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Mapping Calcium-Sensitive Regions in GCAPs by Site-Specific Fluorescence Labelling

  • Karl-Wilhelm KochEmail author
  • Jens Christoffers
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1929)

Abstract

Signal transduction processes that are under control of changes in cytoplasmic Ca2+-concentration involve Ca2+-sensor proteins, which often undergo pronounced conformational transitions triggered by Ca2+. Consequences of conformational changes can be the structural rearrangement of single amino acids, exposition of small patches of several amino acids, or the movement of whole protein regions or domains. Furthermore, these conformational changes can lead to the exposure or movement of posttranslationally attached acyl groups. These processes could then control the function of target proteins, for example, by Ca2+-dependent protein–protein interaction. Fluorescence spectroscopy allows for mapping these Ca2+-sensitive regions but needs site-specific fluorescence labelling. We describe the application of a new group of diaminoterephthalate-derived fluorescence probes targeting either cysteines in guanylate cyclase-activating proteins, named GCAPs, or azide moieties in covalently attached acyl groups. By monitoring Ca2+-dependent changes in fluorescence emission, we identify Ca2+-sensitive protein regions in GCAPs and correlate conformational changes to protein function.

Key words

Neuronal calcium sensor proteins GCAP Fluorescence Myristoylation Diaminoterephthalates 

Notes

Acknowledgments

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (GRK 1885).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Biochemistry Group, Department of Neuroscience, Faculty VIUniversity of OldenburgOldenburgGermany
  2. 2.Organic Chemistry, Faculty VInstitut für Chemie, University of OldenburgOldenburgGermany

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