Download FRET Results Conclusions Introduction Protein Interaction

Fluorescence Resonance Energy Transfer (FRET)
Studies of Protein Interaction
Sarah F Martin*, Mike H Tatham, Ron T Hay and Ifor DW Samuel
Biophotonics Collaboration, University of St Andrews
FRET Results
donor CFP
acceptor YFP
Figure 1: a) Absorption (---) and emission
(__) spectra of CFP and YFP b) Schematic
level diagram of FRET from CFP to YFP
When fluorescently tagged proteins interact, the distance
between the attached fluorescent probes is effectively
reduced. This leads to energy transfer and related changes in
the combined fluorescence spectra.
direct excitation of
YFP-Ubc9
100
control: CFP adding
YFP-Ubc9
FRET signal: CFPSUMO adding YFPUbc9
0
420
440
460
480 500 520 540
wavelength [nm]
560
580
CFP
YFP
We report here in vitro FRET studies of protein binding, and
show how they can be used to give quantitative results. While
FRET is commonly detected visually and in vivo, we aim to
develop quantitative and versatile screening assays.
Figure 2: Ribbon diagram of tagged proteins showing FRET from
CFP to YFP
Increasing YFP-Ubc9:
CFP-SUMO ratio
from 0:1 to 7:1
Figures 3 and 4: Fluorescence microscopy photograph of CFP-SUMO in mouse cells (Rakel Fernandez) and
schematic interactions of Ubc9, SUMO1 and RanGAP1 in human cells.
0.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
free YFP-Ubc9 [micromolar]
0.1
530nm
0
2
4
1
2.6
Analysis by deconvolution
and single-exponential fit
(IBH Das6 software)
2.4
0.1
475nm
0.01
6 8 10 12 14
time [ns]
2.2
2.0
0
2
4
475nm
1.8
0
1
2
3
4
5
6
7
YFP-Ubc9 / CFP-SUMO relative concentration
6 8 10 12 14
time [ns]
2
1
475nm
0
8
0
5
10
free YFP-Ubc9 [microM]
15
Figure 6. Time-resolved FRET measurements at 530nm and 475nm and binding curve with kd=0.6µM.
A B C
5
170
4
a) Add Ubc9 to conjugate
2
3 4
5 6
83
62
CFPSUMO &
YFPRanGAP1
130
b) Add SENP1 to deconjugate
CFP-SUMO1 &
YFP-RanGAP1
1
175
kDa
2
150
SENP1
Ubc9
0
47.5
6
1
110
100
200
300
time [min]
400
500
32.5
YFPRanGAP1
CFPSUMO
Figure 7. Steady state FRET measurements and SDS electrophoresis gel of the conjugation of YFP-RanGAP1
to CFP-SUMO1 following the addition of Ubc9, and the deconjugation by SENP1.
Protein Interaction
In this work, we study the small ubiquitin-like modifier SUMO1 and its interactions with further
proteins of the SUMO cycle, including enzymes (Ubc9), substrate (RanGAP1) and proteases
(SENP1), which are important in many biological processes. Conventionally studied using
NMR (see figure 3), and isothermal calorimetry (ITC) (see figure 4), FRET enables a very
sensitive real-time examination of these molecular interactions.
• SUMO1 and Ubc9 share one binding site. Their binding constant kd
is estimated to be 0.25µM. Kd is defined for a reaction of the type
A+B⇔AB as the product of the initial concentrations cA*cB divided by
the concentration of the end product cAB, and is easily determined from
the hyperbolic form of a binding curve displaying concentrations of
bound versus free protein:
Y = (Bmax x X) / (kd + X),
where Bmax indicates the saturation of the reaction.
We can fit this equation to experimental signals that indicate the amount of protein bound at
varying ratios of two (or more) proteins, e.g. by adding increasing amounts of one component to
the other. ITC will measure the heat change, and hence the total reaction taking place – with
FRET, we can isolate information of specific interactions from within a complex manycomponent system at a much higher resolution.
