Sequential bioluminescence resonance energy transfer–fluorescence resonance energy transfer-based ratiometric protease assays with fusion proteins of firefly luciferase and red fluorescent protein

2011 ◽  
Vol 414 (2) ◽  
pp. 239-245 ◽  
Author(s):  
Bruce R. Branchini ◽  
Justin C. Rosenberg ◽  
Danielle M. Ablamsky ◽  
Kelsey P. Taylor ◽  
Tara L. Southworth ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Firefly Luciferase ◽  
Red Fluorescent Protein ◽  
Bioluminescence Resonance Energy Transfer ◽  
Fluorescence Resonance

scholarly journals Visualization of Pit-1 Transcription Factor Interactions in the Living Cell Nucleus by Fluorescence Resonance Energy Transfer Microscopy

1998 ◽  
Vol 12 (9) ◽  
pp. 1410-1419 ◽  
Author(s):  
Richard N. Day
Keyword(s):  
Estrogen Receptor ◽  
Transcription Factor ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Fluorescence Resonance

Abstract The pituitary-specific transcription factor Pit-1 forms dimers when interacting with specific DNA elements and has been shown to associate with several other nuclear proteins. Recently, techniques have become available that allow visualization of protein-protein interactions as they occur in single living cells. In this study, the technique of fluorescence resonance energy transfer (FRET) microscopy was used to visualize the physical interactions of Pit-1 proteins fused to spectral variants of the jellyfish green fluorescent protein (GFP) that emit green or blue light [blue fluorescent protein (BFP)]. An optimized imaging system was used to discriminate fluorescence signals from single cells coexpressing the BFP- and GFP-fusion proteins, and the contribution of spectral overlap to background fluorescence detected in the FRET images was established. Energy transfer signals from living cells expressing a fusion protein in which GFP was tethered to BFP by short protein linker was used to demonstrate acquisition of FRET signals. Genetic vectors encoding GFP- and BFP-Pit-1 proteins were prepared, and biological function of the fusion proteins was confirmed. FRET microscopy of HeLa cells coexpressing the GFP- and BFP-Pit-1 demonstrated energy transfer, which required the two fluorophores to be separated by less than 100 Å. Biochemical studies previously demonstrated that Pit-1 physically interacts with both c-Ets-1 and the estrogen receptor. FRET imaging of cells coexpressing BFP-Pit-1 and GFP-Ets-1 demonstrated energy transfer between these fusion proteins, a result consistent with their association in the nucleus of these living cells. In contrast, there was no evidence for energy transfer between the BFP-Pit-1 and an estrogen receptor-GFP fusion proteins. It is likely that the FRET imaging approach described here can be applied to many different protein-partner pairs in a variety of cellular contexts.

scholarly journals Physical Association between the Adipocyte Fatty Acid-binding Protein and Hormone-sensitive Lipase

2004 ◽  
Vol 279 (50) ◽  
pp. 52399-52405 ◽  
Author(s):  
Anne J. Smith ◽  
Mark A. Sanders ◽  
Brian R. Thompson ◽  
Constantine Londos ◽  
Fredric B. Kraemer ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fatty Acid ◽  
Fluorescence Resonance Energy Transfer ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Hormone Sensitive Lipase ◽  
Fatty Acid Binding ◽  
Hormone Sensitive ◽  
Fluorescence Resonance

Previousin vitrostudies have established that hormone sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP) form a physical complex that presumably positions the FABP to accept a product fatty acid generated during catalysis. To assess AFABP-HSL interaction within a cellular context, we have used lipocytes derived from 293 cells (C8PA cells) and examined physical association using fluorescence resonance energy transfer. Transfection of C8PA cells with cyan fluorescent protein (CFP)-HSL, yellow fluorescent protein (YFP)-adipocyte FABP, or YFP-liver FABP revealed that under basal conditions each protein was cytoplasmic. In the presence of 20 μmforskolin, CFP-HSL translocated to the triacylglycerol droplet, coincident with BODIPY-FA labeled depots. Fluorescence resonance energy transfer analysis demonstrated that CFP-HSL associated with YFP-adipocyte FABP in both basal and forskolin-treated cells. In contrast, little if any fluorescence resonance energy transfer could be detected between CFP-HSL and YFP-liver FABP. These results suggest that a pre-lipolysis complex containing at least AFABP and HSL exists and that the complex translocates to the surface of the lipid droplet.

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