Molecular scissors under light control.

L ight plays a key role in the living world. It serves as a source of energy through photosynthesis, makes sight possible, and regulates circadian rhythms. During evolution, organisms from bacteria to higher mammals elaborate various mechanisms to sense and respond to light, which manipulates their behavior. The use of light as a trigger is particularly attractive as a means of controlling biological processes at will, because light is noninvasive and can be manipulated both temporally (from microseconds) and spatially (microns) (1). In this issue of PNAS, Schierling et al. (2) demonstrate the possibility of controlling the enzymatic activity of the restriction endonuclease PvuII by light. Restriction endonucleases are molecular scissors that recognize short DNA sequences usually 4–8 bp in length and cut the phosphodiester bond within or close to their target sites to generate a doublestranded break. To manipulate the catalytic activity of the orthodox restriction endonuclease PvuII by light, a previous study exploited a well-known feature of the photosensitive azobenzene derivative to reversibly isomerize between trans and cis forms under the influence of light (Fig. 1A) (3). Under illumination by UV light at 366 nm, the azobenzene in trans configuration is converted to cis isomer, resulting in an end-to-end distance change. This geometry change of the azobenzene moiety is reversible, because illumination by blue light (or thermal relaxation) results in a cis to trans configuration transition. To act as a reversible photoswitch that controls protein activity, one isomeric form of the azobenzene should be able to lock the enzyme in the inactive “off” state, and the other form should be able to lock the enzyme in the active “on” state (Fig. 1A). For this to occur, the bifunctional azobenzene derivative must be attached to the protein structural components, to allow transmittal of the change in switch conformation to the substrate-binding site or the catalytic center. Because the attachment sites, which ensure tight coupling between cross-linker photoisomerization and protein conformational change, are difficult to predict, the optimal switch position must be identified experimentally. Schierling et al. (2) used a Cys-selective azobenzene derivative to install the cross-linker onto proteins bearing two surface-exposed Cys residues. Cys residues can be easily introduced into the protein via site-directed mutagenesis, enabling scanning for suitable cross-linker attachment positions on the protein surface. In the case of the PvuII restriction endonuclease, Schierling et al. engineered more than 30 protein variants, each containing two or four Cys residues for the bifunctional azobenzene cross-linker attachment. Specifically, eight different sets of Cys residue pairs located on the surface of the PvuII dimer or a single chain variant (scPvuII) were probed as a cross-linker attachment sites to identify positions where the installed switch has