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, E
E = (R0/r)j/[(R0/r)j + 1]
 R0, Förster Distance
 r, distance between
the centers of the
chromophores
 j, exponent of
distance dependence

 FRET found to be r6
dependent
11
Förster Distance, R0
5
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.
2.
3.
4.
Donor has a high quantum yield
There is substantial spectral overlap
The dipoles of the donor and acceptor
can align properly
The donor and acceptor are at a proper
distance2
Why use FRET?

FRET occurs over biologically relevant
distances (1-10nm)10
No Binding:
SUMO1
Binding:
414nm
CYPET
414nm
CYPET
SUMO1
UBC9
YPET
475nm
UBC9
YPET
530nm
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
Sal1
Not1
Nhe1
Sal1
Not1
Nhe1
Sal1
Not1
HIS
PCR2.0
PCR2.0
UBC9/SUMO1
CYPET/YPETSUMO1/UBC9
PET28B
CYPET/YPETSUMO1/UBC9
Protein Expression and Purification
Isopropyl β-D-1thiogalactopyranoside
used to induce
expression
Purification using Ni2+NTA affinity
chromatography and
High Performance
Liquid Chromatography
Proteins stored at -800C in 20mM
NaCl, 50mM Tris-HCl pH 7.4, and
5mM Dithiothreitol7
Concentrations determined using a
Bradford Protein Assay
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
1000000
Emssion [a.u.]

800000
600000
400000
200000
0
450
470
490
510
530
wavelength [nm]
550
FRET Data
Fluorescence emission at 530nm of the
multi-well plate assay
CYPET-SUMO1+YPET-UBC9
CYPET+YPET-UBC9
Buffer+YPET-UBC9
530nm Emission [a.u.]

1200000
1000000
800000
600000
400000
200000
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
YPET-UBC9 [μM]
Steady-State FRET

FRET Data after subtraction of
CYPET+YPET-UBC9 control data
530nm Emission [a.u.]
800000
700000
600000
500000
400000
300000
200000
100000
0
0.0
1.0
2.0
3.0
4.0
5.0
YPET-UBC9 [μM]
6.0
7.0
8.0
Calculating Kd
Saturation level corresponds to 1.0 μM CYPETSUMO1 bound
 Converted Fluorescence signal into bound protein
concentration
 Plot of Bound Protein versus Free Protein
Bound Protein [μM]

0.8
0.6
0.4
[BP] = Bmax [FP]
Kd + [FP]
0.2
0
0
Fitted with binding
hyperbola for one
binding site using
MATLAB’s curve fitting
tool8
 Kd was calculated to be
.088 μM +/- .029 μM

1
1
2
3
4
5
6
Free YPET-UBC9 [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 Work
 Determine Kd using BIACORE
 Calculating Kd in vivo1
 Calculating Kd with inhibitors
References
1.
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3.
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8.
9.
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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