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Calmodulin-dependent Signaling

Calmodulin-dependent Signaling

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.
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...

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Pull-down of Calmodulin-binding Proteins

Pull-down of Calmodulin-binding Proteins

Published on: January 23, 2012

Distribution of separations between groups in an engineered calmodulin.

R F Steiner¹, S Albaugh, M C Kilhoffer

¹Department of Chemistry and Biochemistry, University of Maryland (Baltimore County), 5401 Wilkens Avenue, 21228, Baltimore, Maryland.

Journal of Fluorescence

|November 19, 2013

Summary

This summary is machine-generated.

Researchers engineered calmodulin (VU-9-CaM) with tryptophan and nitrotyrosine. Time-domain fluorescence measurements revealed the distribution of separations between these groups, aligning with crystallographic data.

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Published on: July 27, 2016

Area of Science:

Biochemistry
Molecular Biology
Biophysics

Background:

Calmodulin is a crucial calcium-binding protein involved in cellular signaling.
Understanding calmodulin's structural dynamics is key to deciphering its regulatory functions.
Engineered protein variants offer unique tools for studying molecular interactions.

Purpose of the Study:

To characterize the dynamic structural changes in engineered calmodulin (VU-9-CaM).
To investigate the spatial distribution of Ca(2+)-binding domains III and IV using fluorescence energy transfer.
To validate structural models of calmodulin through biophysical measurements.

Main Methods:

Site-directed mutagenesis to introduce tryptophan and tyrosine residues at specific positions.
Conversion of tyrosine to nitrotyrosine to create a Förster Resonance Energy Transfer (FRET) pair.
Time-domain fluorescence spectroscopy to measure energy transfer dynamics.
Analysis of fluorescence decay data to determine inter-residue distances.

Main Results:

Successful preparation of engineered calmodulin (VU-9-CaM) with a tryptophan donor and nitrotyrosine acceptor.
Fluorescence measurements provided parameters characterizing the distribution of separations between domains III and IV.
The determined mean separation between the donor and acceptor groups was in good agreement with crystallographic data.

Conclusions:

The engineered calmodulin (VU-9-CaM) serves as a reliable system for studying domain dynamics.
Time-domain fluorescence measurements effectively probe the conformational landscape of calmodulin.
The findings support the accuracy of existing crystallographic models for calmodulin structure and dynamics.