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Related Experiment Videos

A flagellum-specific calcium sensor.

Kathryn T Buchanan1, James B Ames, Sofya H Asfaw

  • 1Department of Pathology and Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.

The Journal of Biological Chemistry
|September 9, 2005
PubMed
Summary

The flagellar calcium-binding protein (FCaBP) in Trypanosoma cruzi localizes to the flagellum via a calcium-dependent mechanism. This study reveals distinct calcium-binding affinities and conformational changes, impacting protein interactions and membrane association.

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • The flagellar calcium-binding protein (FCaBP) in Trypanosoma cruzi mediates flagellar membrane association through calcium-modulated, conformation-dependent interactions.
  • This localization mechanism resembles neuronal calcium sensors, involving calcium-dependent conformational shifts that regulate membrane binding and partner association.

Purpose of the Study:

  • To investigate calcium-dependent conformational changes in FCaBP.
  • To determine the role of these changes in flagellar targeting.
  • To identify FCaBP-interacting proteins and their calcium-dependent association.

Main Methods:

  • Site-directed mutagenesis of EF-hand calcium-binding sites (EF-3 and EF-4) in FCaBP.
  • Analysis of recombinant mutant proteins to define calcium-binding affinities (Kd) using techniques like NMR spectrometry.

Related Experiment Videos

  • Expression of FCaBP mutants in T. cruzi epimastigotes and FCaBP affinity chromatography to identify interacting proteins.

    • Main Results:

    • EF-3 was identified as the high-affinity calcium-binding site (Kd ≈ 9 µM), and EF-4 as the low-affinity site (Kd ≈ 120 µM).
    • Mutations disrupted calcium-induced conformational changes, with the E151Q mutation nearly completely abolishing the change and the double mutant completely disrupting it.
    • Expressed FCaBP mutants localized to the flagellum, indicating calcium-dependent interactions independent of intrinsic calcium binding.
    • Several interacting proteins were identified, showing calcium-dependent associations with differential reliance on FCaBP's calcium-binding capacity.

    Conclusions:

    • FCaBP exhibits distinct calcium-binding affinities and undergoes calcium-dependent conformational changes crucial for its function.
    • Flagellar targeting of FCaBP involves calcium-dependent interactions that are not solely reliant on its intrinsic calcium-binding sites.
    • These findings contribute to understanding the broader calcium acyl switch mechanism in protein regulation.