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Quantitative analysis of multi‐protein interactions using FRET: Application to the SUMO pathway
Author(s) -
Martin Sarah F.,
Tatham Michael H.,
Hay Ronald T.,
Samuel Ifor D.W.
Publication year - 2008
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.073369608
Subject(s) - förster resonance energy transfer , isothermal titration calorimetry , chemistry , protein–protein interaction , biophysics , small molecule , fkbp , plasma protein binding , fluorescence , computational biology , biochemistry , biology , physics , quantum mechanics
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.