We have investigated the kinetics of the conformational changes of a new type of molecular beacons called tripartite
molecular beacons. The rate constants corresponding to the opening and closing of the beacons have been obtained from
fluorescence correlation spectroscopy experiments. We found that both rate constants are larger for the tripartite
molecular beacon than for the corresponding molecular beacon. This paper outlines the importance of using very low
excitation intensities for this type of measurements, and of considering the fact that the beacon still emits a residual
background fluorescence in its closed form. We also report on the exploration of several strategies to improve the
precision of the measurements by increasing the characteristic time associated with the diffusion of the beacons so that
it would not be confused with the relaxation time associated with the conformational changes.
Using fluorescence correlation spectroscopy we measured the apparent mobility of a nuclear transport cargo (a streptavidin labeled with a nuclear localization signal) both in the cytoplasm and the nucleus of living cells, and we compared it to the mobility of a streptavidin labeled with mutations of the nuclear localization signal known not to support nuclear import, and with the mobility of a set of inert molecules (dextrans) of different sizes. In the cytoplasm, the mobility of the transport cargo is found to be significantly reduced compared to its mobility in the nucleus, or to the mobility of the streptavidins labeled with a mutant nuclear localization signal. This can be partly explained by the fact that the transport cargo forms a complex with two nuclear import mediator proteins (importin α and importin β) in the cytoplasm, but could also be partly due to specific interactions of this cargo with the cell cytoskeleton.