A PHOTOGENERATED PORE-FORMING PROTEIN

Background: The permeabilization of cells with bacterial pore-forming proteins is an important technique in cell biology that allows the exc hange of small reagents into the cytoplasm of a cell. Another notable technology is the use of caged molecules whose activities are blocked by addition of photoremovable protecting groups. This allows the photo generation of reagents on or in cells with spatial and temporal contro l. Here, we combine these approaches to produce a caged pore-forming p rotein for the controlled permeabilization of cells. Results: 2-Bromo- 2-(2-nitrophenyl)acetic acid (BNPA), a water-soluble cysteine-directed reagent for caging peptides and proteins with the alpha-carboxy-2-nit robenzyl (CNB) protecting group, was synthesized. Glutathione (gamma-G lu-Cys-Gly) was released in high yield from gamma-Glu-CysCNB-Gly by ir radiation at 300 nm. Based on this finding, scanning mutagenesis was u sed to find a single-cysteine mutant of the pore-forming protein staph ylococcal alpha-hemolysin (alpha HL) suitable for caging. When alpha H L-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 activit y. 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, alpha HL-R104C-CNB and o ther caged proteins designed to create pores of various diameters shou ld 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 cel l could be used in studies of neuronal modulation. Further, BNPA shoul d be generally useful for caging cysteine-containing peptides and sing le-cysteine mutant proteins to study, for example, cell signaling or s tructural changes in proteins.