کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1175010 | 961783 | 2009 | 7 صفحه PDF | دانلود رایگان |
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.
Journal: Analytical Biochemistry - Volume 387, Issue 1, 1 April 2009, Pages 64–70