Principles of Fluorescence Spectroscopy

772 SINGLE-MOLECULE DETECTION
Figure 23.26. Single-molecule images from IC5-labeled GroEL (EL) and Cy3-labeled GroES (ES) at the indicated times. The images were obtained
using TIR and an intensified CCD camera. The circles show the positions of GroEL. Reprinted with permission from [55].
bind to glass surfaces. If the BSA is biotinylated then avidin
or streptavidin binds strongly to the surface. Streptavidin
contains four biotin binding sites, providing residual binding
sites for biotinylated GroEL. This method of attachment
is frequently used to attach biomolecules to surfaces.
This system was examined using TIR excitation and an
intensified CCD camera for imaging. The upper left panel
in Figure 23.26 shows the locations of GroEL, as determined
by emission from IC5. The remaining three panels
show images of Cy3-GroES. In the presence of ATP GroES
binds to GroEL and the complex acts to refold proteins. At
first the images of GroES are confusing. Some appear
where GroEL is also located and other GroES molecules
appear where there appeared to be no GroEL. In either case
the GroES has to be immobilized to see its emission. The
different positions of GroES binding can be understood as
due to molecular heterogeneity. The surface contains
GroEL molecules that are labeled with IC5 and GroEL molecules
that are not labeled with IC5. This shows how different
types of reasoning are needed to interpret single-molecule
and ensemble measurements.
The arrows in Figure 23.26 point to one GroEL molecule
where a GroES molecule also binds. The GroES molecule
appears in 2 of the 3 frames. In this case the absence
of GroES in the 80-second frame is not due to blinking, but
rather to dissociation from GroEL. The process of GroES
binding to GroEL can be followed with time (Figure 23.27).
The Cy3-GroES emission appears and disappears as it
binds to and dissociates from GroEL. This observation of
single-molecule binding kinetics provides a unique way to
measure association and dissociation rates for the reaction.
The times when GroES is observed and when it is missing
provides a statistical measure of the rate constants. If a suitably
large number of events are studied the fluctuations in
Cy3-GroES emission can be used to determine the on and
off rates for the reaction.
Histograms of the on and off times are shown in Figure
23.28. The top panel of Figure 23.28 shows a histogram of
the off time, the time duration when emission from Cy3labeled
GroES is not observed. A large number of shortduration
events were observed. A smaller number of events
were observed for longer durations. This type of data can be