Chemistry Reference
In-Depth Information
Figure 9.3 Two basic labeling configurations
used for monitoring protein dynamics and
interactions. Both are based on using FRET to
dynamicallymonitor distances between two sites
of proteins. (A) Monitoring internal protein
dynamics is accomplished by site-specifically
labeling one protein with both donor and
acceptor. As the protein changes conformation,
folds or unfolds, changes in the distance between
D and A are monitored in real time using FRET.
(B) Dynamic monitoring of protein interactions
is accomplished by site-specifically labeling two
interacting molecules, one with D and one with
A. FRET is used to monitor distance changes
between the two sites, and the localization as
described in Figure 9.2 is used for detecting
association of the molecules. (C) Fluorescence
intensities of D (yellow) and A (red) are
monitored as a function of time for a single
molecule. If the molecule is immobilized,
changes in distance between D and A are seen as
anti-correlated changes in the intensity of D and
A (when A goes up, D goes down). (D) For freely
diffusing molecules, observation times are
typically too short to see changes in structure.
Small snapshots of Dand A can be seen, allowing
the formation of histograms of distance
distributions of all molecules. Adapted from [25].
The most general way to measure conformational changes in a protein, DNA,
RNA, etc., either internal
fluctuations or externally-induced changes, is to monitor
the distance between two points in the protein using FRET (Figure 9.3B). For
appropriately chosen labeling sites, single-molecule measurements will allow the
conformational state to be monitored for extended periods (Figure 9.3C). Distances
shorter than 3
-
4 nm can be monitored using electron transfer. Fluorescence polari-
zation may also be used as an observable; in this case, rather than monitoring
distance between two points, the mobility of the
fluorophore or the rotational
movement of the protein is monitored.
We can use some of these same observables for monitoring changes in the
con
guration of interactions (Figure 9.3B). FRETand ETmay be used to dynamically
