteins and membrane proteins.
Highlights
Structural dynamics of proteins, those at cell membranes, underlie many cell-signaling events (Henzler-Wildman and Kern, 2007)
maltose-binding protein (MBP) is a clamshell shaped protein that undergoes a significant closure of the clamshell upon binding its ligand, maltose (Figure 1A; Video 1)
Our criteria for selection of fluorescence resonance energy transfer (FRET) pairs were (1) the sites are solvent exposed, (2) the sites are on rigid secondary structural elements, (3) the distance between the sites is predicted to fall in the working range of transition metal ion FRET (tmFRET) (~10–20 A ), and (4) the distance between sites undergoes a moderate change between apo and holo MBP
Summary
Structural dynamics of proteins, those at cell membranes, underlie many cell-signaling events (Henzler-Wildman and Kern, 2007). Structural rearrangements in membrane proteins can occur in response to binding of extracellular or intracellular ligands, covalent modification (e.g. phosphorylation), changes in membrane voltage, and mechanical forces in the membrane These rearrangements, in turn, regulate enzyme activity, open pores, and transport molecules across the membrane. Electrophysiology gives an excellent readout on rearrangements in the pore domain but is a poor surrogate for probing structural rearrangements and energetics in other regions, for example agonist/antagonist binding sites This state of affairs has left a gap in our ability to link structure and function, as we often do not know the functional state for a particular structure or the structural state(s) that underlie a particular function
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