English
 
Help Privacy Policy Disclaimer
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT
  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.

Item is

Files

show Files

Locators

show

Creators

show
hide
 Creators:
Geiger, Anselm1, Author           
Russo, L., Author
Gensch, T., Author
Thestrup, Thomas1, Author           
Becker, S.2, Author           
Hopfner, K. P., Author
Griesinger, C.2, Author           
Witte, G., Author
Griesbeck, Oliver1, Author           
Affiliations:
1Research Group: Cellular Dynamics / Griesbeck, MPI of Neurobiology, Max Planck Society, ou_1113560              
2Department of NMR Based Structural Biology, MPI for biophysical chemistry, Max Planck Society, ou_578567              

Content

show
hide
Free keywords: -
 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 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.

Details

show
hide
Language(s): eng - English
 Dates: 2012-05-16
 Publication Status: Published in print
 Pages: 10
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1016/j.bpj.2012.03.065
 Degree: -

Event

show

Legal Case

show

Project information

show

Source 1

show
hide
Title: Biophysical Journal
Source Genre: Journal
 Creator(s):
Affiliations:
Publ. Info: Bethesda, MD : Biophysical Society
Pages: - Volume / Issue: 102 (10) Sequence Number: - Start / End Page: 2401 - 2410 Identifier: ISSN: 0006-3495
CoNE: https://pure.mpg.de/cone/journals/resource/954925385117