Quantitative analysis of multi‐protein interactions using FRET: Application to the SUMO pathway

Abstract

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 proteinprotein 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.

Association of a Novel Preribosomal Complex in Trypanosoma brucei Determined by Fluorescence Resonance Energy Transfer

“…Cerulean-L5 showed two absorption (blue) peaks at 424 nm and 433 nm and an emission (green) peak at 476 nm with a shoulder at 502 nm, while eYFP-P34 showed two absorption (red) peaks at 483 nm and 519 nm and a single emission (purple) peak at 530 nm. These results are consistent with the previously reported fluorescence properties of cerulean and eYFP (16,20). As a comparison, L5 and P34 are nonfluorescent in this range (data not shown).…”

“…Emission spectra were collected from 450 nm to 600 nm (excitation, 400 nm; 1-nm slit width; sensitivity, 100). Excitation at 400 nm improves the dynamic range as the excitation of eYFP is at a minimum level (20). Excitation at 400 nm leads to emission of cerulean at 475 nm.…”

“…3A. During the titration of eYFP-P34, emission at 530 nm increased in proportion to the number of bound pairs (20,22).…”

“…The saturation level, determined by an exponential fit, corresponds to 1 M cerulean-L5 bound. Using this relationship between the FRET signal and bound species and a binding stoichiometry of 1:1 (20,23), data were converted into the relative amount of bound cerulean-L5 and eYFP-P34. The free eYFP-P34 is calculated in a subtraction for each point and with the axis rescaled to give the bound protein versus free eYFP-P34 binding curve shown in Fig.…”

In vivo localization and identification of SUMOylated proteins in the brain of His 6 -HA-SUMO1 knock-in mice

“…To generate a KI mouse expressing tagged SUMO1, we opted for a His 6 -HA tag because it is known to leave SUMO1 function in vitro unaffected (34,35) and has been used without negative side effects in multiple cell lines and organisms to purify SUMOylated proteins (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). The KIs show no obvious phenotypic changes; KI brains show normal morphology and cell layering; and KI neurons exhibit normal cytoarchitecture, subcellular organization, and expression and localization of marker proteins.…”