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

MPG-Autoren
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Geiger,  Anselm
Research Group: Cellular Dynamics / Griesbeck, MPI of Neurobiology, Max Planck Society;

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Thestrup,  Thomas
Research Group: Cellular Dynamics / Griesbeck, MPI of Neurobiology, Max Planck Society;

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Becker,  S.
Department of NMR Based Structural Biology, MPI for biophysical chemistry, Max Planck Society;

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Griesinger,  C.
Department of NMR Based Structural Biology, MPI for biophysical chemistry, Max Planck Society;

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Griesbeck,  Oliver
Research Group: Cellular Dynamics / Griesbeck, MPI of Neurobiology, Max Planck Society;

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Zitation

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.


Zitierlink: https://hdl.handle.net/11858/00-001M-0000-000F-A4F4-D
Zusammenfassung
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 Forster 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.