Quantifying Protein-Protein Interactions via FRET

Recombinant CyPet–SUMO1 and YPet–Ubc9 proteins were mixed and diluted with phosphate buffered saline (PBS) to a total volume of 30 μL. The final concentration of CyPet–SUMO1 was fixed to 1 μM and the final concentration of YPet–Ubc9 varied from 0 to 4 μM. The mixtures were transferred into a 384-well plate (Falcon) and the fluorescence emission spectrum of each well was measured with a fluorescence multi-well plate reader (Molecular Devices, FlexstationII³⁸⁴). Two excitation wavelengths were used: 414 nm to excite CyPet, and 475 nm to excite YPet. Excited at 414 nm, CyPet has an emission peak at 475 nm (FL_DD) (see Fig. 2). With FRET, another emission peak at 530 nm (Em_total) can be observed which results from the energy transferred from CyPet to YPet. When the mixture is excited at 475 nm, an emission peak at 530 nm (FL_AA) can be observed which is from the direct excitation of YPet but not CyPet.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

When a mixture of CyPet–SUMO1 and YPet–Ubc9 recombinant proteins was excited at 414 nm, the emission intensity at 530 nm was consisted of three components: the direct emission of CyPet, the sensitized emission of YPet and the direct emission of YPet. Because the sensitized emission from YPet–Ubc9 (Em_FRET) is proportional to the amount of YPet–Ubc9 bound to CyPet–SUMO1 ([YPetUbc9]_bound), we can convert the concentration of YPet–Ubc9 in both free ([YPetUbc9]_free) and bound ([YPetUbc9]_bound) forms to functions of Em_FRET. Therefore, we can derive the relationship between Em_FRET and the total concentration of YPet–Ubc9 ([YPetUbc9]_total), and calculate the K_d between CyPet–SUMO1 and YPet–Ubc9 based on the algorithm we developed. Data were processed in Prism 5 (GraphPad Software).