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

FRET Data Fluorescence emission at 530nm of the

multi-well plate assay

Steady-State FRET FRET Data after subtraction of

CYPET+YPET-UBC9 control data

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