A photogenerated pore-forming protein

Area of Science:

• Biochemistry
• Cell Biology
• Protein Engineering

Background:

• Cell permeabilization using bacterial pore-forming proteins facilitates reagent exchange into the cytoplasm.
• Caged molecules enable spatial and temporal control over reagent generation via photoremovable protecting groups.
• Combining these techniques offers a novel approach for controlled cellular manipulation.

Purpose of the Study:

• To develop a caged pore-forming protein for controlled cell permeabilization.
• To create a tool for precise, light-activated delivery of molecules into cells.

Main Methods:

• Synthesis of 2-Bromo-2-(2-nitrophenyl)acetic acid (BNPA) for caging peptides and proteins.
• Development of a single-cysteine mutant of staphylococcal alpha-hemolysin (alpha HL) for derivatization.
• Utilizing scanning mutagenesis to identify suitable cysteine residues for modification.

Main Results:

• BNPA successfully caged glutathione, releasing it upon 300 nm irradiation.
• Derivatization of alpha HL-R104C with BNPA abolished its pore-forming activity.
• Near-UV irradiation restored pore-forming activity by regenerating the cysteine sulfhydryl group.

Conclusions:

• Caged pore-forming proteins offer precise control over cell permeabilization, targeting single cells or specific membrane regions.
• This technology is valuable for studying neuronal modulation, cell signaling, and protein structural dynamics.
• BNPA is a versatile reagent for caging cysteine-containing peptides and proteins.