Molecular dynamics study of chemically engineered green fluorescent protein mutants: Comparison of intramolecular fluorescence resonance energy transfer rate

2008 ◽  
Vol 75 (1) ◽  
pp. 28-39 ◽  
Author(s):  
Felicity L. Mitchell ◽  
Filipp Frank ◽  
Gabriel E. Marks ◽  
Miho Suzuki ◽  
Kenneth T. Douglas ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Energy Transfer ◽  
Green Fluorescent Protein ◽  
Transfer Rate ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Energy Transfer Rate ◽  
Protein Mutants ◽  
Green Fluorescent

Measurement of proteases using chemiluminescence-resonance-energy-transfer chimaeras between green fluorescent protein and aequorin

2001 ◽  
Vol 357 (3) ◽  
pp. 687-697 ◽  
Author(s):  
Jonathan P. WAUD ◽  
Alexandra BERMÚDEZ FAJARDO ◽  
Thankiah SUDHAHARAN ◽  
Andrew R. TRIMBY ◽  
Jinny JEFFERY ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Green Fluorescent Protein ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dependent Manner ◽  
Cell Extracts ◽  
Donor And Acceptor ◽  
Green Fluorescent

Homogeneous assays, without a separation step, are essential for measuring chemical events in live cells and for drug discovery screens, and are desirable for making measurements in cell extracts or clinical samples. Here we demonstrate the principle of chemiluminescence resonance energy transfer (CRET) as a homogeneous assay system, using two proteases as models, one extracellular (α-thrombin) and the other intracellular (caspase-3). Chimaeras were engineered with aequorin as the chemiluminescent energy donor and green fluorescent protein (GFP) or enhanced GFP as the energy acceptors, with a protease linker (6 or 18 amino acid residues) recognition site between the donor and acceptor. Flash chemiluminescent spectra (20–60 s) showed that the spectra of chimaeras matched GFP, being similar to that of luminous jellyfish, justifying their designation as ‘Rainbow’ proteins. Addition of the protease shifted the emission spectrum to that of aequorin in a time- and dose-dependent manner. Separation of the proteolysed fragments showed that the ratio of green to blue light matched the extent of proteolysis. The caspase-3 Rainbow protein was able to provide information on the specificity of caspases in vitro and in vivo. It was also able to monitor caspase-3 activation in cells provoked into apoptosis by staurosporine (1 or 2μM). CRET can also monitor GFP fluor formation. The signal-to-noise ratio of our Rainbow proteins is superior to that of fluorescence resonance energy transfer, providing a potential platform for measuring agents that interact with the reactive site between the donor and acceptor.

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