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Calmodulin-dependent Signaling

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Calmodulin (CaM) is a calcium-binding protein in eukaryotes that controls various calcium-regulated cellular processes. It has four calcium-binding sites that bind calcium to form the calcium-calmodulin ( Ca2+-CaM) complex. GPCR stimulation increases the calcium levels in the cells that bind to CaM and induces a conformational change.
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Related Experiment Video

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Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

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Structural basis for calcium sensing by GCaMP2.

Qi Wang¹, Bo Shui, Michael I Kotlikoff

¹Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.

Structure (London, England : 1993)

|December 17, 2008

Summary

This summary is machine-generated.

Genetically encoded calcium indicators like GCaMP2 are vital for measuring calcium dynamics. Crystal structures reveal GCaMP2

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Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

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Area of Science:

Biochemistry
Molecular Biology
Biophysics

Background:

Genetically encoded calcium indicators (GECIs) are essential for real-time monitoring of intracellular calcium (Ca2+) dynamics.
GCaMP2, a fusion protein of circularly permuted enhanced green fluorescent protein (cpEGFP), M13 peptide, and calmodulin (CaM), is a widely used GECI for in vivo studies.

Purpose of the Study:

To elucidate the molecular mechanism of Ca2+ sensing in GCaMP2 through structural analysis.
To provide a structural basis for the rational design of improved Ca2+-sensitive fluorescent probes.

Main Methods:

X-ray crystallography to determine the structures of bright and dim states of GCaMP2.
Mutational analysis to identify key interactions within the GCaMP2 protein.
Solution scattering studies (e.g., SAXS) to investigate the Ca2+-free conformation.

Main Results:

Crystal structures revealed distinct bright and dim states of GCaMP2, highlighting a sophisticated Ca2+ sensing mechanism.
In the bright state, calmodulin (CaM) stabilizes the fluorophore in an ionized conformation, similar to EGFP.
Mutational studies confirmed crucial interactions between the fluorophore and the fused M13/CaM peptides.
Solution scattering indicated that the Ca2+-free GCaMP2 exists as a compact, pre-docked state, explaining its rapid signaling kinetics.

Conclusions:

The determined structures provide unprecedented insight into the molecular basis of Ca2+ detection by GCaMP2.
Understanding these structural dynamics facilitates the rational engineering of next-generation GECIs with enhanced sensitivity and kinetics.