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-Nα-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