METAL ION SENSORS AND METHODS OF DETECTING METAL IONS

摘要

A method for constructing an metal ion binding motif by identifying an metal ion binding peptide that binds an metal ion with specificity, ascertaining at least a portion of a nucleic acid sequence encoding the metal ion binding peptide, tailoring the nucleic acid sequence encoding the metal ion binding peptide into an metal ion binding site, identifying a host protein and a relevant portion of the nucleic acid sequence of the host protein, operatively linking the tailored nucleic acid sequence encoding the metal ion binding peptide and the host protein nucleic acid sequence into an metal ion binding motif sequence, and expressing metal ion binding motif sequence, in which the nucleic acid sequence encoding the metal ion binding peptide is tailored so as to achieve the metal ion binding motif with a desired specificity for the metal ion. Also, the proteins encoded by the metal ion binding motif sequence as constructed by the method.

主权项

1. A polypeptide metal ion sensor comprising an engineered red fluorescent polypeptide (RFP) having a heterologous metal ion binding site comprising a plurality of negatively charged residues that in the presence of a metal ion bound thereto comprise a plurality of carboxyl oxygens orientated in a pentagonal bipyrimdal geometry, and wherein the metal ion binding site is in cooperative interaction with a chromophore region of the engineered RFP such that when the sensor does not have a metal ion bound thereto it emits a first fluorescent signal and when the sensor does have a metal ion bound thereto it emits a second fluorescent signal, wherein the first and the second fluorescent signals are distinguishably detectable, and wherein the metal ion sensor has a koff value for the metal ion of at least 10s−1.