Rational Design and Structrual Analysis of Ca2+ Biosensor and Application in Skeletal Muscle Cells

Abstract

Quantitative and real-time detection of Ca2+ signaling in internal Ca2+ store sarcoplasmic reticulum (SR) of skeletal muscle cells is essential to explore the mechanism of various diseases such as malignant hyperthermia, central core diseases, brody diseases and so on highly related with SR calcium abnormal handling. To overcome the limitation of reported genetically encoded Ca2+ sensors based on natural Ca2+ binding proteins of perturbing Ca2+ signaling, we report a novel design of calcium biosensor for the first time by rational de novo engineering a non-natural Ca2+ binding site into a single enhanced green fluorescent protein (EGFP), which can successfully quantitatively reveal the subcellular calcium signaling by fluorescence change. These developed Ca2+ sensors exhibit Kd values measured inside the mammalian cells in situ optimal for the measurement of Ca2+ in the SR. Metal selectivity of the sensors for Ca2+ in competition to excessive biological metal ions such as Mg2+, K+, Na+ has been examined. In addition, these developed sensors can be targeted to the SR of muscle cells, and detected the Ca2+ signaling induced by various agonists and antagonists interacting with SR membrane Ca2+ pumps or receptors. Moreover, they exhibit fast response to Ca2+. Further, their optical and conformational properties have been investigated using various spectroscopic methods, including high resonance resolution NMR. Moreover, more than 70% of the amino acids of the EGFP-based designed sensor have been successfully assigned using heteronuclear-labeled proteins. Our studies further reveal the key factors that contribute to the molecular mechanisms of the fluorescence change upon calcium binding and dynamic properties of our designed Ca2+ sensors.