Visible Light Control over the Cytolytic Activity of a Toxic Pore-Forming Protein.

Enabling control over the bioactivity of proteins with light, along with the principles of photopharmacology, has the potential to generate safe and targeted medical treatments. Installing light sensitivity in a protein can be achieved through its covalent modification with a molecular photoswitch. The general challenge in this approach is the need for the use of low energy visible light for the regulation of bioactivity. In this study, we report visible light control over the cytolytic activity of a protein. A water-soluble visible-light-operated tetra-ortho-fluoro-azobenzene photoswitch was synthesized by utilizing the nucleophilic aromatic substitution reaction for installing a solubilizing sulfonate group onto the electron-poor photoswitch structure. The azobenzene was attached to two cysteine mutants of the pore-forming protein fragaceatoxin C (FraC), and their respective activities were evaluated on red blood cells. For both mutants, the green-light-irradiated sample, containing predominantly the cis-azobenzene isomer, was more active compared to the blue-light-irradiated sample. Ultimately, the same modulation of the cytolytic activity pattern was observed toward a hypopharyngeal squamous cell carcinoma. These results constitute the first case of using low energy visible light to control the biological activity of a toxic protein.

Strategies for enzymological studies and measurements of biological molecules with the cytolysin A nanopore.

Pore-forming toxins are used in a variety of biotechnological applications. Typically, individual membrane proteins are reconstituted in artificial lipid bilayers where they form water-filled nanoscale apertures (nanopores). When a voltage is applied, the ionic current passing through a nanopore can be used for example to sequence biopolymers, identify molecules, or to study chemical or enzymatic reactions at the single-molecule level. Here we present strategies for studying individual enzymes and measuring molecules, also in highly complex biological samples such as blood.