Design, synthesis and solution structure of a helix–loop–helix dimer—a template for the rational design of catalytically active polypeptides

Abstract

A ploypeptide with 42 amino acids, SA-42, has been designed to fold into a ‘hairpin’ helix–loop–helix motif. Its intended use is to serve as a template for the rational design of catalytically active polypeptides. SA-42 is made up of two amphiphilic helices, that are designed to interact through the hydrophobic effect and are connected by a short loop. Evidence is presented that it does adopt the intended motif and that it dimerises in an antiparallel mode at concentrations between 2 µmol dm^–3 and 5.6 mmol dm^–3. The structural evidence was obtained from NMR and CD spectroscopy and the state of aggregation was determined with equilibrium sedimentation ultracentrifugation and CD spectroscopy. Special attention was paid to the design of SA-42 with respect to the expected NMR spectrum. The mean residue ellipticity at 222 nm of SA-42, –25000 deg cm² dmol^–1, compares well with that of other published helix–loop–helices. Two polypeptides that were developed from SA-42 by the replacement of three and five residues, respectively, were recently shown to have catalytical activity.