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. 2011 Apr;39(4):1224-34.
doi: 10.1007/s10439-010-0225-x. Epub 2010 Dec 21.

Development of FRET assay into quantitative and high-throughput screening technology platforms for protein-protein interactions

Yang Song  1 , Vipul Madahar, Jiayu Liao
Affiliations

Development of FRET assay into quantitative and high-throughput screening technology platforms for protein-protein interactions

Yang Song et al. Ann Biomed Eng. 2011 Apr.

Abstract

Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research and is a very powerful tool in elucidating protein interactions in many cellular processes. Ubiquitination and SUMOylation are multi-step cascade reactions, involving multiple enzymes and protein-protein interactions. Here we report the development of dissociation constant (K (d)) determination for protein-protein interaction and cell-based high-throughput screening (HTS) assay in SUMOylation cascade using FRET technology. These developments are based on steady state and high efficiency of fluorescent energy transfer between CyPet and YPet fused with SUMO1 and Ubc9, respectively. The developments in theoretical and experimental procedures for protein interaction K (d) determination and cell-based HTS provide novel tools in affinity measurement and protein interaction inhibitor screening. The K (d) determined by FRET between SUMO1 and Ubc9 is compatible with those determined with other traditional approaches, such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). The FRET-based HTS is pioneer in cell-based HTS. Both K (d) determination and cell-based HTS, carried out in 384-well plate format, provide powerful tools for large-scale and high-throughput applications.

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Figures

Figure 1
Figure 1
Multiple protein–protein interactions are involved in sumoylation conjugation pathway
Figure 2
Figure 2
Design and detection of high sensitive FRET-based detection for protein interactions in sumoylation conjugation cascade. (a) The diagram of FRET-based detection of SUMO1 and its E2 ligase, Ubc9, interaction. (b) Emission spectra of protein mixtures with [CyPet–SUMO1] fixed as 1 μM and [YPet–Ubc9] increased from 0 to 7.5 μM. Excitation wavelength is 414 nm
Figure 3
Figure 3
Spectrum analysis of FRET signals. (a) Dissection of emission spectra from mixture of CyPet–SUMO1 and YPet–Ubc9. (b) Emission spectrum of YPet equations used to determine sensitized FRET emission, to convert sensitized emission intensity to bound concentration, to calculate the concentration of free YPet–Ubc9 and to fit bound concentration against free concentration to get the K d value
Figure 4
Figure 4
Determination of interaction affinity K d between CyPet–SUMO1 and YPet–Ubc9 using FRET technology. (a) Interaction specificity and quantitation among pairs. (b) Plot of FRET-sensitized YPet–Ubc9 emission vs. the total YPet–Ubc9 concentration
Figure 5
Figure 5
Determination of interaction affinity K d by surface plasma resonance measurement. (a) Determination of K d between CyPet–SUMO1 and YPet–Ubc9 using BIAcore, resulting in K d = 0.35 μM. (b) Determination of K d between SUMO1 and Ubc9 using BIAcore, resulting in K d = 0.10 μM
Figure 6
Figure 6
Characterizations of stable cell line for FRET assay. (a) FRET characterization of individual stable cell lines. (b) Fluorescence spectrum of control cells, single transfected stable cell line, and double transfected stable cell line when excited at 414 nm. (c) Fluorescence spectrum of control cells, single transfected stable cell line, and double transfected stable cell line when excited at 465 nm. (d) Fluorescence image analysis of stable cell lines
Figure 7
Figure 7
Reversible interaction of SUMO1 and Ubc9 in living cells. (a) FRET assays of stable HEK293 cell line containing CyPet–SUMO1 and YPet–Ubc9 after different treatments with or without protein interaction disrupting reagents, such as Triton-100 or Urea. (b) Plot of FRET ratio (Em530/Em475) of cells treated with or without disrupting buffers as in (a). (c) Positive hit selection of seven hundred screened compounds
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