The State of Biophysics - Biophysical Journal

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single-molecule measurement techniques that use the abil-
ity to detect and manipulate single molecules have become
widely adopted by the researchers. Here, I will discuss two
major fluorescence-based technologies used for single-
molecule measurements.
Single-molecule localization and tracking
stoichiometry
Imagine a soccer field being imaged from a faraway planet.
If you attach an LED lamp to your favorite soccer player,
because of the fundamental phenomenon called light
diffraction, that lamp may appear to be about the size of
the soccer field to that distant observer. Nevertheless, using
a simple mathematical trick, we can determine the position
of the lamp, or ‘‘localize’’ it, with great precision, say, down
to the size of the player’s shoe. In principle, we can track the
movements of the player and his feet with precision limited
only by how many photons are being detected to form the
image. For example, we can determine the size of his gait
during a run, or his ‘‘step size’’ if you were to use the jargon
of biophysicists. The same trick can be used to localize
individual protein nanomachines and track their positions
over time.
Using this single-molecule localization-and-tracking
approach, researchers have shown that myosin V, which
carries a cargo along a track called the actin filament, moves
in steps of 37 nm in length ). Furthermore, by labeling the
‘‘foot’’ of myosin V with a single dye, a 72-nm step length
was observed, which is double the center-of-mass step
length. The latter finding conclusively showed that myosin
V walks like a human adult, with the two feet taking the
leading position alternatingly, instead of crawling like an
inchworm ). These types of studies also showed that pro-
teins can slide along the length of DNA and determined the
rapidity of their motion and whether the motion was unidi-
rectional or not. As I will discuss below, a protein’s motion
on DNA in search of a target can be very important in keep-
ing the threat of cancer at bay.
In addition, if we label a single protein with one dye and
one dye only, we can deduce how many proteins are work-
ing together at a given time by determining the brightness of
the protein assembly; that is, if it is four times as bright as a
single dye, we can conclude that four copies of the same
protein are needed for a certain function . If multiple
dyes of different colors are used, we can also determine
how many different kinds of proteins are functioning in
the same biological process. This type of ‘‘stoichiometric’’
information is often difficult or tedious to obtain using other
approaches.
However, single-molecule localization alone cannot
follow the internal motion of the molecule. We can see
how quickly a soccer player can change direction and how
fast he runs with a ball, but this alone does not tell us
what makes Maradona a great player instead of a merely
average player. We need to increase the number of degrees
of freedom that are being observed.
Single-molecule FRET: conformational changes
and molecular interactions
One powerful technique that can follow the conformational
changes of a molecule is fluorescence resonance energy
transfer (FRET). ‘‘Conformation’’ is jargon used by biolo-
gists to denote the shape of a molecule. Just as a soccer
player needs to change his bodily shape or posture to run
and handle a ball, proteins need to change their conforma-
tions repeatedly to carry out their duties. In FRET, dyes of
two different colors, say green and red, are attached to
two sites of a protein. Normally, when we excite the green
dye with a laser, we would see only green photons coming
out. But when the two are very close to each other, within
a few nanometers, the two dyes communicate with each
other and the excitation energy is transferred from the green
dye to the red dye such that we now see red photons come
out. The relative ratio of the two colors is then used as a
measure of their distance from each other, and if we know
where the dyes are attached on the protein, we can deduce
conformational changes of the molecule. shows a
cartoon of a rap musician dancing, undergoing conforma-
tional changes between different postures that are detected
as anticorrelated changes in the intensities of green and
red signals.
Let me illustrate the use of FRET to study DNA repair.
Inside every cell of our body there are two meters of
DNA. Because there are ~1014 human cells in our body,
with the estimated renewal of 100 times for an average
cell during our lifetime, our body would need to make about
one light year length of DNA. DNA is under constant threat
of damage. For example, sunlight and smoking can cause
DNA damage that can accumulate and eventually lead to
cancer. If DNA repair did not exist when we need to make
so much DNA, we would die of cancer at a far younger
age than is usually the case.
One major mechanism of DNA repair is called homolo-
gous recombination. When a segment of DNA is broken,
the cell uses another copy of the same DNA as a template
to repair the breakage. To aid this process, a protein filament
is formed around the broken DNA, and this filament then
searches for a matching DNA sequence in a sea of millions
of basepairs of DNA. This is no easy task and is often
compared to finding a needle in a haystack.
How does the cell accomplish this feat rapidly yet accu-
rately? One possibility is to perform a three-dimensional
search. Let the filament land on a random location of the
target DNA, and if there is no sequence match, then disso-
ciate and repeat until a match is found. It is equivalent to
dating random people on the street until a soul mate is
found, which would be exceedingly time consuming if
you live in a big city. Another possibility is to perform
Biophysical Journal 110(5) 1004–1007
Probing Nature’s Nanomachines 1005