Rachel Rattner Dr. Jiayu Liao BRITE Program

1. Fluorescent Resonance Energy Transfer (FRET)-based Method for Dissecting Protein - Protein Interactions in Sumoylation Pathway Rachel Rattner Dr. Jiayu Liao BRITE Program

2. Outline • Introduction • Objective • Approach • Results • Conclusion • Acknowledgements

3. 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

4. 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

5. 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

6. 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

7. 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 serum-free 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

8. 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

9. 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

10. 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

11. 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.

12. Acknowledgements Thank you to Dr. Liao’s Research Team, the BRITE Program, and Jun Wang.