130 4.3  Förster Resonance Energy Transfer

being variants of a Cy3 (green) donor and Cy5 (red) acceptor, but the technique has also been

applied to QD and FP pairs (see Miyawaki et al., 1997).

The use of organic fluorophore FRET pairs comes with the problem that the chem­

ical binding efficiency to a biomolecule is never 100%, and so there is a subpopulation of

unlabeled “dark” molecules. Also, even when both fluorophores in the FRET pair have bound

successively, there may be a subpopulation that are photoinactive, for example, due to free

radical damage. Again, these “dark” molecules will not generate a FRET response and may

falsely indicate the absence of molecular interaction.

Since the emission spectrum of organic dye fluorophores is a continuum, there is a risk of

bleed-​through of each dye signal into the other’s respective detector channel, which is diffi­

cult to distinguish from genuine FRET unless meticulous control experiments are performed.

These issues are largely overcome by alternating laser excitation (ALEX) (Kapanidis et al.,

2005). Here, donor and acceptor fluorophores are excited in alternation with respective fluor­

escence emission detection synchronized to excitation.

One of the most common approaches for using smFRET is with confocal microscope exci­

tation in vitro. Here, interacting components are free to diffuse in aqueous solution in and

out of the confocal excitation volume. The ~10⁻⁶ m length scale of the confocal volume sets

a low upper limit on the time scale for observing a molecular interaction by FRET since the

interacting pair diffuses over this length scale in typically a few tens of milliseconds.

An approach taken to increase the measurement time is to confine interacting molecules

either through tethering to a surface (Ha et al., 2002) or confinement inside a lipid nanovesicle

immobilized to a microscope slide (Benitez et al., 2002). This latter method exhibits less

interaction with surface forces from the slide. These methods enable continuous smFRET

observations to be made over a time scale of tens of seconds.

A significant disadvantage of smFRET is its very limited application for FP fusion systems.

Although certain paired combinations of FPs have reasonable spectral overlap (e.g., CFP/​YFP

for blue/​yellow and GFP/​mCherry for green/​red), R0 values are typically ~6 nm, but since the

FPs themselves have a length scale of a few nanometers, this means that only FRET efficiency

values of ~0.5 or less can be measured since the FPs cannot get any closer to each other due

to their β-​barrel structure. In this regime, it is less sensitive as a molecular ruler compared

to using smaller, brighter organic dye pairs, which can monitor nanoscale conformational

changes.

A promising development in smFRET has been its application in structural determination

of biomolecules. Two-​color FRET can be used to monitor the displacement changes involved

between two sites of a molecule in conformational changes, for example, during power stroke

mechanisms of several molecular machines or the dynamics of protein binding and folding.

It is also possible to use more than two FRET dyes in the same sample to permit FRET effi­

ciency measurements to be made between three or more different types of dye molecule. This

permits triangulation of the 3D position of the dye molecule. These data can be mapped onto

atomic level structural information where available to provide a complementary picture of

time-​resolved changes to molecular structures.

Worked Case Example 4.2: FRET

A single-​molecule FRET experiment was performed on a short linear synthetic DNA con­

struct composed of 18 nucleotide base pairs, which was surface immobilized to a glass

coverslip in an aqueous flow-​cell, to which was attached FRET donor Cy3 (green) fluores­

cent dye molecule at one end and FRET acceptor Cy5 (red) fluorescent dye molecule at the

other end. The construct was excited into fluorescence using ALEX of a 532 and 640 nm

laser at 1 kHz modulation for each laser using TIRF excitation for which the polarization

had been circularized. In one experiment, the average brightness of the green and red dye

molecules was measured at ~7320 and ~4780 counts on an EMCCD detector when both

molecules were emitting at the same time. When a red molecule photobleached during an

acquisition in which both green and red molecules had been emitting, the average green

molecule brightness then changed to ~5380 counts. Similarly, when a green molecule

KEY BIOLOGICAL

APPLICATIONS: FRET

Determining molecular

interactions over a ~0–​5 nm

length scale.