Fluorescence Technologies for Monitoring Interactions Between Biological Molecules In Vitro - Progress in Molecular Biology and Translational Science - page 128

3. MONITORING PROTEIN/PROTEIN AND POLYPEPTIDE
INTERACTIONS
Protein/protein interactions control major pathways in biology and nu-
merous technologies have been developed to better understand these pro-
cesses. 32 Indeed technologies such as cross-linking, pull-downs, and mass
spectrometry require cytosolic extraction by cellular lysis, which may induce
a loss of the natural properties, especially in situ interactions. In addition, dou-
ble or reverse hybrid strategies involve genetic constructs that may interfere
with the real function of the targeted proteins and/or induce detection of false
positives. In order to optimize sensitivity and specificity and to decrease the
invasiveness of other methods, large numbers of both natural and synthetic
fluorescent probes have been developed, yielding a toolbox of probes for
the characterization of protein/protein interactions. 64 The grafting of fluores-
cent probes enables the detection and quantification of the interactions
between protein and partners. A large number of fluorescence approaches
have been developed to improve the knowledge of protein/protein interac-
tions in vitro , fromsteady-state tokinetic investigations, and a combinationof all
of these approaches provides a better understanding of protein/protein and
peptide/protein interactions and multiprotein complex formation.
3.1. Probing protein/protein interactions
An important requirement in setting up fluorescence experiments to
investigate or monitor protein/protein interactions is the selection of an
appropriate probe to follow the interaction/dissociation of complexes in a
noninvasive manner. Ideally, one should monitor changes in intrinsic
protein fluorescence, mainly related to tryptophan. Tryptophan residues
constitute sensitive probes that are often located at protein/protein interfaces
and/or involved in substrate or ligand binding domains. Extrinsic fluores-
cence has also been applied using solvatochromic dyes which are conjugated
to proteins or dyes compatible for FRET between two partners (protein/
protein or protein/ligand). In the latter, it is important to validate that
the presence of the dye does not alter protein structure and/or function.
Protein complex association/dissociation and reversible unfolding can be
followed by size exclusion chromatography, circular dichro¨sm, analytical
centrifugation, and fluorescence spectroscopy. 37 The sensitivity of
fluorescence to its environment has been largely employed to understand