Binding of Zn-chlorin to a synthetic four-helix bundle peptide through histidine ligation.
We have used a two histidine-containing synthetic peptide (Sharp et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 10465-10470) as a scaffold to bind Zn(II) chlorin e6 (ZnCe6) through histidine ligation. Protocols for the preparation and purification of the peptide using an Escherichia coli expression system are presented. Size-exclusion chromatography and circular dichroism measurements indicate that the peptide self-assembles into a four-helix bundle protein. Two variants of the peptide lacking either one or both of the histidine residues were used to demonstrate the stoichiometry of ZnCe6 binding. Comparison of the titration profiles determined by UV-vis spectroscopy for the purified one- and two-histidine peptides suggests that the two-histidine peptide can bind two ZnCe6. The binding stoichiometry of ZnCe6 was verified by gel chromatography and native gel electrophoresis using the peptide variant lacking histidine residues as the control. Like many other chlorophyll analogue molecules, ZnCe6 can be photooxidized. The light-induced electron transfer between the ZnCe6-peptide complex and the added phenyl-p-benzoquinone was measured using time-resolved EPR spectroscopy and shown to be faster and have a higher yield than the electron transfer between unbound ZnCe6 and quinone. The implications of constructing a ZnCe6-peptide complex in terms of artificial photosynthesis are discussed.
Razeghifard
M Reza
MR
Photobioenergetics, Research School of Biological Sciences, The Australian National University, Canberra, ACT 0200, Australia. razeghifard@rsbs.anu.edu.au
Wydrzynski
Tom
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D004579:Electron Transport; D018257:Helix-Loop-Helix Motifs; D006639:Histidine / Q000737:chemistry*; D008024:Ligands; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry*; D010782:Photolysis; D011166:Porphyrins / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D020449:Repetitive Sequences, Amino Acid; D015032:Zinc / Q000737:chemistry*
D008024:Ligands; D010455:Peptides; D011166:Porphyrins; C006969:chlorin; D006639:Histidine; D015032:Zinc