Förster Resonance Energy Transfer (FRET)-based Method for
Determining Protein-Protein Interaction Affinity for the SUMO Pathway
Timothy Chen, Vipul Madahar, Yang Song, Dr. Jiayu Liao
Department of Bioengineering, University of California, Riverside
Department of Bioengineering, University of California, Berkeley
Method
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 Kd. Here we used an optical
method based on steady-state Förster resonance energy
transfer (FRET) to determine the Kd. 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.
cDNA cloning
No Binding:
414nm
SUMO1
Binding:
CYPET
475nm
UBC9
414nm
SUMO1
CYPET
UBC9
YPET
YPET
530nm
Figure 2. FRET occurs upon Protein binding2
Objective
We wanted to calculate the dissociation constant, Kd
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 Kd in vivo. Our technique also allows us to
introduce inhibitors to our proteins and determine the Kd
with different inhibitors bound.
Digestion of vector
and insert at
restriction sites
Transformation into
E. Coli Top 10
DNA extraction by
Miniprep,
Characterization and
digestion of PCR 2.0
Emssion [a.u.]
Amplified by PCR
with Forward/
Reverse Primers
Ligation of vector
and fluorescent
insert
600000
400000
0.5
[BP] = Bmax [FP]
Kd + [FP]
0.25
0
0
200000
1
2
3
4
5
6
Free YPET-UBC9 [μM]
0
450
PCR product
electrophoresis and
gel extraction
DNA extraction by
Miniprep and
Characterization
Gel extraction and
Ligation onto
PET28B
TOPO cloning onto
PCR 2.0
Transformation into
Escherichia Coli
Top 10
Transformation into
E. Coli BL21, and
Characterization
Sal1
Not1
Nhe1 Sal1
Not1
Nhe1 Sal1
Not1
HIS
PCR2.0
PCR2.0
UBC9/SUMO1
CYPET/YPETSUMO1/UBC9
PET28B
CYPET/YPETSUMO1/UBC9
Figure 3. cDNA cloning onto PET28B expression vector
Protein Expression and Purification
All plasmids were expressed in Escherichia coli BL21.
Cells were induced to express proteins using Isopropyl βD-1-thiogalactopyranoside (IPTG). Proteins were purified
using Ni2+-NTA affinity chromatography and High
Performance Liquid Chromatography (HPLC). Proteins
were stored at -800C 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.
Figure 4. A
photograph of
CYPET (left) and
YPET (right) after
purification by HPLC
490
510
530
550
wavelength [nm]
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.
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 CYPETSUMO1+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 Kd.5
Figure 6.
Fluoresence
emission at
530 nm of the
multi-well
assay.
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 384well 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
470
CYPET-SUMO1+YPET-UBC9 CYPET+YPET-UBC9
Buffer+YPET-UBC9
1200000
530nm Emission [a.u.]
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.
Increasing
YPET-UBC9
800000
Purchased cDNA
from commercial
sources
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.
0.75
1000000
1000000
800000
600000
MATLAB’s curve fitting tool was used to fit the nonlinear
regression and Kd was found to be .36 μM +/- .19 μM and
Bmax was found to be .73 μM +/- .1 μM.
Conclusion
Our calculated value for Kd is in the same range as that
calculated from the previous paper’s FRET experiment
whose Kd = .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 Kd 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 Kd with
them to determine the best inhibitor. This FRET-based
method opens up many possibilities.
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200000
References
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]
800000
530nm Emission [a.u.]
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.
Results
Bound Protein [μM]
Introduction
Figure 7. SteadyState FRET. The
FRET data for
CYPET-SUMO1 and
YPET-UBC9 after
subtraction of the
CYPET+YPETUBC9 control.
700000
600000
500000
400000
300000
200000
100000
0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
YPET-UBC9 [μM]
7.0
8.0
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 multiprotein 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.
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.