Chemistry Reference
In-Depth Information
The dynamic information from single-molecule spectroscopy can complement the
fine structural detail obtained using crystallography and NMR for structure
determination.
We will review in detail a few recent case studies which illustrate the power of
single-molecule approaches, and brie
y survey the growing amount of work using
thesemethods. For each example, we will examine how the authors of the papers used
many of the principles and procedures outlined in Sections 9.2 and 9.3 to obtain the
information sought.
9.2
Fluorescence Spectroscopy as a Tool for Dynamic Measurements of Molecular
Conformation and Interactions
There are ways to perform
finer measurements of distance, measurements of faster
timescales, and less invasive ways to measure protein dynamics, but
uorescence
makes up for all of these in sensitivity. Fluorescence can be measured from single
molecules, and this has caused increased interest in the methods available to extract
the information needed from
fluorescence techniques.
9.2.1
Jablonski Diagram (Intensity, Spectrum, Lifetime, Polarization)
The Jablonski diagrams shown in Figure 9.1 is a representation of the energy levels of
a
fluorescent molecule, or
uorophore [9]. The ground state S
0
is shown as a thick
black line. Also part of S
0
is a series of vibronic energy levels. These differ from the
ground state only in vibrational energy of the
uorophore. The
first excited state S
1
differs from S
0
by an energy E
S1
-
fluorophores used in single-molecule
studies operate in the visible region, so the energy is 1.8
-
3.1 eV, or using E
E
S0
. The
ΒΌ
h
n
a
l
a
is between 400 and 700 nm. A photon is absorbed by the
fluorophore with
energy E, which excites the molecule fromS
0
to S
1
. The rate at which the
uorophore
is excited depends on the intensity of the incident light and the absorption cross-
section of the
fluorophore at the wavelength of the incident light. Typically, absorp-
tion cross-sections are quoted as molar extinction coef
cients (in M
1
cm
1
).
The excitation of the
fluorophore from S
0
to S
1
with rate k
e
can be to any of the
vibronic energy levels of S
1
. The vibrational energy is quickly dissipated (
hc/
l
a
,
1 ps),
and the
fluorophore remains in the lowest vibronic energy level of S
1
. At this point,
the
fluorophore waits in the excited state until one of four processes occurs. First, the
fluorophore may emit a photon; this process has a rate k
r
. Second, the
uorophore
may de-excite non-radiatively, with rate k
nr
. Third, the
uorophore may undergo
intersystemcrossing to a triplet state with rate k
ISC
, whichmay also emit a red-shifted
phosphorescence photon with rate k
Ph
. Fourth, the
uorophore may undergo
photobleaching with rate k
bl
, which chemically modi
es the
fluorophore so that it
no longer
uoresces. The
uorescence rate k
r
is maximized for good
uorophores,
while the other processes are minimized.
