Fluorescent sensor for monitoring calcium dynamics
AssigneeJenny YANG
InventorsJenny YANG, Xiaonan DENG
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Abstract
The present disclosure relates to engineered protein metal ion sensors and methods of measuring metal ions. Disclosed herein are polypeptide metal ion sensors comprising engineered green-fluorescent polypeptides and engineered red-fluorescent polypeptides and methods of detecting metal ions. The polypeptide metal ion sensors disclosed herein can provide for ultrafast kinetics, larger absorption changes, and/or a greater fluorescence dynamic range.
Claims31
-28. (canceled)
A polypeptide metal ion sensor comprising an engineered red-fluorescent polypeptide having a heterologous metal ion binding site, wherein said engineered red-fluorescent polypeptide is a variant amino acid sequence of SEQ ID NO:
- 11 having the amino acid substitutions corresponding to A145E, K198D, and/or R216D and, when having a metal ion species bound thereto, exhibits an elevated fluorescence output compared to the polypeptide SEQ ID NO:
- 11 binding to the same metal ion species.
A method of detecting metal ions in a biological sample, comprising:
- (i) providing a polypeptide metal ion sensor comprising an engineered red-fluorescent polypeptide having a heterologous metal ion binding site, wherein said engineered red-fluorescent polypeptide is a variant of amino acid sequence SEQ ID NO:
- 11 and having the amino acid substitutions corresponding to A145E, K198D, and/or R216D and, when having a metal ion species bound thereto, exhibits an elevated fluorescence output compared to the polypeptide SEQ ID NO:
- 11 binding to the same metal ion species;
- (ii) delivering the polypeptide metal ion sensor or an expression vector having a nucleic acid sequence encoding said metal sensor to a biological sample;
- (iii) detecting a first spectroscopic signal emitted by said sensor;
- (iv) generating a physiological or cellular change in the biological sample;
- and (v) detecting a second spectroscopic signal emitted by said sensor after step (iii).
(canceled)
(canceled)
-56. (canceled)
A polypeptide metal ion sensor comprising an engineered red-fluorescent polypeptide having a heterologous metal ion binding site, wherein said engineered red-fluorescent polypeptide is a variant amino acid sequence of SEQ ID NO:
- 11 having the amino acid substitutions corresponding to A145E, K198D, and/or R216E and an amino acid substitution at residue K163.
Dependencies are inferred from the claim text.