Chemistry Reference
In-Depth Information
(R/R
0
)
6
)
Figure 9.8 Accurate E measurements may be
performed at the single-molecule level.
cyan crosses. FRET model (E
¼
1/1
þ
2
i
measured by nsALEX versus distance between
attachment points for Dand A for dsDNAwith 7-,
12-, 17-, 22-, and 27-base pair separations (black
squares). h
h
E
with measured R
0
¼
2/3 (solid
red) and simulation accounting for linkers and
the measured slower rotational diffusion
(t
69 Å and
hk
i¼
r
r
3ns; solid black) are
shown. Simulations were adjusted to R
0
¼
¼
3
:
0nsand t
¼
1
:
i calculated from calibrated single-
molecule intensity ratios rather than
fluorescence lifetime information are shown as
E
62 Å
to match gray squares. Reproduced with
permission from [44].
to be timed. By comparing the observed folding and unfolding trajectories for
proteins exhibiting complex [88] and simple two-state [87] folding pathways, the
roughness of the free energy surfaces can be probed using single-molecule spec-
troscopy (Figure 9.9).
A second approach to facilitating immobilized protein-folding studies of single
molecules was to passivate the surface in a better way. Nienhaus and colleagues
introduced surface coatings made of ultrathin networks of isocyanate-terminated
star-shaped polyethylene oxide (PEO) molecules, cross-linked at their ends via urea
groups to minimize the intertwining of a denatured polypeptide chain with the PEO-
polymer. Immobilizing proteins on this surface allowed single-molecule protein-
folding trajectories to be monitored [89]. Repeated changes in denaturant concen-
trations demonstrated that folding and unfolding were completely reversible on the
specialized surface.
9.4.1.7 Probing Biomolecular Dynamics via Fluorescence Quenching and Electron
Transfer
Fluorescence quenching by electron transfer or other processes promises to provide a
method of measuring contacts between two sites at short distances at the single-
molecule level. Correlations calculated from these
fluctuating signals would reveal
the time scales of the
fluctuations directly. Folding and unfolded of DNA hairpins
were monitored using FCS on a donor-quencher system [90]. An ensemble method
was used to provide information on time scales of unfolded protein and nucleic acid
fluctuations [91, 92]. Electron transfer from native
fluorophores was used to monitor
the
fluctuations of an enzyme at the Angstrom scale [93]. The greatest weakness in
