Mg2+ is an essential metal that regulates the structure and function of biological macromolecules, including enzymes and nucleic acids, and cellular metabolites. Mg2+ is integral to supporting nerve and muscle function, the immune system and bone health, and Mg2+ deficiency and dysregulation of Mg2+ homeostasis are associated with the development of cardiovascular disease, diabetes and osteoporosis. Despite the well-accepted role of Mg2+ in preserving cellular and organismal health, the activity and regulation of Mg2+ at the single cell level is ill-defined. Genetically encoded fluorescent sensors can be used to illuminate the localization and behavior of metal ions in cells with remarkable spatiotemporal resolution. However, there are a dearth of fluorescent tools that can be used to interrogate Mg2+ homeostasis in cells. The overall goal of the current study is to develop a robust fluorescent Mg2+ sensor platform with an elevated Mg2+ response and enhanced selectivity toward Mg2+ versus competing metal ions. In this study, we engineered three fluorescent Mg2+ sensor platforms that contain the same two fluorescent proteins, ECFP and cpVenus173, but distinct Mg2+-binding domains. The Mg2+-binding domain couples metal binding with changes in fluorescent signal; therefore, each sensor is likely to demonstrate a unique Mg2+ response. In an effort to tune the Mg2+ affinity of each sensor, we developed mutants of each sensor by targeting amino acids that are directly involved in Mg2+ coordination. Each of the Mg2+ sensor constructs were expressed in bacteria, purified using immobilized metal affinity chromatography and underwent photophysical characterization using a spectrofluorometer.

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Evan Pratt

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