Subject(s)
Peptides/chemical synthesis , Proteins/chemical synthesis , Amino Acid Sequence , Animals , Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/radiation effects , Cysteine/chemistry , Indicators and Reagents , Macromolecular Substances , Molecular Probes/chemical synthesis , Molecular Probes/chemistry , Molecular Probes/radiation effects , Oligopeptides/chemistry , Oligopeptides/radiation effects , Peptides/chemistry , Peptides/radiation effects , Photochemistry , Photolysis , Proteins/chemistry , Proteins/radiation effects , Recombinant Proteins/chemistry , Recombinant Proteins/radiation effects , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Sulfur/chemistryABSTRACT
BACKGROUND: The permeabilization of cells with bacterial pore-forming proteins is an important technique in cell biology that allows the exchange 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 photogeneration of reagents on or in cells with spatial and temporal control. Here, we combine these approaches to produce a caged pore-forming protein 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-nitrobenzyl (CNB) protecting group, was synthesized. Glutathione (gamma-Glu-Cys-Gly) was released in high yield from gamma-Glu-CysCNB-Gly by irradiation at 300 nm. Based on this finding, scanning mutagenesis was used to find a single-cysteine mutant of the pore-forming protein staphylococcal alpha-hemolysin (alpha HL) suitable for caging. When alpha HL-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, alpha HL-R104C-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.
