Chapter 4 – Making Light Work Harder in Biology  131

photobleached during an acquisition in which both green and red molecules had been

emitting, the average red molecule brightness then changed to ~6450 counts.

a

Explain these observations and estimate the Förster radius for Cy3–​Cy5, stating any

assumptions you make.

A molecular machine that was known to undergo a conformational change by a

“lever arm” component of the whole molecular complex upon hydrolyzing ATP had

two protein subunits X and Y labeled with Cy3 (green) and Cy5 (red) fluorescent dyes,

respectively, in a single-​molecule ALEX FRET experiment in vitro, one attached to the

lever arm and the other attached on a nonlever component of the complex, here,

involving diffusion of the molecular machine complex through a confocal excitation

volume. In one experiment, a “loose” short linker was used between the dyes and the

proteins allowing free rotation of the dyes. For this, addition of ATP resulted in chan­

ging the average green and red channels’ signals from ~5560 and ~6270 counts,

respectively, before the addition of ATP and ~7040 and ~5130 counts in the first ~5

ms after the addition of ATP.

b

Calculate the average speed in microns per second of the molecular conformational

change due to ATP hydrolysis stating any assumptions you make.

In a second experiment, a “tight” linker has prevented any free rotation between

the dyes and the protein subunits, and a chemical cross-​linker was added to pre­

vent any relative translational change between the two dye loci. For this, addition

of ATP resulted in changing the average green and red channels’ signals from

roughly the same values, respectively, as for the part (b) experiment earlier before

the addition of ATP, but ~6100 and ~5800 counts in the first ~5 ms after the add­

ition of ATP.

c

Explain these observations, and comment on previous speculation that the molecular

machine might operate via either a “pincer” or a “twister” type of action.

Answers

a

The average distance between Cy3 and Cy5 molecules is ~18 × 0.34 =​ 6.12 nm

(see Chapter 2), which is within the typical “FRET ruler” scale of ~0–​10 nm; thus,

one might expect FRET to be occurring when both molecules are emitting. When

the donor (green) molecule bleaches, the FRET transfer from green to red is

stopped and so the acceptor (red) molecule brightness goes down. Similarly, if

the acceptor molecule (red) bleaches first, then no energy is subsequently trans­

ferred from the green to the red molecule by FRET, and so the green molecule

brightness goes up. Assuming isotropic emissions of the dyes in the time scale

of sampling, no polarization dependence of fluorescence excitation for TIRF, that

the height of the dyes molecules are the same in evanescent field (i.e., construct is

sitting “flat”), and that there is minimal bleed-​through between the acceptor and

donor detector channels, the FRET efficiency can then be calculated using either

the donor intensity as

1

4780

−

=

(

counts)/6450 counts)

0.36

or the acceptor intensity as

(7320

5380

−

=

counts)/(5380 counts)

0.36

Thus, from Equation 4.7 the Förster radius is

( .

/

6 12

1 6

nm)/(1/0.36

1)

5.6 nm

−

=