Engineering of a 129-residue tripod protein by chemoselective ligation of proline-II helices

A 129-residue tripod protein was designed, synthesized, and biophysically characterized. This receptor-adhesive modular protein contained three 30-residue proline-II helices linked to a 9-residue proline-II helix through thioether bonds. Coupling of 6-maleimidohexanoic acid succinimido ester to cis-N^α-Boc-4-amino-L-proline furnished in 77% yield the maleimido acid cis-N-Boc-4-(6-maleimidohexanamido)-L-proline (Boc-Prm), which was used in the solid-phase synthesis of the linker peptide CH3-CO-Pro3-Prm3-Pro3-***NH2. The leg peptide, the 40-residue thiol Gly-Arg-Gly-Asp-Ser-Pro-Gly-Tyr-Gly-Pro30-Cys-NH2, was also made by solid-phase synthesis. The tripod protein was prepared by Michael addition of the thiol groups of three leg peptides to the three maleimide groups of the linker peptide. By ¹³C NMR spectrometry, the linker peptide was a proline-II helix, as indicated by the presence of only trans Pro-Pro resonances for its β and γ carbons. By circular dichroic spectroscopy, the model peptide CH3-CO-Pro9-NH2, the linker peptide, the leg peptide, and the tripod protein each contained substantial proline-II helix, as indicated by a strong negative band at 205 nm and a weak positive band at 226 nm. Since the Pro30 proline-II helix of each leg is about 93 Å long, two Arg-Gly-Asp sites on different legs of the tripod protein could be as much as ≈250 Å apart. © 1995 Elsevier Science Ltd.

Duke Scholars

Author Dewey G. McCafferty Chemistry