Analysis of Protein Interactions In Vivo with Fluorescence Resonance Energy Transfer (FRET)

2006 ◽  
Vol 2006 (28) ◽  
pp. pdb.prot4581-pdb.prot4581 ◽  
Author(s):  
M. Damelin ◽  
P. Silver
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Resonance

scholarly journals Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach

2007 ◽  
Vol 407 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Michael Russwurm ◽  
Florian Mullershausen ◽  
Andreas Friebe ◽  
Ronald Jäger ◽  
Corina Russwurm ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Systematic Approach ◽  
Dynamic Range ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Binding Domains ◽  
Fluorescence Resonance

The intracellular signalling molecule cGMP regulates a variety of physiological processes, and so the ability to monitor cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create FRET (fluorescence resonance energy transfer)-based cGMP indicators from two known types of cGMP-binding domains which are found in cGMP-dependent protein kinase and phosphodiesterase 5, cNMP-BD [cyclic nucleotide monophosphate-binding domain and GAF [cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA] respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive. However, the GAF-derived sensors were unable to be used to study cGMP dynamics because of slow response kinetics to cGMP. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with an EC50 between 500 nM and 6 μM. These indicators possess excellent specifity for cGMP, fast binding kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of these new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics as measured by radioimmunoassays.

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