The coiled coil stem loop miniprotein as a presentation scaffold

The coiled coil helical dimer found in naturally occurring proteins is conformationally stable and can tolerate significant sequence variation on the solvent-exposed surfaces of the helices. We are interested in exploring the use of a de novo designed coiled coil stem loop miniprotein (CCSL) as a template for presenting (1) helical and loop sequences from heterologous proteins and (2) constrained libraries of peptides. Towards this end, we synthesized a 56 residue prototype CCSL and verified its structure by extensive biophysical characterization. CCSL variants with altered sequences in the solvent-exposed helical positions were found to fold similarly to the prototype design. Based on the results with the CCSL produced by peptide synthesis, we assembled a synthetic cDNA for the CCSL prototype and expressed the CCSL miniprotein on filamentous phage. This genetic construction can be used to introduce random peptide libraries into regions within the scaffold loop and helices in order to identify key side chains of native proteins involved in binding, to establish structural models for their pharmacophores and to identify novel peptide recognition mimics of macromolecular ligands and their receptors.