Achieving photo-control of protein conformation and activity: producing a photo-controlled leucine zipper

Faraday Discuss. 2003:122:89-103; discussion 171-90. doi: 10.1039/b200897a.

Abstract

We have recently developed a technique that has great potential in producing proteins with photo-control of conformation and consequently activity (J. R. Kumita, O. S. Smart and G. A. Woolley, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 3803-3808). The method is based on incorporating two cysteine residues into the sequence of a polypeptide. An azobenzene derivative is subsequently used to produce an intramolecular cross-link between the cysteine sulfhydryl groups. In previous work photo-isomerisation of the azobenzene moiety has been used to control the helicity of a monomeric peptide. In the experiments described here this method has been applied to the coiled coil leucine zipper peptide GCN4-p1. The aim was to produce a variant of GCN4-p1 whose helicity and consequently dimerisation is under direct photo-control. We have produced a modified GCN4-p1 incorporating two cysteine residues. The mutations introduced are shown to interfere with the ability of the uncross-linked peptide to form a coiled coil. After the peptide was cross-linked with the azobenzene derivative more normal coiled-coil behaviour was restored. Irradiation of the peptide producing a conformational change in the azobenzene cross-linker was accompanied by an increase in the helicity of the peptide. The work presented here highlights the potential of the use of photo-isomerisable cross-linkers to control protein activity through induced conformational change. In addition, the methodology has the potential to provide a fast trigger for the initiation of protein conformational changes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Azo Compounds / chemistry
  • Cross-Linking Reagents / chemistry
  • Leucine Zippers*
  • Photochemistry / methods*
  • Protein Conformation / radiation effects
  • Protein Engineering / methods*
  • Structure-Activity Relationship

Substances

  • Azo Compounds
  • Cross-Linking Reagents
  • azobenzene