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Calcium Indicators Based on Calmodulin–Fluorescent Protein Fusions

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Creative Biolabs offers promising methods for constructing Calcium Indicators based on Calmodulin-Fluorescent Protein Fusions to study Ca2+ concentration changes within the spatial and temporal context.

Ca2+ signals in the cytosol and organelles are very important for many cellular events, as calcium can act in signal transduction resulting from activation of ion channels or as a second messenger caused by indirect signal transduction pathways: i.e.g the G protein-coupled receptors. However, Ca2+ ions are also often extremely localized and difficult to measure.

Therefore, sensitive calcium indicators are strongly needed nowadays. Such indicators based on Calmodulin-fluorescent protein fusions are protein-based Ca2+ sensors made of Ca2+–calmodulin (CaM) complexes and green fluorescent protein (GFP) mutants, shortly named “chameleon”. CaM, as a ubiquitous protein, plays a key role in Ca2+-mediated signal transduction: after Ca2+ influx, it can bind various cellular proteins with one or more CaM recognition sequences and regulate Ca2+ cascades. The most important advantage of this technique is that they can be expressed in single cells and targeted at the specific organelles or tissues to measure localized Ca2+ changes using a digital fluorescence microscope. A number of yellow chameleons (YC), which use the GFP mutants CFP–YFP (cyan fluorophore partners and yellow fluorophore partners), have been created with various Ca2+ binding affinities, subcellular localizations, and dynamic ranges. Technically, a chameleon with a larger dynamic range is a more effective in vivo Ca2+ indicator. And the effective range can be assessed by the Ca2+-binding curve. These indicators can be genetically modified to target specific intracellular locations.

Besides, specific mutations in calmodulin can be used to tune the Ca2+ affinities and measure free Ca2+ concentrations in the range 10-8 to 10-2 M. Free Ca2+ dynamics can be visualized in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells, which have been transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 uM at rest, and 1 to 50 uM after Ca2+ mobilization.

We offer the following methods for construction of Calcium Indicators:

  1. YC2.1: CaM bound to the myosin light chain kinase CRS peptide (MLCKp).
  2. YC3.1: E104Q mutation of CaM to lower Ca2+ affinity;
  3. YC2nu: target to the nucleus;
  4. YC6.1: CaM bound to CaM-dependent kinase kinase CRS peptide (CKKp) to improve dynamic range.

Creative Biolabs offers promising tools in the field of protein engineering aided by NMR and crystallographic structural studies.


Figure 1. Crystal Structures of GCaMP2•Ca2+. (Qi Wang et al., 2008)

List of References:
1. Qi Wang, Bo Shui. Structural Basis for Calcium Sensing by GCaMP2. Structure 2008, 16(12): 1817–1827.


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