Protein interaction affinity determination by quantitative FRET technology

Abstract

The dissociation constant, K(d) , is an important parameter for characterizing protein-protein interaction affinities. SUMOylation is one of the important protein post-translational modifications and it involves a multi-step enzymatic cascade reaction, resulting in peptide activation and substrate conjugation. Multiple covalent and non-covalent protein-protein interactions are involved in this cascade. Techniques involving Förster resonance energy transfer (FRET) have been widely used in biological studies in vitro and in vivo, and they are very powerful tools for elucidating protein interactions in many regulatory cascades. In our previous studies, we reported the attempt to develop a new method for the determination of the K(d) by FRET assay using the interaction of SUMO1 and its E2 ligase, Ubc9 as a test system. However, the generality and specifications of this new method have not been fully determined. Here we report a systematic approach for determining the dissociation constant (K(d) ) in the SUMOylation cascade and for further sensitivity and accuracy testing by the FRET technology. From a FRET donor to acceptor concentration ratio range of 4-40, the K(d) s of SUMO1 and Ubc9 consistently agree well with values from surface plasmon resonance and isothermal titration calorimetry. These results demonstrate the high sensitivity and accuracy of the FRET-based K(d) determination approach. This technology, therefore, can be used in general for protein-protein interaction dissociation constant determination.

FRET Based Quantification and Screening Technology Platform for the Interactions of Leukocyte Function-Associated Antigen-1 (LFA-1) with InterCellular Adhesion Molecule-1 (ICAM-1)

“…However, the emission peak intensity at 570 nm (F DA ) has several components. The analysis of this typical FRET spectrum was done following the procedure reported by Song et al [49]. In brief, the emission intensity at 570 nm consisted of the contributions from (i) the emission of unquenched Alexa Fluor 488, (ii) the direct emission of Alexa Fluor 555, and (iii) the emission of Alexa Fluor 555 due to the non-radiative energy transfer from Alexa Fluor 488 (F FRET ).…”

“…This F FRETmax was eventually used to obtain the dissociation constant (K d ) of the interaction between D1-D2-Fc and LFA-1 via equation (5) given below, derived from the general law of mass action of protein-protein interactions.Song etal. [49] have reported a systematical methodology to deduce K d from steady-state FRET measurements. Following their procedure and assuming one-to-one interaction between D1-D2-Fc and LFA-1, K d was obtained by fitting the datasets with equation (6),where “A” denotes the concentration of the acceptor, Alexa Fluor 555-D1-D2-Fc, which was varied from 0 to 1.6µM; “D” is the total concentration of donor, Alexa Fluor 488-LFA-1, which was kept fixed at 100 nM.…”

“…However, an earlier obstacle in successful development of these assays was to extract the FRET emission signal at the acceptor emission wavelength from a mixed emission spectrum which contains the unquenched donor emission, direct acceptor emission and the actual FRET emission signal. Recently, Song et al [48], [49] developed a theoretical and experimental procedure to overcome this obstacle and obtained the dissociation constant (K d ) of the interaction between SUMO 1 and Ubc9. In this method, correlation of donor and acceptor emissions were used to obtain the absolute fluorescence signal contributions (due to unquenched donor, direct acceptor and FRET emission signal) at the acceptor emission wavelength in one single assay [49].…”

“…Recently, Song et al [48], [49] developed a theoretical and experimental procedure to overcome this obstacle and obtained the dissociation constant (K d ) of the interaction between SUMO 1 and Ubc9. In this method, correlation of donor and acceptor emissions were used to obtain the absolute fluorescence signal contributions (due to unquenched donor, direct acceptor and FRET emission signal) at the acceptor emission wavelength in one single assay [49].…”

Genetically Encoded Fluorescent Biosensors for Live-Cell Visualization of Protein Phosphorylation

“…The bimolecular sensor class is quite large and includes many sensors designed on an ad-hoc basis for studies to determine when and where two proteins of interest interact, including heteromeric binding of receptors for peptide hormones (Almabouada et al, 2013), various neurotransmitters (Borroto-Escuela et al, 2012), SNAREs (Degtyar et al, 2013) and immune signaling molecules (Hashimoto-Tane et al, 2010). In addition to providing information about the location and timing of protein interactions, bimolecular sensors can be used to determine what portions of the proteins are required for interaction (Borroto-Escuela et al, 2012) or to characterize the dissociation kinetics of the two proteins (Song et al, 2012). …”