Multipartite Fluorogenic Sensors for Monitoring Tyrosine Phosphatase Activity.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Fluorogenic substrates are crucial for assaying enzyme activity, particularly protein tyrosine phosphatases (PTPs).
• Developing genetically encodable sensors offers a powerful approach for real-time monitoring of enzyme activity within living systems.

Purpose of the Study:

• To develop genetically encodable sensors for PTP activity.
• To utilize the Fluorescence-Activating and absorption Shifting Tag (FAST) protein for PTP activity detection.

Main Methods:

• Utilized the FAST protein, which becomes fluorescent upon binding a small molecule dye.
• Demonstrated FAST protein's ability to sense PTP-mediated dephosphorylation of phosphorylated dye molecules.
• Assessed PTP1B activity using phosphorylated 4-hydroxybenzylidene rhodanine (pHBR) in the presence of FAST.

Main Results:

• Phosphorylated pHBR, in conjunction with FAST, enabled sensitive PTP activity assays, detecting concentrations as low as 100 pM PTP1B.
• The FAST system exhibited a kcat of 19±1 s⁻¹ and a KM of 93±3 μM for PTP1B.
• Phosphorylation of a split FAST protein's C-terminal peptide abrogated fluorescence, which was restored upon PTP-mediated dephosphorylation.

Conclusions:

• The FAST protein serves as a viable sensor for PTP activity by detecting the dephosphorylation of specific substrates.
• Genetically encodable FAST-based sensors represent a promising advancement for studying PTPs in biological contexts.