A single circularly permuted GFP sensor for inositol-1,3,4,5-tetrakisphosphate based on a split PH domain

Area of Science:

• Biochemistry
• Molecular Biology
• Fluorescence Spectroscopy

Background:

• Inositol phosphates play crucial roles in cellular signaling.
• Accurate detection of specific inositol phosphates like Ins(1,3,4,5)P4 is vital for understanding cellular processes.
• Existing detection methods may lack sensitivity or specificity.

Purpose of the Study:

• To develop a novel, highly specific fluorescent sensor for Ins(1,3,4,5)P4 detection.
• To enable ratiometric fluorescence detection for enhanced signal-to-noise ratio.
• To leverage structure-based design for sensitive molecular detection.

Main Methods:

• Construction of a fluorescent sensor using a split PH domain and a circularly permuted GFP.
• Structure-based design to link ligand binding to GFP conformational changes.
• Ratiometric fluorescence measurements upon excitation of distinct GFP chromophore states.

Main Results:

• The sensor successfully detected Ins(1,3,4,5)P4 with high affinity and selectivity.
• Ligand binding induced a measurable change in the protonation state of the GFP chromophore.
• Ratiometric fluorescence changes correlated with Ins(1,3,4,5)P4 concentration.

Conclusions:

• A novel split PH domain-based fluorescent sensor for Ins(1,3,4,5)P4 was successfully developed.
• The sensor provides a sensitive and selective method for ratiometric fluorescence detection.
• This tool facilitates further research into the biological roles of Ins(1,3,4,5)P4.