| Photo-regulatable protein “switches” could be applied to a wide variety of systems and would provide a powerful tool for probing biological function without removing the protein from its native environment. Our approach has been to chemically synthesize photo-isomerizable cross-linking reagents and then use these to reversibly alter the three dimensional structure of a target protein. A bifunctional iodoacetamide derivative of azobenzene was synthesized, characterized and used to intramolecularly cross-link two cysteine residues in a variety of engineered peptides. Reversible photo-control of peptide secondary structure in systems with cysteine spacings of (i, i+7), (i, i+4) and (i, i+11) and with a variety of different amino acids located between the cysteines (positions i+3,1+4) were studied. Photo-isomerization of the azobenzene group altered the peptide's helicity in the predicted direction. In the (i, i+7)Cys and (i, i+4)Cys peptides, the trans conformation was disordered whereas the cis conformation was predominantly helical. In the (i, i+11)Cys peptide, the trans resulted in a stable helical structure and the cis conformation was disordered. This photo-isomerizable cross-linking strategy was then applied to the GCN4-bZIP fragment of the yeast transcriptional activator, GCN4. GCN4-bZIP contains a leucine zipper segment and a DNA binding segment. The cross-linker was successfully introduced into the 33 residue leucine zipper segment and the 58 residue GCN4-bZIP fragment. Circular dichroism analysis for both systems demonstrated an increase in helical character upon isomerization, and DNA binding activity of cross-linked GCN4-bZIP is destabilized (relative to the uncross-linked peptide), in the trans conformation, while in the cis conformation, DNA binding increases (to binding similar or slightly higher than uncross-linked peptide). We have thus successfully designed a photo-controlled DNA transcription factor. |
