1. Introduction The determination of protein-protein interaction affinity is very important in studying cell signaling pathways such as the SUMO pathway, and the best measurement of protein interaction is through the determination of the equilibrium binding constant K d. Here we used an optical method based on steady-state Förster resonance energy transfer (FRET) to determine the K d. FRET occurs over distances between 1-10nm. This is useful because FRET can be used to determine if proteins interact due to its distance dependence. 2 FRET also has many advantages over conventional techniques because protein concentrations, a vital part of equilibrium constant calculations, can be accurately determined through absorbance, and also small testing volumes can be used, allowing the assay to be developed into a multi-well plate assay. In our experiment we fused the fluorescent proteins CYPET and YPET with SUMO1 and UBC9, respectively. SUMO1 and UBC9 are known interacting proteins in the SUMO pathway, while CYPET and YPET are able to form a FRET pair with CYPET as the donor and YPET as the acceptor. Timothy Chen, Vipul Madahar, Yang Song, Dr. Jiayu Liao Department of Bioengineering, University of California, Riverside Department of Bioengineering, University of California, Berkeley Conclusion Our calculated value for K d is in the same range as that calculated from the previous paper’s FRET experiment whose K d =.59 μM. 4 With this we have established that the use of FRET is an accurate and convenient method for determining the dissociation constant. It is superior to traditional techniques because we can use small volumes in a high throughput assay. Also, concentration is more accurately determined through emission and absorption of our fluorescently tagged proteins Future Work We also want to go beyond K d in vitro calculations and start measuring them in vivo. Previous publications have attempted this, but they have not taken into account the presence of endogenous proteins and how they will affect the calculations. 1 With our in vitro measurement as a standard, we can accurately compare our in vivo calculations to it to determine its accuracy. Another thing we can do is introduce inhibitors and calculate the K d with them to determine the best inhibitor. This FRET-based method opens up many possibilities. Acknowledgements We would like to thank Dr. Victor Rodgers, Denise Sanders, Jun Wang, Hong Xu, Harbani Malik, Yan Liu, Monica Amin, Steven Bach, Richard Lauhead, Randall Mello, Farouk Bruce, Sylvia Chu, Yongfeng Zhou, the Bioengineering Research Institute for Technological Excellence, and the National Science Foundation. Objective We wanted to calculate the dissociation constant, K d between SUMO1 and UBC9 as these are major players in the SUMO pathway. We want to validate and establish FRET-based techniques as legitimate methods to calculate equilibrium constants. We also wanted to calculate it in vitro to have standards for when we calculate K d in vivo. Our technique also allows us to introduce inhibitors to our proteins and determine the K d with different inhibitors bound. Method cDNA cloning Protein Expression and Purification All plasmids were expressed in Escherichia coli BL21. Cells were induced to express proteins using Isopropyl β-D-1-thiogalactopyranoside (IPTG). Proteins were purified using Ni 2+ -NTA affinity chromatography and High Performance Liquid Chromatography (HPLC). Proteins were stored at -80 0 C in 20mM NaCl, 50mM Tris-HCl pH 7.4, and 5mM Dithiothreitol (DTT). Protein concentrations were determined using a Bradford Protein Assay, using Bovine Serum Albumin as the standard. Multi-well Plate Assay Measurements were done using a spectrofluorometer using bottom excitation and collection. The YPET- UBC9 dilution series was dispensed in triplicate into Falcon 384-well black, clear bottom plates. The final concentrations ranged from 0.0 μM – 7.5 μM. Each well was filled with 15 μL of YPET-UBC9 and topped with 5 μL of 4 μM CYPET-SUMO1, CYPET, or buffer for a total volume of 20 μL. 4 Results We used the emission at 530nm for our results because that is the peak of YPET emission. We subtracted the CYPET+YPET-UBC9 data from the CYPET- SUMO1+YPET-UBC9 data in order to account for nonspecific interactions and direct excitation. We then used data from direct excitation of YPET-UBC9 to obtain a standard and determined the concentration of bound protein based on the 1:1 binding ratio of UBC9 and SUMO1 and the standard. 