Protein-protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET)

2000 ◽  
Vol 80 (3) ◽  
pp. 293-303 ◽  
Author(s):  
Hoi Yeung Li ◽  
Enders Kai On Ng ◽  
Simon Ming Yuen Lee ◽  
Masayo Kotaka ◽  
Stephen Kwok Wing Tsui ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Protein Interaction ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Green Fluorescent Proteins ◽  
Protein Protein Interaction ◽  
Fluorescence Resonance ◽  
Green Fluorescent

scholarly journals Intramolecular Fluorescence Resonance Energy Transfer between Fused Autofluorescent Proteins Reveals Rearrangements of the N- and C-terminal Segments of the Plasma Membrane Ca2+ Pump Involved in the Activation

2007 ◽  
Vol 282 (49) ◽  
pp. 35440-35448 ◽  
Author(s):  
Gerardo R. Corradi ◽  
Hugo P. Adamo
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fluorescent Proteins ◽  
Average Distance ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Green Fluorescent Proteins ◽  
Fluorescence Resonance ◽  
Green Fluorescent

The blue and green fluorescent proteins (BFP and GFP) have been fused at the N- and C-terminal ends, respectively, of the plasma membrane Ca2+ pump (PMCA) isoform 4xb (hPMCA4xb). The fusion protein was successfully expressed in yeast and purified by calmodulin affinity chromatography. Despite the presence of the fused autofluorescent proteins BFP-PMCA-GFP performed similarly to the wild-type enzyme with respect to Ca2+-ATPase activity and sensitivity to calmodulin activation. In the autoinhibited state BFP-PMCA-GFP exhibited a significant intramolecular fluorescence resonance energy transfer (FRET) consistent with the location of the fluorophores at an average distance of 45Å. The FRET intensity in BFP-PMCA-GFP decreased when the enzyme was activated either by Ca2+-calmodulin, partial proteolysis, or acidic lipids. Moreover, FRET decreased and became insensitive to calmodulin when hPMCA4xb was activated by mutation D170N in BFP-PMCA(D170N)-GFP. The results suggest that the ends of the PMCA are in close proximity in the autoinhibited conformation, and they separate or reorient when the PMCA achieves its final activated conformation.

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