Single-molecule FRET methods to study the dynamics of proteins at work

Abstract

Feynman commented that "Everything that living things do can be understood in terms of the jiggling and wiggling of atoms". Proteins can jiggle and wiggle large structural elements such as domains and subunits as part of their functional cycles. Single-molecule fluorescence resonance energy transfer (smFRET) is an excellent tool to study conformational dynamics and decipher coordinated large-scale motions within proteins. smFRET methods introduced in recent years are geared toward understanding the time scales and amplitudes of function-related motions. This review discusses the methodology for obtaining and analyzing smFRET temporal trajectories that provide direct dynamic information on transitions between conformational states. It also introduces correlation methods that are useful for characterizing intramolecular motions. This arsenal of techniques has been used to study multiple molecular systems, from membrane proteins through molecular chaperones, and we examine some of these studies here. Recent exciting methodological novelties permit revealing very fast, submillisecond dynamics, whose relevance to protein function is yet to be fully grasped.

Keywords: Allostery; Fluorescence correlation spectroscopy; Molecular machines; Protein conformational dynamics; Single-molecule FRET.

Figure 1. Probing proteins with smFRET spectroscopy: slow (a–c) vs fast (d–g) dynamics.

(a) Slow conformational dynamics involve a high free-energy barrier. (b) Two different methods to tether a single molecule to a surface, using direct tethering or vesicle encapsulation. (c) smFRET trajectory of an immobilized molecule. Top panel: donor (green) and acceptor (red) intensities; bottom panel: FRET efficiency (gray) and state assignments from a HMM analysis (blue and orange). (d) Fast conformational dynamics involve a low free-energy barrier. (e) Spectroscopy of freely diffusion molecule. Top: cartoon of a focal volume with a molecule passing through. Bottom: fluorescence bursts emanating from excited molecules. Inset; FRET efficiency histogram (orange) that is broader than shot noise (red), suggesting fast dynamics. (f) A photon-by-photon single-molecule trajectory, with donor and acceptor photons in green and red, respectively. Blue and orange lines are state assignments from an H²MM analysis. (g) Cross-correlation function of donor and acceptor fluorescence. The initial increase in the signal (shaded area) indicates conformational dynamics on the microsecond timescale. smFRET, single-molecule fluorescence resonance energy transfer; HMM, Hidden Markov Models

Figure 2. Membrane protein conformational dynamics measured with smFRET.

(a) Single-molecule dynamics of a surface-tethered SBD of the ABC importer GlnPQ probed by confocal scanning microscopy. (b) Single molecule trajectories at different ligand concentrations. Figure 2 A-B reprinted from Ref. [81] with permission from Nature publishing group. (c) Dynamics of the G-protein-coupled receptor mGluR, reprinted from Ref. [85] with permission. Top: filtered fluorescence cross-correlation curves, indicating microsecond conformational dynamics in the wild type (grey) but not in the constitutively active mutant (blue). Bottom: kinetic model of mGluR dynamics. SBD, substrate-binding domain; smFRET, single-molecule fluorescence resonance energy transfer; ABC, ATP-binding cassette.

Figure 3. Dynamics of soluble protein machines measured with smFRET.

(a–c) The disaggregation machine ClpB, reprinted from Ref. [70] with permission. (a) Schematic of ClpB and its dynamic M domain (green), which toggles between three states on the microsecond timescale (b). (c) DnaK binding tunes the ratio between the two major states of the M domain and in turn disaggregation activity. (d–e) The chaperone Hsp90, reprinted from Ref. [47] with permission. (d) ATP-binding related dynamics of Hsp90 are studied using three-color smFRET. (e) Top: single molecule trajectories in three channels upon donor excitation. Middle: FRET efficiency trajectories in different states of Hsp90, as depicted in the bottom cartoons. (f) Adenylate kinase, reprinted from Ref. [33] with permission. Rates of domain closing and opening (cherry and green, respectively) as a function of substrate concentration. Inset: structure with attached labels. (g) F₁-ATPase, Reprinted from Ref. [91] with permission. Simultaneous measurement of rotational motion of the ɣ-shaft using a bead (top panel, black line) and of FRET between αβ dimers (bottom panel, donor and acceptor in green and red). smFRET, single-molecule fluorescence resonance energy transfer.