Depsipeptide substrates for sortase-mediated N-terminal protein ligation

Area of Science:

• Biochemistry
• Chemical Biology
• Molecular Biology

Background:

• Efficient chemical modification of proteins under mild conditions is crucial.
• Sortase-mediated peptide ligation is a known strategy for protein terminus modification.
• Traditional sortase A ligation is reversible, often requiring excess reagents and leading to lower yields.

Purpose of the Study:

• To develop an irreversible sortase-mediated ligation strategy for complete protein modification.
• To synthesize and utilize novel depsipeptide substrates for enhanced sortase A activity.
• To achieve efficient protein labeling under mild conditions with catalytic amounts of sortase.

Main Methods:

• Utilized sortase A (SrtA) enzyme for protein ligation.
• Synthesized novel depsipeptide substrates containing an ester linkage (threonine-glycolic acid).
• Appended an N-terminal FITC fluorophore via a thiourea linkage to the substrate.

Main Results:

• The use of depsipeptide substrates rendered the SrtA-mediated ligation irreversible.
• Complete protein labeling was achieved with a single N-terminal glycine residue under mild conditions in 4-6 hours.
• Efficient labeling was accomplished using a small excess of substrate and catalytic quantities of sortase.
• Depsipeptide substrate synthesis typically takes 2-3 days.

Conclusions:

• Depsipeptide substrates offer an irreversible and efficient alternative for sortase A-mediated protein ligation.
• This method allows for complete protein modification with reduced reagent requirements.
• The protocol is suitable for labeling proteins under mild, biologically relevant conditions.