Achieving photo-control of protein conformation and activity: producing a photo-controlled leucine zipper.
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.
Kumita
Janet R
JR
Department of Chemistry, The University of Toronto, 80 St. George Street, Toronto M5S 3H6, Canada.
Flint
Daniel G
DG
Woolley
G Andrew
GA
Smart
Oliver S
OS
D001391:Azo Compounds / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D016350:Leucine Zippers; D010777:Photochemistry / Q000379:methods*; D011487:Protein Conformation / Q000528:radiation effects; D015202:Protein Engineering / Q000379:methods*; D013329:Structure-Activity Relationship
D001391:Azo Compounds; D003432:Cross-Linking Reagents; C009850:azobenzene