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  Correlating calcium binding, Förster resonance energy transfer, and conformational change in the biosensor TN-XXL.

Geiger, A., Russo, L., Gensch, T., Thestrup, T., Becker, S., Hopfner, K. P., et al. (2012). Correlating calcium binding, Förster resonance energy transfer, and conformational change in the biosensor TN-XXL. Biophysical Journal, 102(10), 2401-2410. doi:10.1016/j.bpj.2012.03.065.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-000F-9FEC-E Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-47C0-6
Genre: Journal Article

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 Creators:
Geiger, A., Author
Russo, L., Author
Gensch, T., Author
Thestrup, T., Author
Becker, S.1, Author              
Hopfner, K. P., Author
Griesinger, C.1, Author              
Witte, G., Author
Griesbeck, O., Author
Affiliations:
1Department of NMR Based Structural Biology, MPI for biophysical chemistry, Max Planck Society, ou_578567              

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 Abstract: Genetically encoded calcium indicators have become instrumental in imaging signaling in complex tissues and neuronal circuits in vivo. Despite their importance, structure-function relationships of these sensors often remain largely uncharacterized due to their artificial and multimodular composition. Here, we describe a combination of protein engineering and kinetic, spectroscopic, and biophysical analysis of the Förster resonance energy transfer (FRET)-based calcium biosensor TN-XXL. Using fluorescence spectroscopy of engineered tyrosines, we show that two of the four calcium binding EF-hands dominate the FRET output of TN-XXL and that local conformational changes of these hands match the kinetics of FRET change. Using small-angle x-ray scattering and NMR spectroscopy, we show that TN-XXL changes from a flexible elongated to a rigid globular shape upon binding calcium, thus resulting in FRET signal output. Furthermore, we compare calcium titrations using fluorescence lifetime spectroscopy with the ratiometric approach and investigate potential non-FRET effects that may affect the fluorophores. Thus, our data characterize the biophysics of TN-XXL in detail and may form a basis for further rational engineering of FRET-based biosensors.

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Language(s): eng - English
 Dates: 2012-05-162012
 Publication Status: Published in print
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 Rev. Method: Peer
 Identifiers: DOI: 10.1016/j.bpj.2012.03.065
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Title: Biophysical Journal
Source Genre: Journal
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Pages: - Volume / Issue: 102 (10) Sequence Number: - Start / End Page: 2401 - 2410 Identifier: -