Detection of transient protein-protein interactions by bimolecular fluorescence complementation: the Abl-SH3 case.
Protein-protein interactions are essential in most biological processes. Many proteomic approaches have succeeded in the identification of strong and obligatory interactions but the study of weak and transient protein-protein interactions is still a challenge. The aim of the present study was to test the ability of bimolecular fluorescence complementation to detect and discriminate in vivo weak intracellular protein interactions. As a test case, the interaction of the SH3 domain from the c-Abl tyrosine kinase with both natural and designed targets has been chosen. The reassociation of functional yellow fluorescent protein (YFP) from its fragments requires previous binding between the SH3 domain and its partners; but once this occurs, the complex is trapped, turning transient SH3 interactions into stable, easily detectable ones. The method is very sensitive and can be implemented for proteomic analysis of weak protein interactions using flow cytometry. The fluorescence emission is dependent on the strength of the interaction, in such a way that it can be used, at least qualitatively, to screen for best binding candidates among similar proline-rich peptides. In addition, it is illustrated how this method can be used to gain structural insights into particular c-Abl SH3 interactions.
Morell
Montse
M
Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Espargaró
Alba
A
Avilés
Francesc X
FX
Ventura
Salvador
S
D000818:Animals; D019313:BRCA1 Protein / Q000378:metabolism; D004586:Electrophoresis; D005434:Flow Cytometry; D005453:Fluorescence; D008164:Luminescent Proteins / Q000378:metabolism; D008958:Models, Molecular; D010455:Peptides / Q000378:metabolism; D010544:Peroxidases / Q000378:metabolism; D054464:Peroxiredoxins; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D016315:Proto-Oncogene Proteins c-abl / Q000737:chemistry* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D018909:src Homology Domains
D019313:BRCA1 Protein; D008164:Luminescent Proteins; D010455:Peptides; D010544:Peroxidases; D054464:Peroxiredoxins; D016315:Proto-Oncogene Proteins c-abl