Principles of Fluorescence Spectroscopy

784 SINGLE-MOLECULE DETECTION
Figure 23.49. Calcium sensor based on a GFP donor–acceptor pair
linked by calmodulin (CaM) and the M13 peptide. The panels show
the single-molecule RET efficiency. Reprinted with permission from
[10, 89].
23.10.4. Single-Molecule Calcium Sensor
Single-molecule detection has been extended to single-molecule
sensors. 89 A single-molecule sensor was designed according
using two GFPs as a donor–acceptor pair, linked by
calcium-sensitive proteins (Figure 23.49). The linker consisted
of calmodulin (CaM) and the M13 peptide. In the
presence of calcium CaM expresses a hydrophobic region
that binds the M13 peptide, bringing the GFPs closer
together. 90 Single molecules of a similar sensor were ob-
served using a confocal scanning microscope. The individual
molecules were examined at various concentrations of
calcium. Histograms were constructed showing the number
of times a particular transfer efficiency was observed. These
histograms show the closer average distance of the donor
and acceptor in the presence of calcium. In this case the distributions
are rather wide, with overlap of the high- and
low-calcium histograms. This suggests that this sensor
adopts more than two conformations in the presence of calcium.
23.10.5. Motions of Molecular Motors
Motion occurs constantly in cells and tissues, and are due to
a variety of proteins. Intracellular transport of organelles,
mRNA, and other molecules is due in part to kinesin.
Kinesin is a dimeric protein that moves along actin filaments
while it consumes ATP. Single-molecule imaging has
been used to follow the motion of kinesin 91 and other proteins.
92–95 For kinesin there were two possible modes of
motion: hand-over-hand and inchworm motion (Figure
23.50). Previous studies had shown that the central stalk in
kinesin does not rotate during motion, so this mechanism
was not considered.
To study kinesin motions one of the actin-binding
domains was labeled with Cy3 and the other left unlabeled.
91 Actin filaments were immobilized on the slides to
provide a binding site for kinesin. Upon addition of ATP the
Cy3 spot was found to move in discrete steps (Figure 23.50,
lower panel). Several different kinesin mutants were labeled
and studied, and all displayed similar step sizes. The size of
the steps allowed selection of the hand-over-hand model for
kinesin motion. The average step size of 17 nm was consistent
with the size of two actin molecules. The inchworm
mode of motion would have resulted in a smaller 8.3-nm
step size.
23.11. ADVANCED TOPICS IN SMD
23.11.1. Signal-to-Noise Ratio in
Single-Molecule Detection
We have now seen examples of SMD. These experiments
all had one feature in common, which is careful design of
the sample and optical system to achieve an adequate S/N
ratio. In order to detect a single molecule the signal from
the molecule must be larger than the fluctuations in the
background signal. Additionally, all single-molecule exper-