Chemistry Reference
In-Depth Information
Figure 9.6 Probing single-molecule protein
folding kinetics under non-equilibrium
conditions. (A) Top: view of the mixing region of
the microfluidic laminar mixing device, with the
denaturant concentration indicated by a false
color code. The laser beam (light blue) and
collected fluorescence (yellow) are shown
100
mixing time. Within the first 100ms of mixing,
the expanded chain collapsed into a more
compact coil structure, indicated by the slight
increase in mean E. The collapsed denatured
species reconfigure into the compact folded
state on a timescale of a few seconds. Note that
the mean E of the collapsed denatured
subpopulation does not vary with time (red line)
nor final denaturant concentration (data not
shown). Reproduced with permission from [83].
m from the center inlet. Bottom: cross-
section of the mixing region. (B) Histograms of
measured FRET-efficiency as a function of the
m
conditions. They found that the unfolded population becomes more compact
rapidly (within 100ms), matching the size of unfolded protein in their previous
measurements. This validates the previous detection of change in size of the
unfolded protein at lower denaturant concentrations, and allowed the study of this
more compact unfolded state at denaturant concentrations down to 0M. Subsequent
to this fast compaction of the unfolded protein, the population of folded proteins
increases as the population of unfolded proteins decreases. Rather than a shift in the
position of the peak in the E distribution that accompanies the fast compaction of the
unfolded state, the folding process is seen as changes in the relative populations of
two states (Figure 9.6).
Such non-equilibrium studies hold great promise in elucidating the nature of the
protein folding process.
9.4.1.5 Monitoring Conformational Dynamics using Fluorescence Lifetime
In the protein-folding experiments described above, the only information recorded
from the molecules was the intensity of the signal from the donor and acceptor. What
is obtained from suchmeasurements is a distance measurement between donor and
