Transcript Fluorescent Resonance Energy Transfer (FRET)

Fluorescent Resonance Energy
Transfer (FRET)-based Method for
Dissecting Protein - Protein
Interactions in Sumoylation Pathway
Rachel Rattner
Dr. Jiayu Liao
BRITE Program
Outline
Introduction
 Objective
 Approach
 Results
 Conclusion
Acknowledgements


Introduction – FRET Assays
Fluorescent Resonance Energy Transfer
• Used to quantify molecular dynamics
• protein-DNA interactions
• protein conformational changes
• protein-protein interactions
• One molecule is labeled as a donor
and the other as an acceptor
• When they dissociate, the donor
emission is detected upon the donor
excitation
http://www.nature.com/nrm/journal/v4/n7/slideshow/nrm1153_F2.html
Introduction – Sumoylation Pathway
SUMO
• Small ubiquitin-like modifier
• Affects the function/metabolism
of cellular proteins through
posttranslational modifications
Sumoylation – involved in:
• Apoptosis
• Transcriptional regulation
• Protein stability
• Nuclear transport
http://www.rcsb.org/pdb/explore.do?structureId=1U4A
Objective

Develop FRET technology for dissecting SUMO
network in living cells

Clone genes in the sumoylation pathway with
fluorescent protein tags

Establish mammalian expression systems of
fluorescent fusion proteins

Understand the regulation/function of various protein
interactions in living cells using FRET technology
Approach
Construct fusion protein of SUMO-CyPet using molecular
cloning
1. Amplified SUMO2/3 using PCR
2. Cloned into PCRII TOPO bacterial vector
3. Digested with SalI and NotI
4. Ligated into a PCRII plasmid with CyPet
5. Digested SUMO2/3-CyPet with NotI and NheI
6. Ligated inserts into PCDNA3.1hygro
Approach
Transfect SUMO2/3-CyPet PCDNA3.1hygro
plasmids into mammalian cells
1. FuGENE 6 transfection reagent formed
a complex with plasmid DNA
2. Transported the DNA complex into
animal cells
3. Diluted FuGENE 6 reagent with serumfree medium
4. Added 1µg plasmid DNA to FuGENE 6
reagent
5. Added complex to the mammalian cells
6. Incubated for 24 to 48 hours
http://www.amaxa.com/stable-transfection.html
Approach
FRET Assays
1. Harvested cells by scrubbing the plates in PBS
2. Transferred into a 384 well polypropylene plate
3. Excited cells at 414 nm (excitation wavelength of
CyPet)
4. Scanned emission spectrum from 455 nm to 555
nm for YPet emission
5. Recorded emission at 535 nm (YPet emission
wavelength) as energy was transferred from CyPet
to YPet
Results
The SUMO-CyPet interaction emitted at 475 nm, as shown in the graph.
http://www.nature.com/nrm/journal/v4/n7/slideshow/nrm1153_F2.html
Results
If two molecules are close enough to interact with each other, the
second molecule can be excited by the first molecule. The right panel
showed the result that the SUMO2-CyPet construct interacted with
SENP2, which emits at both 475 nm and 510 nm.
http://www.nature.com/nrm/journal/v4/n7/slideshow/nrm1153_F2.html
Conclusion

By using a Fluorescent Resonance Energy Transfer
(FRET) technique, the SUMO2-SENP2 interaction was
successfully determined in living cells.

Construct of SUMO3 was made by utilizing PCR
amplification in conjunction with TOPO TA cloning.

After restriction enzyme digestion, the SUMO3 was
ligated into a mammalian vector to generate a fusion
construct with CyPet.

The constructs will be used with FRET technology to
study interactions with other proteins similar to that of the
SUMO2-SENP2 interaction in the near future.
Acknowledgements
Thank you to Dr. Liao’s Research Team, the BRITE Program, and Jun Wang.