1. Timothy Chen, Vipul Madahar, Yang Song, Dr. Jiayu Liao Department of Bioengineering, University of California, Riverside August 20, 2009

2. Objective We wanted to calculate the dissociation constant, Kd, between proteins in the SUMO pathway using Förster Resonance Energy Transfer.

4. Calculating K d  K d is the dissociation constant  SUMO1 + UBC9 ↔ SUMO1-UBC9 Kd = [SUMO1] [UBC9] [SUMO1-UBC9]  K d is the concentration at which half the protein is free, and half is bound

5. Förster Resonance Energy Transfer (FRET)  Based on the principles published by Theodore Förster in 1948 5  FRET involves the transfer of energy between oscillating dipoles of similar resonance frequency 3

6. Transfer Effeciency, E 11  E = (R 0 /r) j /[(R 0 /r) j + 1]  R 0, Förster Distance  r, distance between the centers of the chromophores  j, exponent of distance dependence FRET found to be r 6 dependent

7. Förster Distance, R 0 5  ĸ 2, Dipole Orientation Factor  Q 0, Quantum Yield of the energy donor in the absence of energy transfer  J, spectral overlap 4  n, refractive index of the solvent

8. Dipole Orientation Factor, ĸ 2  Ranges from 0 to 4  Typically assumed to be 2/3 when both molecules can freely diffuse in solution 5

9. FRET 1. Donor has a high quantum yield 2. There is substantial spectral overlap 3. The dipoles of the donor and acceptor can align properly 4. The donor and acceptor are at a proper distance 2

10. SUMO1 UBC9 CYPET YPET No Binding: 414nm 475nm SUMO1 UBC9 CYPET YPET Binding: 414nm 530nm  FRET occurs over biologically relevant distances (1-10nm) 10 Why use FRET?  Small quantities can be used  Concentrations can be accurately determined 7  No radioactive materials are required  Can be developed into an in vivo method 1

12. cDNA cloning UBC9/SUMO1 Sal1Not1 PCR2.0 CYPET/YPET- SUMO1/UBC9 Sal1Not1 PCR2.0 Nhe1 PET28B Sal1Not1Nhe1 CYPET/YPET- SUMO1/UBC9 HIS

13. Protein Expression and Purification Isopropyl β-D-1- thiogalactopyranoside used to induce expression Proteins stored at -80 0 C in 20mM NaCl, 50mM Tris-HCl pH 7.4, and 5mM Dithiothreitol 7 Concentrations determined using a Bradford Protein Assay Purification using Ni 2+ - NTA affinity chromatography and High Performance Liquid Chromatography

14. Multi-well Plate Assay  Measurements done in spectrofluorometer using bottom excitation and collection  Used Falcon 384-well black, clear bottom plates  YPET-UBC9 dispensed in triplicate from concentrations of 0.0 μM – 7.5 μM  Wells topped off with either 4μM CYPET+UBC9, 4μM CYPET, or buffer 7

16. Proof of Concept  Increasing YPET-UBC9 concentration from 0.0 μM – 5.0 μM  CYPET-SUMO1 concentration remains constant at 1.0 μM Increasing YPET-UBC9

17. FRET Data  Fluorescence emission at 530nm of the multi-well plate assay

18. Steady-State FRET  FRET Data after subtraction of CYPET+YPET-UBC9 control data

19. Calculating K d  Saturation level corresponds to 1.0 μM CYPET- SUMO1 bound  Converted Fluorescence signal into bound protein concentration  Plot of Bound Protein versus Free Protein  Fitted with binding hyperbola for one binding site using MATLAB’s curve fitting tool 8  K d was calculated to be.088 μM +/-.029 μM [BP] = B max [FP] Kd + [FP] Free YPET-UBC9 [μM] Bound Protein [μM]

20. Conclusion  Our Kd =.088 μM +/-.029 μM  The previous publication’s FRET experiment calculated K d =.59 μM +/-.09 μM. (Martin, 2008) 7  Isothermal Calorimetry (ITC) calculated Kd =.082 μM +/-.023 μM (Puck, 2007) 9  Future Work Determine K d using BIACORE Calculating K d in vivo 1 Calculating K d with inhibitors

21. 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. Dos Remedios, Cristobal G., and Pierre D.J. Moens. "Fluorescence Resonance Energy Transfer Spectroscopy Is a Reliable "Ruler" for Measuring Structural Changes in Proteins." Journal of Structural Biology 115 (1995): 175-85. Print. 3. "FRET Introductory Concepts." Olympus FluoView Resource Center. Web. 31 July 2009.. 4. Haughland, Richard P., Juan Yguerabide, and Lubert Stryer. "DEPENDENCE OF THE KINETICS OF SINGLET-SINGLET ENERGY TRANSFER ON SPECTRAL OVERLAP." Chemistry 63 (1969): 23-30. Print. 5. Lakowicz, Joseph R. Principles of Fluorescence Spectroscopy. 3rd ed. New York: Springer, 2006. Print. 6. 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. 7. 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. 8. 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. 9. Puck, Knipscheer, Vsn Dijk J. Willem, Olsen V. Jesper, Mann Matthias, and Sixma K. Titia. "Noncovalent interaction between Ubc9 and SUMO promotes SUMO chain formation." EMBO 26.11 (2007): 2797-807. Print. 10. 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. 11. Stryer, Lubert. "FLUORESCENCE ENERGY TRANSFER AS A SPECTROSCOPIC RULER." Ann. Rev. Biochem. 47 (1978): 819-46. Print. 12. Stryer, Lubert, and Richard P. Haughland. "ENERGY TRANSFER: A SPECTROSCOPIC RULER." Biochemistry 58 (1967): 719-26. Print.

22. Acknowledgements  Special Thanks to Jun Wang, Dr. Victor Rodgers, Denise Sanders, Hong Xu, Harbani Malik, Yan Liu, Farouk Bruce, Sylvia Chu, Yongfeng Zhou, Monica Amin, Steven Bach, Richard Lauhead, Randall Mello, the Bioengineering Research Institute for Technological Excellence, and the National Science Foundation