Biology Reference
In-Depth Information
3
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
1.8
1.6
1.5
1.4
cdk2/cyclin A
cdk7/cyclin H
cdc2/cyclin A
1.4
1.3
1.2
0
0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1
Time (s)
1.2
0
500
1000
1500
2000
2500
0
2
4
6
8
10
12
Cyclin (nM)
Time (s)
Figure 4.13 Monitoring protein/protein interaction using fluorescently labeled sub-
strates. (A) Binding titration of cyclin to their Cdk partners. The binding was followed
by Mant-ATP fluorescence enhancement. A fixed concentration of protein kinase
(Cdk2, Cdc2, or Cdk7) previously saturated with Mant-ATP was titrated with increasing
amounts of cyclin. The enhancement of Mant-ATP fluorescence was monitored at
450 nm upon excitation at 340 nm. Dissociation constants were obtained by fitting
the data with a quadratic equation. (B) Kinetics of binding of Cdk2 to cyclin A and cyclin
H. kinetics of binding of Cdk2 (0.1 mM) to cyclins (0.4 mM). Cyclin A (red) and cyclin H
(blue). Excitation was performed at 350 nm, and fluorescence emission of Mant-ATP
was monitored through a cutoff filter (408 nm). Curves were fitted using a double-
exponential term.
3.2. Probing peptide/protein interactions
Protein/protein interaction studies provide a better understanding of the dif-
ferent parameters that rule the association of protein complexes. Analyses of
these parameters enable the development of shorter peptides with similar in-
teraction properties. These peptides may then associatewith complexes in the
same fashion as the protein partners and induce activation and/or inhibition
of the whole complexes. This approach has been extensively developed to
design novel inhibitors of specific protein/protein interactions as well as bio-
sensors of protein complexes. 75-78 In parallel to the inhibitor, activator, or
biosensor approaches, short peptides have also been developed for drug
delivery. From the fluorescence point of view, as for protein/protein
interactions, the intrinsic tryptophan fluorescence cannot be systematically
used to monitor association since the cargo protein has to be devoid
of tryptophan residues. In this case, the approach allows for the recording
of intrinsic tryptophan fluorescence spectra of peptides in the presence of
increasing amounts of cargo protein. Thus
interactions may induce
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