Monitoring the interference of protein-protein interactions in vivo by bimolecular fluorescence complementation: the DnaK case.
Many cellular processes depend on protein-protein interactions. The identification of molecules able to modulate protein contacts is of significant interest for drug discovery and chemical biology. Nevertheless, finding antagonists of protein interactions that work efficiently within the cell is a challenging task. Here, we describe the novel use of bimolecular fluorescence complementation (BIFC) to detect compounds that block the interaction of target proteins in vivo. In the BIFC method, each interaction partner is fused to a complementary fragment of a fluorescent protein and interactions are detected by fluorescence restoration after reporter reassembly. Here, we demonstrate that the inhibition of specific intracellular protein interactions results in a concomitant decrease in fluorescence emission. We also show that integration of BIFC with flow cytometry might provide an effective means to detect interaction modulators by directly reading out changes in the reporter signal. The in vivo application of this approach is illustrated through monitoring the inhibition of the interaction between the Escherichia coli Hsp70 chaperone and a short peptidic substrate by pyrrhocoricin-derived antibacterial peptides.
Morell
Montse
M
Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Czihal
Patricia
P
Hoffmann
Ralf
R
Otvos
Laszlo
L
Avilés
Francesc X
FX
Ventura
Salvador
S
D023181:Antimicrobial Cationic Peptides / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005434:Flow Cytometry; D005453:Fluorescence; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D019476:Insect Proteins / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*
D023181:Antimicrobial Cationic Peptides; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D019476:Insect Proteins; C087710:pyrrhocoricin protein, Pyrrhocoris apterus; C063842:dnaK protein, E coli