• Ubc9 catalyses the covalent linking of
SUMO1 to RanGAP1, a substrate located at
Human Cell
the cell’s nuclear membrane. We can easily
Ubc9
monitor the speed of this conjugation for
Binds to SUMO:
varying concentrations of Ubc9.
noncovalent temporary
• SENP1 cleaves 4 amino acids off the Nconnection
RanGAP1
terminus of SUMO1, hence breaking its
SENP1
Protease
covalent link to RanGAP1. We can study this
SUMO
with both a tagged RanGAP1-SUMO1
nucleus
Transfers SUMO1 to
complex, or simply by tagging both ends of
link to RanGAP1 on
SUMO1.
nuclear membrane
0.5
bound CFP-SUMO [microM]
1
FRET signal [au]
SUMO
Ubc9
1.0
600
3
50Ǻ
1.5
bound CFP-SUMO [micromolar] .
intensity [au]
150
50
buffer
CFP
CFP-SUMO
dilution of CFPSUMO
Figure 5. Steady state FRET measurements of the binding of Ubc9 to SUMO1 (see also figure 2).
a) A schematic overview of the experiment, b) spectra and c) the resulting binding curve with kd=0.6µM.
0.01
S0
200
lifetime [ns]
S5
S4
S3
S2
S1
250
YFPUbc9
ln (nomralised photon count)
S5
S4
S3
S2
S1
Complexes of CFP-SUMO, YFP-Ubc9 and YFP-RanGAP were expressed and purified.
Steady-state and time-resolved experiments were performed taking into account dilution, direct
excitation and non-specific interactions. The results are compiled in figures 5-7.
CFP-SUMO
Protein interactions regulate essential cellular functions, ranging from subcellular transport and
cell structure formation to DNA transcription and translation to name just a few. Failure of the
sophisticated cross-talk within complex protein cycles can lead to diseases such as Alzheimers,
diabetes and certain forms of cancer.
The study of these interactions has been significantly advanced by the discovery of the genetic
structure of the green fluorescent protein (GFP) in the jellyfish Aequorea Victoria (1992),
enabling fluorescent labelling. Furthermore, the introduction of mutations into GFP has allowed
the generation of fluorescent probes with altered spectral properties (e.g. cyan CFP and yellow
YFP), that facilitate the use of fluorescence resonance energy transfer (FRET) to indicate the
proximity of tagged molecules.
Energy transfer is observed as a decrease in emission
1
CFP
intensity and fluorescence lifetime of a higher energy (bluer
YFP
“donor”), and an increase in emission and lifetime of a lower
0.5
energy (redder “acceptor”) fluorophore. For this dipoledipole coupling to occur, the emission spectrum of the donor
0
probe must overlap considerably the absorption spectrum of
350
400
450
500
550
600
wavelength [nm]
the acceptor probe, which is the case for CFP and YFP (see
figure 1a).
Also, the two fluorophores must lie in close proximity (10100Ǻ).
ln (nomralised photon count)
absorbance / intensity [au]
Introduction
Conclusions
• Fluorescence resonance energy transfer (FRET) is a powerful, versatile and sensitive tool and
an exciting field of biophotonics research.
• In this work we used SUMO1, Ubc9 and RanGAP1, proteins involved for example in
subcellular transport within human cells, and the fluorescent proteins CFP and YFP that are a
spectrally suitable FRET pair.
•We demonstrate FRET between CFP-SUMO1 and YFP-Ubc9 arising from the binding of
Ubc9 to SUMO1. This interaction clearly brings YFP and CFP into the proximity required for
energy transfer, and the resulting FRET signal is proportional to the amount of protein bound.
Not only can we confirm previous work on the binding of these two proteins, we can also
quantitatively reproduce the associated binding constant kd with both steady-state and timeresolved experiments.
• The Ubc9-catalysed conjugation of YFP-RanGAP to CFP-SUMO, and the subsequent
cleaving of the complex were also monitored by FRET. This provides a real-time conjugation
and protease cleaving assay, with a sensitivity and simplicity novel to biomolecular science.
Conventional kinetics assays use gel electrophoresis – a more labour intensive method yielding
results at both far lower resolution and sensitivity.
• FRET requires no specific equipment beyond a fluorometer, gives a strong signal suitable for
the analysis of small amounts of tagged protein, and reports only the information from
specifically the tagged parts of a many-component experiment – making it ideal for the study of
two proteins in the presence of further interacting enzymes. This, for the first time, enables the
quantitative study of complex systems in vitro, and makes FRET an ideal high-throughput drug
screening assay.
• The versatility of this FRET-based technique is expected to yield fresh insight into the
specificity of protein modification by SUMO and other ubiquitin-like proteins. Future work will
explore the new possibilities available to studying protein interactions.
Protein Structures from the RCSB Protein Data Bank at www.rcsb.org/pdb
* School of Physics and Astronomy, North Haugh, St Andrews KY16 9SS [email protected]