Chemistry Reference
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acceptor time-averaged over the measurement time of 100
s to 1ms. As demon-
strated by Schuler et al. [81], this is insuf
cient time resolution to detect any structural
fluctuations of the unfolded protein. For a folded protein, there is in general one
structure, with only small
fluctuations from this structure. There may be changes to
the structure due to interactions with other proteins, DNA, or small molecules, but
these are very different from the unfolded protein. The unfolded state of a protein is
an ensemble of many random conformations of the protein chain that are inter-
converting rapidly due to thermal
m
fluctuations.
fluorescence lifetime of FRET have been used for many years at
the ensemble level to monitor distributions of distances within unfolded and folded
proteins [84]. Using time-correlated single photon counting (TCSPC), the time
between a laser pulse and the
fluorescence photon is timed to 4
-
50-ps accuracy.
Fluorophores generally exhibit single exponential lifetime
uorescence decays,
which are shortened by the process of FRET. Shorter lifetimes indicate higher FRET
ef
ciency, and longer lifetimes indicate lower FRETef
ciency. If the donor exhibits a
single-exponential
fluorescence lifetime decay with FRET, there is a single distance
between the donor and acceptor. Multi-exponential decays for the donor undergoing
FRET indicate multiple distances. By analyzing such decays, it is possible to resolve
distance distributions down to the nanosecond time scale.
In Laurence et al. [44], we introduced several methodological improvements to
single-molecule protein-folding studies that provide additional information on
fluorescence lifetime and polarization. In this study, we were able to combine
nearly all of the observables available for a single FRET pair, and use them
advantageously. Following Seidel and collaborators, we increased the number of
detection channels to four, further dividing the donor and acceptor
uorescence
emission by polarization, and we used TCSPC to measure the
fluorescence lifetime
of the donor and acceptor. Lastly, we introduced alternating laser excitation (ALEX)
using two interlaced, pulsed lasers, one laser exciting the donor, and one laser
exciting the acceptor. For the acceptor, there are lifetime curves both for excitation
by the donor laser (via FRET) and by the acceptor laser (via direct excitation). Using
the ALEX procedure allows us to distinguish between subpopulations with donor-
only, acceptor-only, folded and unfolded proteins. After this procedure of single-
molecule sorting, we select all bursts emanating from the unfolded protein, and
form
fluorescence lifetime decays for that subpopulation. We then globally
fit all of
the lifetime curves, obtaining information on distance distributions along with the
polarization information.
Using this analysis, we determined the mean and width of the distance distribu-
tions of dsDNA, ssDNAwith various salt concentrations, and two unfolded proteins.
For random polymer
fluctuations there is a limit to the possible widths of the
distributions. Distribution widths for the nucleic acids were below or at this limit,
but, interestingly, the unfolded proteins exhibited larger
uctuations than possible by
random polymer
uctuations. These excess
fluctuations increased as the denatur-
ant decreased. We proposed that these
fluctuations are due to transient formation of
residual structure, although transient compaction of the unfolded state is another
possible explanation [85] (Figure 9.7).
Measurements of
