Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 771
Figure 23.24. Single-molecule enzyme kinetics of ATPase activity. The enzyme was myosin subfragment 1 (S1). The measurements were performed
using objective-type TIR. Reprinted with permission from [54].
ATPase the Cy3 bound to S1 was photobleached to eliminate
its emission. This location was then observed after
addition of Cy3-ATP. The intensity shows transient increases
when Cy3-ATP/ADP is immobilized on the enzyme. The
intensity returns to background when the Cy3-ADP dissociates
from the protein. This experiment was performed
using objective-type TIR. The small thickness of the illuminated
area was important to reduce the background intensity
from freely diffusing Cy3-ATP.
23.6.3. Single-Molecule Studies of a
Chaperonin Protein
The concept shown in Figure 23.23 can be used to study
any association reaction. Suppose the surface contains a
green capture protein that binds to a red protein. Green dots
will be seen whenever there is a capture protein. Red dots
will only appear when this protein binds to the capture protein.
Co-localization of green and red spots will indicate the
presence of a complex on the surface. This concept has
been applied to the study of chaperonin GroEL.
Chaperonins are a class of proteins that promote folding
reactions in cells. They can facilitate folding of newly
synthesized proteins or the refolding of denatured proteins.
A well-known chaperonin is GroEL from (Escherichia
coli). GroEL is a large cylindrical protein containing 12
subunits (Figure 23.25). GroEL was labeled with an indocyanine
dye IC5 and bound to a glass surface. 55 Bovine
(BSA) and human serum albumin (HSA) spontaneously
Figure 23.25. Chaperonin GroEL and GroES. GroEL is labeled
with IC5 and GroES is labeled with Cy3. Reprinted with permission
from [55].