timothy chen, vipul madahar, yang song, dr. jiayu liao department of bioengineering, university of...
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Timothy Chen, Vipul Madahar, Yang Song, Dr. Jiayu Liao
Department of Bioengineering, University of California, Riverside
August 20, 2009
Objective
We wanted to calculate the dissociation constant, Kd, between proteins in the SUMO pathway using Förster Resonance Energy Transfer.
Calculating Kd
Kd is the dissociation constant
SUMO1 + UBC9 ↔ SUMO1-UBC9
Kd = [SUMO1] [UBC9]
[SUMO1-UBC9] Kd is the concentration at which half the
protein is free, and half is bound
Förster Resonance Energy Transfer (FRET) Based on the
principles published by Theodore Förster in 19485
FRET involves the transfer of energy between oscillating dipoles of similar resonance frequency3
Transfer Effeciency, E11
E = (R0/r)j/[(R0/r)j + 1]
R0, Förster Distance
r, distance between the centers of the chromophores
j, exponent of distance dependenceFRET found to be r6
dependent
Förster Distance, R05
ĸ2, Dipole Orientation Factor Q0, Quantum Yield of the energy donor in
the absence of energy transfer J, spectral overlap4
n, refractive index of the solvent
Dipole Orientation Factor, ĸ2
Ranges from 0 to 4 Typically assumed
to be 2/3 when both molecules can freely diffuse in solution5
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 distance2
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 determined7
No radioactive materials are required Can be developed into an in vivo method1
cDNA cloning
UBC9/SUMO1
Sal1 Not1
PCR2.0
CYPET/YPET-SUMO1/UBC9
Sal1 Not1
PCR2.0
Nhe1
PET28B
Sal1 Not1Nhe1
CYPET/YPET-SUMO1/UBC9
HIS
Protein Expression and Purification
Isopropyl β-D-1-thiogalactopyranoside used to induce expression
Proteins stored at -800C in 20mM NaCl, 50mM Tris-HCl pH 7.4, and 5mM Dithiothreitol7
Concentrations determined using a Bradford Protein Assay
Purification using Ni2+-NTA affinity chromatography and High Performance Liquid Chromatography
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 buffer7
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
Calculating Kd 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 tool8
Kd was calculated to be .088 μM +/- .029 μM
0 1 2 3 4 5 60
0.2
0.4
0.6
0.8
1
[BP] = Bmax [FP] Kd + [FP]
Free YPET-UBC9 [μM]
Bou
nd P
rote
in [
μM
]
Conclusion Our Kd = .088 μM +/- .029 μM The previous publication’s FRET
experiment calculated Kd = .59 μM +/- .09 μM. (Martin, 2008)7
Isothermal Calorimetry (ITC) calculated Kd = .082 μM +/- .023 μM (Puck, 2007)9
Future WorkDetermine Kd using BIACORECalculating Kd in vivo1
Calculating Kd with inhibitors
References1. 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. <http://www.olympusfluoview.com/applications/fretintro.html>.
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.
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