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TP‐2Rho Is a Sensitive Solvatochromic Red‐Shifted Probe for Monitoring the Interactions between CDK4 and Cyclin D
Author(s) -
Pellerano Morgan,
NaudMartin Delphine,
Peyressatre Marion,
Prével Camille,
TeuladeFichou MariePaule,
Morris May,
MahuteauBetzer Florence
Publication year - 2016
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201500641
Subject(s) - cyclin dependent kinase complex , protein subunit , peptide , fluorescence , polarity (international relations) , chemistry , solvatochromism , substrate (aquarium) , protein kinase a , biophysics , biochemistry , kinase , biology , cyclin dependent kinase 2 , ecology , physics , quantum mechanics , solvent , cell , gene
Understanding the intricate steps of protein kinase regulation requires characterization of protein–protein interactions between the catalytic subunit, its regulatory partners and the substrate. Fluorescent probes are useful tools with which to study such interactions and to gain insight into their affinities and specificities. Solvatochromic probes, which display changes in their fluorescence emission in response to changes in the polarity of the medium, are particularly attractive. Here we describe conjugation of a switchable fluorescent dye, TP‐2Rho, to peptide and protein derivatives of cyclin‐dependent kinase 4 (CDK4) and its application to characterization of the interactions between the catalytic subunit of this kinase, its regulatory partner cyclin D1 and a peptide substrate. We demonstrate the sensitivity of TP‐2Rho in relation to of those other dyes used for monitoring peptide–protein and protein–protein interactions. Moreover, we show that TP‐Rho‐labelled peptides can be introduced into living cells to probe endogenous CDK4/cyclin D.