A photogenerated pore-forming protein Original Research Article

Results: 2-Bromo-2-(2-nitrophenyl)acetic acid (BNPA), a water-soluble cysteine-directed reagent for caging peptides and proteins with the α-carboxy-2-nitrobenzyl (CNB) protecting group, was synthesized. Glutathione (γ-Glu-Cys-Gly) was released in high yield from γ-Glu-Cys-CNB-Gly by irradiation at 300 ran. Based on this finding, scanning mutagenesis was used to find a single-cysteine mutant of the pore-forming protein staphylococcal α-hemolysin (αHL) suitable for caging. When αHL-R104C was derivatized with BNPA, pore-forming activity toward rabbit erythrocytes was lost. Near UV irradiation led to regeneration of the cysteine sulfhydryl group and the restoration of pore-forming activity. Conclusions: Caged pore-forming proteins are potentially useful for permeabilizing one cell in a collection of cells or one region of the plasma membrane of a single cell. Therefore, αHL-Rl04C-CNB and other caged proteins designed to create pores of various diameters should be useful for many purposes. For example, the ability to introduce reagents into one cell of a network or into one region of a single cell could be used in studies of neuronal modulation. Further, BNPA should be generally useful for caging cysteine-containing peptides and single-cysteine mutant proteins to study, for example, cell signaling or structural changes in proteins.