Fluorescence Technologies for Monitoring Interactions Between Biological Molecules In Vitro - Progress in Molecular Biology and Translational Science - page 131

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Figure 4.12 Monitoring acetonitrile-mediated RT dissociation kinetics using a
noncovalent external probe and the implication for inhibitor sensing. Heterodiomeric
form or RT (0.5 mM) can be dissociated using acetonitrile and dissociation kinetics
followed in the presence of 0.8 mM bis-ANS. The kinetics of dissociation was monitored
by following the fluorescence resonance energy transfer between tryptophan of RT and
bis-ANS. Tryptophan excitation was performed at 290 nm, and the increase of bis-ANS
fluorescence emission at 490 nmwas detected through a 420 nm cutoff filter, by adding
10% acetonitrile in the absence (black line) or presence of a dimerization inhibitor(gray
line). Data were fitted according to a single-exponential equation (adapted from
Agopian et al.). 41

or the protein/protein interaction results in conformational changes within
the substrate binding site locatedon one of the subunits. The substrate binding
site is located at the interface 37 or formed by the associationof the two subunits
as in the case of the dimeric polymerase RT, 70 Mdm2/P53 interfaces, 71 or
protease. 72 Peptide beacon/biosensor (HIV and caspase biosensors) 73 or fluo-
rescently labeled nucleic acid 40 has been used tomonitor protease or RNA or
DNA polymerase activation as discussed in Section 3 .
Monitoring protein/protein interactions does not necessarily require an
interface substrate binding site. Several studies have shown that protein/
protein interactions induce a marked conformational change in the closed
environment of the catalytic site. As reported in Fig. 4.13 , binding of cyclin
A to Cdk2 results in an important fluorescence change in Mant-ATP bound
to the Cdk moiety. 28 This change in fluorescence has been used to probe
cyclin/Cdk interactions at the steady-state and pre-steady-state levels and
to discriminate between the different Cyclin partners ( Fig. 4.13 ). 27 Similar
studies were also performed to understand the interaction of the ras onco-
gene with the GAP exchange factor. 74