Laura L. Kiefer

Site-specific mutants of carbonic anhydrase for fluorescence energy-transfer-based metal-ion biosensing

In order to gain wavelength and analyte flexibility, we have recently altered the transduction approach of our fluorescence-based biosensor. Briefly, binding of metal ions such as zinc to the active site of carbonic anhydrase is transduced by metal-dependent binding of a colored inhibitor to a fluorescent derivative of the enzyme; in the absense of metal the inhibitor does not bind and the label fluorescence is unquenched, but at higher metal concentrations the inhibitor binds, energy transfer occurs with moderate efficiency and the fluorescent label exhibits reduced intensity and lifetime. Inasmush as Forster energy transfer is distance dependent the position of the fluorescent label on the surface of the enzyme has some impact on the performance of the sensor. We designed, produced, and expressed site-selective mutants of carbonic anhydrase which could be unambiguously derivatized with suitable fluorescent labels, and which gave much improved responses to zinc ion compared with randomly derivatized wild type enzyme.