Single-molecule biophysics: at the interface of biology, physics and chemistry

Abstract

Single-molecule methods have matured into powerful and popular tools to probe the complex behaviour of biological molecules, due to their unique abilities to probe molecular structure, dynamics and function, unhindered by the averaging inherent in ensemble experiments. This review presents an overview of the burgeoning field of single-molecule biophysics, discussing key highlights and selected examples from its genesis to our projections for its future. Following brief introductions to a few popular single-molecule fluorescence and manipulation methods, we discuss novel insights gained from single-molecule studies in key biological areas ranging from biological folding to experiments performed in vivo.

Figure 1

Components and scope of single-molecule science.

Figure 2

Overview of single-molecule methods and their main applications and strengths for measuring molecular properties.

Figure 3

Overview of single-molecule instrumentation. Six examples for typical single-molecule instrumentation. Components that occur in more than one type of instrumentation are often labelled only in the panel where they appeared first for better clarity of the picture. (a) Confocal geometry and dual-colour detection; focal volume (fV), objective (Ob), excitation with a laser (ex), dichroic mirror (DC), pinhole (Ph), point detector (PD), emission filter (emf). (b) Prism-type TIRFM. (c) Through-objective TIRFM; evanescent wave (eW), area detector/camera (C). (d) AFM; position-sensitive detector (psd), molecule (M). (e) Optical tweezer; light source (LS), polysterene bead (pb). (f) Magnetic tweezer; magnet (Mag), magnetic bead (mb).

Figure 4

Protein folding studies. (a) Schematic energy landscape for protein folding. (b) FRET efficiency histograms showing folded and denatured subpopulations for CI2. (i) 3 M GdmCl, (ii) 4 M GdmCl and (iii) 6 M GdmCl. Adapted from Deniz et al. (2000). (c) FCS correlation function for Alexa488-labelled prion domain of Sup 35 yeast prion protein, displaying nanosecond time-scale conformational fluctuations in addition to a slower diffusion decay component. Adapted from Mukhopadhyay et al. (2007). (d) Progressive shift of FRET efficiency during the unfolding of monomeric prion domain. (i) 0 M GdmCl, (ii) 0.5 M GdmCl and (iii) 2 M GdmCl. Figure 4b and d clearly show the difference between a natively folded globular protein (CI2) and a natively unfolded yeast prion. Adapted from Mukhopadhyay et al. (2007).

Figure 5

Single-molecule RNA dynamics. (a) FRET time traces for two-way junction hairpin ribozymes showing static heterogeneity in the unfolding rates for three different immobilized ribozyme molecules. From Zhuang et al. 2002 Science 296, 1473. Reprinted with permission from AAAS. (b) FRET histogram for an undocked state mimic of the natural four-way junction hairpin ribozyme, showing broadening consistent with rapid fluctuations (50–100 μs) between extended undocked (EU) and quasi-docked (QD) conformations. Simulated histograms are shown for forward/reverse (k₁/k₋₁) rate constants of 20×10³/6.8×10³ s⁻¹ (1), 41×10³/14×10³ s⁻¹ (2) and 10×10³/3.4×10³ s⁻¹ (3) with a corresponding equilibrium constant of 3 in favour of the quasi-docked state in all cases. Adapted from Pljevaljčić et al. (2004). (c) Optical tweezers pulling of the Tetrahymena thermophila ribozyme showing eight different unfolding transitions (marked with arrows from a to h). From Onoa et al. 2003 Science 299, 1892. Reprinted with permission from AAAS.

Figure 6

Memory effects in single-molecule enzyme activity. (a) Fluorescence image (8×8 μm) of single cholesterol oxidase (Cox) molecules immobilized in a film of agarose gel. The emission is from the fluorescent FAD, which is tightly bound to the enzyme active site. Each individual peak is attributed to a single Cox molecule. (b) Real-time observation of enzymatic turnovers of a single Cox molecule catalysing oxidation of cholesterol molecules (the emission intensity trajectory is recorded (shown in counts per channel (ct/ch)) with a time resolution of 13.1 ms) with a 0.2 mM cholesterol concentration and 0.25 mM saturated oxygen concentration. The time trajectory exhibits stochastic blinking behaviour as FAD interconverts between oxidized (fluorescent) and reduced (non-fluorescent) states, each on–off cycle corresponding to an individual enzymatic turnover. (c) Turnover waiting times (x- and y-axes are waiting times from 0 to 1 s) for (i) sequential turnovers and waiting times separated by (ii) 10 turnovers of Cox molecules. The diagonal population density in (i), which is missing from (ii), shows a memory effect that decays within 10 turnovers. From Lu et al. 1998 Science 282, 1877. Reprinted with permission from AAAS.

Figure 7

Single-molecule studies of molecular motors. (a) (i) Structure of F₁-ATPase viewed from the F₀ side. (ii) The observation system and laser dark-field microscopy set-up for observation of gold beads attached to the ATPase γ-subunit. Only light scattered by the beads was detected (DFC: dark-field condenser). (iii) Time courses of stepping rotation of 40 nm beads at 20 μM ATP concentration. Adapted by permission from Macmillan Publishers Ltd: Nature, Yasuda et al. 2001 Nature 41, 898, copyright 2001. (b) (i) Donor-labelled Rep binds to a 3′ ssDNA tail and translocates towards the acceptor, and fluorescence intensity traces for a 3′ (dT)₈₀ tail at (ii) 22°C and (iii) 37°C. Adapted by permission from Macmillan Publishers Ltd: Nature, Myong et al. 2005 Nature 437, 1321, copyright 2005. (c) Single base-pair stepping by RNA polymerase using ultrastable optical tweezers. (i) The dual optical tweezer passive force clamp. Strong trap, T_strong; weak trap, T_weak; and trap stiffness k=dF/dx. (ii) Base-pair stepping as a function of time under 18 pN of assisting load, median-filtered at 50 (grey) and 750 ms (black), with the distance between horizontal lines representing a 1 bp separation. Adapted by permission from Macmillan Publishers Ltd: Nature, Abbondanzieri et al. 2005 Nature 438, 460, copyright 2005.