Abstract
A novel, sensible, and specific fluorescent biosensor of human calmodulin (hCaM), namely hCaM L39C-mBBr/V91C-mBBr, was constructed. The biosensor was useful for detecting ligands with opposing fluorescent signals, calcium ions (Ca(2+)) and CaM inhibitors in solution. Thus, the device was successfully applied to analyze the allosteric effect of Ca(2+) on trifluoroperazine (TFP) binding to CaM (Ca(2+)K(d) = 0.24 μM ± 0.03 with a stoichiometry 4.10 ± 0.15; TFPK(d) ∼ 5.74-0.53 μM depending on the degree of saturation of Ca(2+), with a stoichiometry of 2:1). In addition, it was suitable for discovering additional xanthones (5, 6, and 8) with anti-CaM properties from the fungus Emericella 25379. The affinity of 1-5, 7, and 8 for the complex (Ca(2+))(4)-CaM was excellent because their experimental K(d)s were in the nM range (4-498 nM). Docking analysis predicted that 1-8 bind to CaM at sites I, III, and IV as does TFP.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Antipsychotic Agents / analysis
-
Antipsychotic Agents / chemistry
-
Binding Sites
-
Biosensing Techniques*
-
Bridged Bicyclo Compounds / chemistry
-
Calcium / analysis*
-
Calmodulin / antagonists & inhibitors*
-
Calmodulin / chemical synthesis
-
Calmodulin / chemistry
-
Calmodulin / genetics
-
Calmodulin / metabolism*
-
Calmodulin / pharmacology
-
Emericella / chemistry*
-
Emericella / growth & development
-
Emericella / metabolism
-
Fluorescent Dyes / chemical synthesis*
-
Fluorescent Dyes / chemistry
-
Fluorescent Dyes / metabolism
-
Fluorescent Dyes / pharmacology
-
Humans
-
Ligands
-
Models, Molecular
-
Molecular Conformation
-
Patents as Topic
-
Trifluoperazine / metabolism
-
Xanthones / analysis
-
Xanthones / chemical synthesis
-
Xanthones / chemistry
-
Xanthones / isolation & purification
-
Xanthones / metabolism*
-
Xanthones / pharmacology*
Substances
-
Antipsychotic Agents
-
Bridged Bicyclo Compounds
-
Calmodulin
-
Fluorescent Dyes
-
Ligands
-
Xanthones
-
hCaM L39C-mBBr-V91C-mBBr
-
Trifluoperazine
-
Calcium
-
monobromobimane