Quantitative analysis of multi-protein interactions using FRET: application to the SUMO pathway.
Protein-protein binding and signaling pathways are important fields of biomedical science. Here we report simple optical methods for the determination of the equilibrium binding constant K(d) of protein-protein interactions as well as quantitative studies of biochemical cascades. The techniques are based on steady-state and time-resolved fluorescence resonance energy transfer (FRET) between ECFP and Venus-YFP fused to proteins of the SUMO family. Using FRET has several advantages over conventional free-solution techniques such as isothermal titration calorimetry (ITC): Concentrations are determined accurately by absorbance, highly sensitive binding signals enable the analysis of small quantities, and assays are compatible with multi-well plate format. Most importantly, our FRET-based techniques enable us to measure the effect of other molecules on the binding of two proteins of interest, which is not straightforward with other approaches. These assays provide powerful tools for the study of competitive biochemical cascades and the extent to which drug candidates modify protein interactions.
Martin
Sarah F
SF
Biophotonics Collaboration, School of Physics and Astronomy, University of St. Andrews, St. Andrews KY16 9SS, United Kingdom.
Tatham
Michael H
MH
Hay
Ronald T
RT
Samuel
Ifor D W
ID
D000818:Animals; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D025842:SUMO-1 Protein / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D000098843:Ubiquitin-Conjugating Enzyme UBC9
C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; D025842:SUMO-1 Protein; C094493:ran-binding protein 2; D049452:Green Fluorescent Proteins; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9