Synthesis and characterization of a long, rigid photoswitchable cross-linker for promoting peptide and protein conformational change

Azobenzene-based photoswitchable compounds can be use to photocontrol a variety of biochemical systems. In some cases, their effectiveness may be limited by the size of the conformational change that the switch undergoes. To produce an azobenzene photoswitch that undergoes a large end-to-end distance change upon isomerization, we synthesized 3,3′-diazene-1,2-diylbis[6-[2-sulfonato-4- (chloroacetylamino)phenylethynyl]benzene sulfonic acid] (DDPBA). This long, rigid, water-soluble, thiol-reactive cross-linker undergoes an end-to-end distance change of ∼ 13 Å upon isomerization. DDPBA was successfully cross-linked to peptides through cysteine side chains. The photoswitch undergoes trans-to-cis photoisomerization maximally when irradiated at 400 nm, although the efficiency of production of the cis isomer is lower than for simpler azobenzens. Under steady-state illumination conditions, the percentage of cis form produced increases as temperature increases; ∼ 56% cis is obtained at 60°C. Thermal relaxation occurs with a half-life of approximately 75 min at room temperature. When DDPBA was attached to an α-helical peptide with two cysteine residues at i and i+14 positions, an increase in helix content was observed after photoirradiation. When cross-linked to another peptide with two cysteine residues spaced at i and i+21 positions, a decrease in helix content after trans-to-cis isomerization was observed. Due to the small percentage of cis form produced under the experimental conditions, the CD signal changes were small. However, the large structural change upon photoisomerization provided by this cross-linker can potentially be used to photoswitch other biochemical systems.