Sequential activation and deactivation of protein function using spectrally differentiated caged phosphoamino acids

Area of Science:

• Biochemistry
• Chemical Biology
• Molecular Biology

Background:

• Photolabile caging groups, such as 1-(2-nitrophenyl)ethyl (NPE), are used to study biological processes like protein phosphorylation.
• Previous studies were limited to uncaging only one phosphopeptide per experiment.

Purpose of the Study:

• To develop a method for sequentially uncaging two different phosphopeptides in a single system.
• To enable the interrogation of multiple phosphorylation events simultaneously.

Main Methods:

• Synthesis of [7-(diethylamino)coumarin-4-yl]methyl (DEACM)-caged phosphorylated serine, threonine, and tyrosine building blocks.
• Incorporation of these building blocks into peptides using Fmoc-based solid-phase peptide synthesis.
• Sequential uncaging using two different wavelengths of light: 420 nm for DEACM and 365 nm for NPE.

Main Results:

• Selective release of DEACM-caged phosphopeptides with 420 nm light without affecting NPE-caged peptides.
• Subsequent release of NPE-caged phosphopeptides with 365 nm light.
• Demonstration of the sequential uncaging approach by controlling Wip1 phosphatase activity.

Conclusions:

• A versatile sequential uncaging approach for studying multiple phosphorylation events has been established.
• This method allows for precise temporal control over biological processes using light.
• The developed building blocks facilitate the incorporation of photocaged phosphoresidues into peptides and proteins.