High-resolution, single-molecule measurements of biomolecular motion

Abstract

Many biologically important macromolecules undergo motions that are essential to their function. Biophysical techniques can now resolve the motions of single molecules down to the nanometer scale or even below, providing new insights into the mechanisms that drive molecular movements. This review outlines the principal approaches that have been used for high-resolution measurements of single-molecule motion, including centroid tracking, fluorescence resonance energy transfer, magnetic tweezers, atomic force microscopy, and optical traps. For each technique, the principles of operation are outlined, the capabilities and typical applications are examined, and various practical issues for implementation are considered. Extensions to these methods are also discussed, with an eye toward future application to outstanding biological problems.

Figure 1

Centroid tracking of (a) particles and (b) single fluorescent molecules. Rotation can be monitored by measuring the motion of an attached, micron-sized particle. A single fluorophore attached to a protein of interest allows direct visualization of motion. By measuring the center of the fluorescence distribution, the fluorophore can be localized to ~1.5 nm.

Figure 2

FRET mechanism and sensitivity. (Left) Excitation light (blue) excites the donor fluorophore ( yellow). When the acceptor fluorophore (red ) moves close to the excited donor, resonant energy transfer occurs, generating fluorescence centered at the acceptor wavelength (red lines). (Right) The efficiency of energy transfer is depicted with the region of sensitivity highlighted.

Figure 3

The effect of external force on the energy landscape. The natural energy landscape is altered by applying a constant force to produce a perturbed energy landscape. This perturbation changes the height of the energy barrier, ΔE, by an amount equal to Fδ.

Figure 4

Brownian noise reduction in a limited bandwidth. Halving the viscous drag on the observed particle reduces mean-squared fluctuations in the measurement bandwidth by a factor of two. Stiffening the molecular handles by a factor of two reduces mean-squared fluctuations by a factor of four within this same region.

Figure 5

Measured distances are reduced by system compliance. (a) Force is applied by an elastic probe to a molecule (green) tethered by a molecular handle, and length changes are measured from the displacement of the force probe (blue cross). (b) The handle acts as a spring in series with the probe. (c) When molecular motion through a distance dL occurs, a portion of this displacement stretches the handle, reducing the observed displacement, dx, by an amount that depends on the relative stiffness of the molecule and the probe, as shown.

Figure 6

Magnetic tweezers. A superparamagnetic bead experiences a force in an inhomogeneous magnetic field and thereby applies load to a single molecule attached to its surface.

Figure 7

Atomic force microscopy. A cantilever exerts tension on a molecule of interest attached to the tip. Motions are measured by recording with a position-sensing detector the deflection of a laser beam reflected off the cantilever, and force is modulated by adjusting the position of the sample (or probe) piezoelectrically.

Figure 8

Comparison of the (a) surface-based assay, (b) dumbbell-based assay using one optical trap and a micropipette, and (c) dumbbell-based assay using two optical traps. Force is recorded by measuring the displacement of the bead from the center of the optical trap using light scattered by the bead.