DE NOVO DESIGN OF [[alpha]]-HELICAL PROTEINS: BASIC RESEARCH TO MEDICAL APPLICATION. Hodges, R.S., Dept. Biochemistry and the Protein Engineering Network of Centres of Excellence, Univ. Alberta, Edmonton, AB, Canada, T6G 2H7.
The two-stranded [[alpha]]-helical coiled-coil is not only an ideal model protein system to determine quantitatively the relative contributions that all the noncovalent interactions have in controlling protein conformation, folding and stability but is nature's way of creating a rod-like molecule for structural and regulatory roles. This universal dimerization motif has been found in a diverse group of over 200 proteins (muscle proteins, DNA binding proteins, viral proteins, enzymes and receptor proteins). We have shown that the minimum polypeptide chain length of coiled-coils is 3-heptads when maximally stabilized by hydrophobic and intra- and interchain electrostatic interactions. By controlling electrostatic interactions we have been able to design peptides which will not form homodimers (exist as random coils) but can form extremely stable heterodimers (Kd of ~ 1 nM). Our knowledge base has allowed us to use this heterodimerization system for a series of medical applications:
1) as synthetic vaccine delivery vehicles;
2) as a two-stage targeting and drug delivery system;
3) as a dimerization motif for antibody reductants and receptor domains;
4) as a cloning/expression/detection and purification system for peptides and proteins;
5) as a dimerization domain for biosensor applications.
In addition, we have utilized a small interchain disulfide bridged and intrachain lactam bridged homodimeric coiled-coil as a template for displaying combinatorial peptide libraries in a helical conformation. Lastly, the most difficult de novo design challenge is the design of nonhomodimerizing inhibitor molecules of coiled-coil function (both homo- and heterodimers). We have been able to control parallel and antiparallel association of polypeptide chains into either two-stranded or 4-stranded coiled-coils. These designs lay the ground-work for the next generation of pH, salt and metal-ion induced folding of proteins with unique biological activities.