An alternative assay to discover potential calmodulin inhibitors using a human fluorophore-labeled CaM protein

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Mario Figueroa Saldivar, Adjunct Faculty (Creator)
The University of North Carolina at Greensboro (UNCG )
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Abstract: This article describes the development of a new fluorescent-engineered human calmodulin, hCaM M124C–mBBr, useful in the identification of potential calmodulin (CaM) inhibitors. An hCaM mutant containing a unique cysteine residue at position 124 on the protein was expressed, purified, and chemically modified with the fluorophore monobromobimane (mBBr). The fluorophore-labeled protein exhibited stability and functionality to the activation of calmodulin-sensitive cAMP phosphodiesterase (PDE1) similar to wild-type hCaM. The hCaM M124C–mBBr is highly sensitive to detecting inhibitor interaction given that it showed a quantum efficiency of 0.494, approximately 20 times more than the value for wild-type hCaM, and a large spectral change (~80% quenching) when the protein is in the presence of saturating inhibitor concentrations. Two natural products previously shown to act as CaM inhibitors, malbrancheamide (1) and tajixanthone hydrate (2), and the well-known CaM inhibitor chlorpromazine (CPZ) were found to quench the hCaM M124C–mBBr fluorescence, and the IC50 values were comparable to those obtained for the wild-type protein. These results support the use of hCaM M124C–mBBr as a fluorescence biosensor and a powerful analytical tool in the high-throughput screening demanded by the pharmaceutical and biotechnology industries.

Additional Information

Analytical Biochemistry, 387 (1), pp. 64-70
Language: English
Date: 2009
Calmodulin, Fluorophore-labeled CaM protein, Fluorescence assay, CaM inhibitors

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