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state model protein Chymotrypsin Inhibitor 2 (CI2). The ability to monitor long time
trajectories (
1ms) was sacri ced, but the FRET ef ciency E was calculated for
each protein as it diffused through the optically isolated detection volume. The
distribution at low denaturant contained a single peak at high E indicating a compact,
folded conformation. The distribution at high denaturant also contained a single
peak, but now at low E, indicating a more extended, unfolded conformation. At the
midpoint of the sigmoidal curve, when 50% of the protein is unfolded, both peaks
were present in the E distribution, indicated two interconverting subpopulations in
solution. A good correspondence between the denaturation curves from ensemble
and single-molecule measurements was found (Figure 9.5).
Simple proteins often have two states as extensively documented by many
experiments over the years. We chose a simple, a two-state model protein folder
precisely to demonstrate the validity of single-molecule level measurements. Even so,
for many researchers we have talked to, the initial response to the single-molecule
experiments showing folded and unfolded subpopulations is satisfaction that there
really are two well-separated, interconverting subpopulations. The more direct
>
Figure 9.5 FRET efficiency distributions of chymotrypsin inhibitor
2 (CI2) at three different denaturant concentrations. Top: 3M
GdmCl (native conditions), middle: 4M GdmCl (close to the
midpoint of unfolding), bottom: 6MGdmCl (strongly denaturing
conditions). The bimodal distribution of the FRET-efficiency
clearly indicates the two-state nature of the unfolding process.
Reproduced with permission from [80].
 
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