Analysis of in vitro SUMOylation using bioluminescence resonance energy transfer (BRET)

We demonstrated in vitro small ubiquitin-like modifier (SUMO)-mediated modification (SUMOylation) of RanGTPase activating protein-1 (RanGAP1) by using bioluminescence resonance energy transfer (BRET) for studying protein interactions. Renilla luciferase ( Rluc) was fused to SUMO, and RanGAP1, the bi...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2009-05, Vol.382 (3), p.530-534
Main Authors: Kim, Young-Pil, Jin, Zongwen, Kim, Eunkyung, Park, Sunyoung, Oh, Young-Hee, Kim, Hak-Sung
Format: Article
Language:English
Subjects:
Animals
Bacterial Proteins - analysis
Bacterial Proteins - genetics
BRET
Cell Line
Electrophoresis, Polyacrylamide Gel
Energy transfer
Fluorescence Resonance Energy Transfer - methods
FRET
GTPase-Activating Proteins - metabolism
Humans
Luciferase
Luciferases, Renilla - analysis
Luciferases, Renilla - genetics
Luminescent Proteins - analysis
Luminescent Proteins - genetics
Protein Interaction Mapping - methods
RanGAP
Recombinant Fusion Proteins - analysis
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
SUMO
SUMO-1 Protein - genetics
SUMO-1 Protein - metabolism
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Summary:We demonstrated in vitro small ubiquitin-like modifier (SUMO)-mediated modification (SUMOylation) of RanGTPase activating protein-1 (RanGAP1) by using bioluminescence resonance energy transfer (BRET) for studying protein interactions. Renilla luciferase ( Rluc) was fused to SUMO, and RanGAP1, the binding partner of SUMO, was fused to enhanced yellow fluorescence protein (EYFP). Upon binding of SUMO and RanGAP1, BRET was observed between EYFP (donor) and Rluc (acceptor) in the presence of E1 (Aos1/Uba2) and E2 (Ubc9) enzymes, whereas mutation (K524A) of RanGAP1 at its SUMO binding site prevented significant energy transfer. Comparing BRET and fluorescence resonance energy transfer (FRET) efficiencies using this in vitro model system, we observed that BRET efficiency was 3-fold higher than FRET efficiency, due to the lower background signal intensity of EYFP in the BRET system. Consequently, BRET system is expected to be useful for in vitro analysis of SUMOylation as well as studying other protein interactions.
ISSN:0006-291X
1090-2104
DOI:10.1016/j.bbrc.2009.03.055