Analysis of Three Architectures for Controlling PTP1B with Light

Area of Science:

• Biochemistry
• Molecular Biology
• Optogenetics

Background:

• Photosensory domains enable optical control of protein function.
• Integrating these domains into light-sensitive proteins (chimeras) is challenging.
• Comparing different chimera architectures is crucial for optimizing optogenetic tools.

Purpose of the Study:

• To compare three distinct architectures for light-controlled protein tyrosine phosphatase 1B (PTP1B).
• To evaluate the effectiveness of fusion, insertion, and split constructs for optogenetic applications.
• To assess the correlation between in vitro and in vivo performance of optogenetic designs.

Main Methods:

• Constructed three PTP1B chimera architectures: fusion, insertion, and split.
• Assessed PTP1B activity in vitro using kinetic assays.
• Validated optical control in mammalian cells, monitoring activity and localization.
• Tested insertion architecture on other phosphatases like TCPTP and SHP2.

Main Results:

• All three PTP1B designs enabled optical control in vitro and in cells.
• Fusion and insertion architectures demonstrated superior dynamic range and native localization.
• In vitro and in cell photoresponses were linearly correlated.
• Insertion architecture successfully controlled PTP1B and TCPTP, but not SHP2.

Conclusions:

• Protein tyrosine phosphatases (PTPs) tolerate domain insertions well.
• In vitro screening is a reliable method for evaluating optogenetic designs.
• Fusion and insertion architectures are promising for developing light-controlled PTPs.