Covalent control of polypeptide folding. Induction of helix-loop-helix motifs by bridging

Abstract

A de novo designed helix-loop-helix polypeptide motif is induced by introduction of an interhelical carbon bridge by a site selective reaction. The proposed structure of the bridged polypeptide is based on CD spectroscopy and previous structural studies of polypeptides with similar amino acid sequences. The polypeptide used for the bridging experiments is PE42Dcap, a de novo designed polypeptide with 42 amino acid residues that has been designed to fold into a helix-loop-helix motif and to dimerise to form four-helix bundles in aqueous solution. PE42Dcap is engineered to perform acyl transfer reactions with bifunctional esters yielding an interhelical, covalent bridge between two lysine residues. The active residues in PE42Dcap are His7, His11 and Lys15 in helix I and His26, His30 and Lys34 in helix II. Reaction of PE42Dcap with disuccinimidyl glutarate at pH 4.1 in sodium acetate buffer and 5 vol% 2,2,2-trifluoroethanol gave the pure bridged polypeptide DSG-PE42Dcap in an isolated yield of 35%.

CD spectroscopy on the precursor polypeptide PE42Dcap gave the absolute mean residue ellipticity −4800 deg cm² dmol⁻¹ at 222 nm in aqueous solution at pH 5.1. This shows that the helical content in the polypeptide is low. The introduction of the interhelical carbon bridge increased the absolute mean residue ellipticity to −20100 deg cm² dmol⁻¹, showing that the bridge has dramatically increased the helical content and that DSG-PE42Dcap is mainly present as helix-loop-helix motifs. The mean residue ellipticity of DSG-PE42Dcap shows concentration dependence that indicates that this bridged polypeptide is mainly monomeric at micromolar concentrations and dimeric above 100 μM.

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