Chromophore Formation of Fluorescence Proteins and Its Application of Developing Ca2+ Sensors

Abstract

Ca2+ regulates numerous biological processes through spatio-temporal changes of cytosolic Ca2+ concentration and subsequent interactions with Ca2+ binding proteins in living cells. There is a strong need to develop Ca2+ sensors capable of real-time quantitative Ca2+ measurements in specific subcellular environments without using natural Ca2+ binding proteins participating in signaling transduction.1, 2 Taking advantage of fluorescence proteins (FPs) as a useful tool, we created a series of Ca2+ sensors by engineering a sensitive location of single FP with different color.3 Both spectroscopic properties including extinction coefficient, quantum yield, and pKa and metal binding properties of engineered FPs were identified with different spectroscopic methods including absorbance, fluorescence, and circular dichroism. The engineered Ca2+ sensors exhibit a ratiometric fluorescence and absorbance changes upon Ca2+ binding with affinities corresponding to the Ca2+ concentration found in the ER (Kd values range from 0.4 - 2 mM). The developed Ca2+ sensors have applied to monitor Ca2+ changes occurring in various subcellular compartments including ER and mitochondria of various mammalian cells upon response of different agonists. These sensors were engineered into virus transfection system for its application to monitor cellular Ca2+ signaling during muscle contraction and neuron events.