rachel rattner dr. jiayu liao brite program
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
Fluorescent Resonance Energy Transfer (FRET)-based Method for
Dissecting Protein - Protein Interactions in Sumoylation Pathway
Rachel RattnerDr. Jiayu Liao
BRITE Program
Outline
Introduction Objective Approach Results
Conclusion Acknowledgements
Introduction – FRET Assays
http://www.nature.com/nrm/journal/v4/n7/slideshow/nrm1153_F2.html
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
Introduction – Sumoylation Pathway
http://www.rcsb.org/pdb/explore.do?structureId=1U4A
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
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
ApproachConstruct 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
ApproachTransfect SUMO2/3-CyPet PCDNA3.1hygroplasmids into mammalian cells
1. FuGENE 6 transfection reagent formed a complex with plasmid DNA 2. Transported the DNA complex into animal cells3. Diluted FuGENE 6 reagent with serum-free medium4. Added 1µg plasmid DNA to FuGENE 6 reagent5. Added complex to the mammalian cells6. Incubated for 24 to 48 hours
http://www.amaxa.com/stable-transfection.html
ApproachFRET Assays
1. Harvested cells by scrubbing the plates in PBS2. 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.