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Biophys. J. BioFAST: First Published December 9, 2005. doi:10.1529/biophysj.105.073536
© 2005 by the Biophysical Society.

A more recent version of this article appeared on March 1, 2006.
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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change

Marco Mank 1, Dierk F Reiff 1, Nicola Heim 1, Michael W Friedrich 1, Alexander Borst 1 and Oliver Griesbeck 1*

1 Max-Planck-Institut für Neurobiologie

* To whom correspondence should be addressed. E-mail: griesbeck{at}neuro.mpg.de.

Submitted on August 30, 2005
Revised on September 22, 2005
Accepted on 7 November 2005


  Abstract
Genetically encoded calcium biosensors have become valuable tools in cell biology and neuroscience but some aspects such as signal strength and response kinetics still need improvement. Here we report the generation of a FRET-based calcium biosensor employing troponin C as calcium binding moiety that is fast, stable in imaging experiments and shows a significantly enhanced fluorescence change. These improvements were achieved by engineering magnesium and calcium binding properties within the C-terminal lobe of troponin C and by the incorporation of circularly permuted variants of the green fluorescent protein. This sensor named TN-XL shows a maximum fractional fluorescence change of 400 % in its emission ratio and linear response properties over an expanded calcium regime. When imaged in vivo at presynaptic motoneuron terminals of transgenic fruit flies, TN-XL exhibits highly reproducible fluorescence signals with the fastest rise and decay times of all calcium biosensors known so far.

Key Words: Biosensor, FRET, GFP, Imaging, calcium




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Copyright © 2005 by the Biophysical Society.