Designed Red Fluorescent Calcium Sensor with Ultrafast Kinetic Properties
2012
Transient change of cytosolic calcium level leads to physiological actions, which are modulated by the intracellular calcium store, as well as membrane calcium channels and vesicles/endosomes. To probe calcium responses in such high calcium environments simultaneously with the calcium transients in intracellular plasma, there is a pressing need to develop a calcium sensor featured with 1) fast kinetic properties, 2) a different spectral window from other calcium indicators, and 3) pH-independent fluorescence change. In this study, we first report our development of fast red calcium sensors using our protein design instead of using endogenous calcium binding proteins or natural calcium binding motifs. Calcium binding results in fluorescence signal increase with a Kd in the submilimolar level. The red calcium sensors with fluorescence emission in the near Infrared region allow us to monitor calcium signaling together with GFP-based calcium indicators. The pH stability of the red sensors was dramatically enhanced, with pKa below 5, compared to the GFP-derived calcium sensors. High resolution NMR has been applied to probe the mechanism of calcium-induced conformational change of designed calcium binding proteins. Our results show the developed calcium sensors are able to monitor calcium dynamics at several cellular environments responding to perturbations of extracellular calcium signaling.
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