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Fluorescent Binding Protein Sensors for Detection and Quantification of Biochemicals, Metabolites, and Natural Products.

ABSTRACT: Membrane transporters and soluble binding proteins recognize particular nutrients, metabolites, vitamins, or ligands. By modifying genetically engineered single cysteine residues near the active sites of such proteins with extrinsic maleimide fluorophores,the approaches that we report create sensitive fluorescent sensors that detect, quantify, and monitor molecules that are relevant to the biochemistry, physiology, microbiology, and clinical properties of pro- and eukaryotic organisms. This protocol was validated in: J Biol Chem (2022), DOI: 10.1016/j.jbc.2022.101651 Graphical abstract.

SUBMITTER: Newton S

PROVIDER: S-EPMC9724013 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Fluorescent Binding Protein Sensors for Detection and Quantification of Biochemicals, Metabolites, and Natural Products.

Newton Salete S Klebba Phillip M E PME

Bio-protocol 20221120 22

Membrane transporters and soluble binding proteins recognize particular nutrients, metabolites, vitamins, or ligands. By modifying genetically engineered single cysteine residues near the active sites of such proteins with extrinsic maleimide fluorophores,the approaches that we report create sensitive fluorescent sensors that detect, quantify, and monitor molecules that are relevant to the biochemistry, physiology, microbiology, and clinical properties of pro- and eukaryotic organisms. <b>This p ...[more]

PMID: 36532683

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Project description:Treatment of aqueous zinc solutions with incremental additions of a ditopic fluorescent sensor of the Zinpyr family, based on pyridine/pyrazine-containing metal recognition units, affords a fluorescence titration curve with a sharp maximum at a sensor:Zn(2+) ratio of 0.5 (Zhang, X-a.; Hayes, D.; Smith, S. J.; Friedle, S.; Lippard, S. J. J. Am. Chem. Soc.2008, 130, 15788-15789). This fluorescence response enables the quantification of readily chelatable zinc in biological samples by a simple titration protocol. In the present work a new set of ditopic fluorescence zinc sensors functionalized with pyridine/pyrazine-containing metal chelating units is described, and through detailed studies the principles governing the characteristic OFF-ON-OFF fluorescence behavior and quantification capabilities of the family are delineated. Incorporation of carboxylate/ester groups in the 6 position of the fluorescein allows for control of the spatial distribution of the sensor for selective extra- or intracellular imaging of mobile zinc, without introducing significant changes in zinc-binding properties. A combination of spectrophotometric and potentiometric measurements provided a complete description of the H(+)- and Zn(2+)-binding properties of the compounds and their correlation with the observed fluorescence profile. The first zinc-binding event has an apparent affinity, K(1)', of 1.9 × 10(9)-3.1 × 10(9) M(-1), whereas for coordination of the second Zn(2+) ion, responsible for fluorescence turn-on, the apparent formation constant, K(2)', is 5.5 × 10(7)-6.9 × 10(7) M(-1). A detailed chemical and mathematical analysis of the system demonstrated that the difference in emission efficiencies of the dimetalated (LZn(2)) vs monometalated (LZn) and metal-free (L) forms, a consequence of the combined quenching effects of the two metal-chelating units, significantly influences the shape of the titration curve. The scope of the titration method was investigated mathematically, and a lower boundary for the range of concentrations that can be determined was established as a function of the magnitude of K(2)'. Our results suggest that the principles governing the response of the ZPP1 series are applicable to other analogues of the Zinpyr family. Moreover, they may guide the design of other ditopic sensors suitable for determining the concentrations of a wide range of mobile metal ions and other chemical signaling agents of relevance in biological systems.

Dataset Information

Fluorescent Binding Protein Sensors for Detection and Quantification of Biochemicals, Metabolites, and Natural Products.

Publications

Fluorescent Binding Protein Sensors for Detection and Quantification of Biochemicals, Metabolites, and Natural Products.

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