Metal ions are key in regulating many biological functions, but their accumulation is associated with neurological diseases. Cellular metal ion concentrations are often detected through exogenous fluorescent dyes, but less invasive detection can be done using genetically-encoded fluorescent metal-binding proteins such as Twitch-2B. Twitch-2B changes conformation when a calcium ion is bound to the active site, initiating an energy transfer between two fluorescent peptides that can be quantified through fluorescent spectroscopy (FRET). Previous analyses have demonstrated that calcium can be accurately measured from concentrations of 70uM to 84.77mM, indicating large detection limits for calcium. By mutating the calcium binding site to preferentially bind alternate metal ions zinc, copper, nickel, iron, or manganese while retaining or increasing the sensitivity of the binding and fluorescent energy transfer, genetically-encoded sensors to detect the <i>in vivo </i>concentrations of these transition metals could be developed. A library containing combinations of mutations at ten different key residues was designed, amplified, and purified to maximize transformation efficiency. Screening controls for potential allosteric interactions that may affect the measured fluorescence of the mutants were performed via fluorescence spectroscopy in the presence of each of the metal ions over a range of conditions with and without calcium. It was determined that wild-type Twitch-2B has slight active site affinity for copper, nickel, and zinc, and there are additional allosteric interactions with manganese, nickel, zinc, and copper, while iron does not demonstrate any significant interactions. The natural active site affinity of Twitch-2B for copper, nickel, and zinc indicates that further mutations are likely to be successful in increasing the affinity. High-throughout screening was performed using a well-plate reader against each of the five metal ions, and mutants that showed successful binding for any of the ions were sequenced. The detection limits of these promising mutant were evaluated via fluorescence spectroscopy titrations, and the viability of these mutants for <i>in vivo </i>detection was determined.

Re-purposing the calcium biosensor Twitch-2B: Creating transition metal detection proteins through FRET screening

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