3 We could then find the concentration of free YPET-UBC9 by subtracting the bound protein from the total concentration, allowing us to plot bound protein versus free protein. We then fitted our data with the binding hyperbola for one binding site to obtain the K d. 5 MATLAB’s curve fitting tool was used to fit the nonlinear regression and K d was found to be.36 μM +/-.19 μM and B max was found to be.73 μM +/-.1 μM. Förster Resonance Energy Transfer (FRET)-based Method for Determining Protein-Protein Interaction Affinity for the SUMO Pathway Purchased cDNA from commercial sources Amplified by PCR with Forward/ Reverse Primers PCR product electrophoresis and gel extraction TOPO cloning onto PCR 2.0 Transformation into Escherichia Coli Top 10 DNA extraction by Miniprep and Characterization Digestion of vector and insert at restriction sites Ligation of vector and fluorescent insert Transformation into E. Coli Top 10 DNA extraction by Miniprep, Characterization and digestion of PCR 2.0 Gel extraction and Ligation onto PET28B Transformation into E. Coli BL21, and Characterization UBC9/SUMO1 Sal1Not1 PCR2.0 CYPET/YPET- SUMO1/UBC9 Sal1Not1 PCR2.0 Nhe1 PET28B Sal1Not1Nhe1 CYPET/YPET- SUMO1/UBC9 HIS Figure 3. cDNA cloning onto PET28B expression vector Figure 1. 6 An example of the FRET process. Upon excitation of the donor, energy is transferred to the acceptor which then shows fluoresces at its emission. Figure 4. A photograph of CYPET (left) and YPET (right) after purification by HPLC Figure 2. FRET occurs upon Protein binding 2 SUMO1 UBC9 CYPET YPET No Binding: 414nm 475nm SUMO1 UBC9 CYPET YPET Binding: 414nm 530nm Increasing YPET-UBC9 Figure 5. Proof of Concept: energy transfer increases as the concentration of YPET-UBC9 increases from 0.0 μM – 5.0 μM while the concentration of CYPET-SUMO1 remains constant at 1.0 μM. Figure 6. Fluoresence emission at 530 nm of the multi-well assay. Figure 7. Steady- State FRET. The FRET data for CYPET-SUMO1 and YPET-UBC9 after subtraction of the CYPET+YPET- UBC9 control. Free YPET-UBC9 [μM ] Bound Protein [μM] Figure 8. Graph of Bound Protein versus Free YPET-UBC9 determine d from fluoresence emission. The hyperbola for one binding site was used to determine Kd. References 1.Chen, Huanmian, Henry L. Puhl III, and Stephen R. Ikeda. "Estimating protein-protein interaction affinity in living cells using quantitative Forster resonance energy transfer measurements." Journal of Biomedical Optics 12 (2007): 054011. Print. 2.Lakowicz, Joseph R. Principles of Fluorescence Spectroscopy. New York: Springer, 2006. Print. 3.Liu, Q., C. Jin, X. Liao, Z. Shen, D. Chen, and Y. Chen. "The binding interface between an E2 (Ubc9) and a ubiquitin homologue (UBL1)." J. Biol. Chem. 274 (1999): 16979-6987. Print. 4.Martin, Sarah F., Michael H. Tatham, Ronald T. Hay, and Ifor D.W. Samuel. "Quantitavtive analysis of multi-protein interactions using FRET: Application to the SUMO pathway." Protein Science 17 (2008): 777-84. Print. 5.Motulski, H. J., and A. Christopoulos. "Fitting models to biological data using linear and nonlinear regression: A practical guide to curve fitting." GraphPad Software, Inc., San Diego, CA. Print. 6.Sapsford, Kim E., Lorenzo Berti, and Igor L. Medintz. "Materials for Fluorescence Resonance Energy Transfer Analysis: Beyond Traditional Donor-Acceptor Combinations." Angew. Chem. 45 (2006): 4562-588. Print. [BP] = B max [FP] Kd + [FP]