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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:
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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