Toward beta-amino acid proteins: a cooperatively folded beta-peptide quaternary structure

Abstract

Folded polymers in nature are assembled from simple monomers and adopt complex folded structures through networks of stabilizing noncovalent interactions. These interactions define secondary and tertiary structure and in most cases specify a unique three-dimensional architecture. Individual secondary or tertiary structures can also associate with one another to form multi-subunit quaternary structures. Nonnatural folded polymers have potential for similar structural versatility. Here we describe a pair of beta3-peptides whose sequences were designed to promote a 14-helix structure in water, favor hetero-oligomer formation, and disfavor nonspecific aggregation. These beta3-peptides assemble noncovalently into a well-defined hetero-oligomer characterized by a defined stoichiometry, a highly stabilized secondary structure, and a cooperative melting transition (TM > 55 degrees C). This work demonstrates that beta3-peptides can assemble into defined, cooperatively folded quaternary structures and constitutes an important step toward designing protein-like assemblies from nonnatural polymers.

Figure 1

Helical net representation of β³-peptides described in this work. β³-homoamino acids are identified by the single letter code used for the corresponding α-amino acids. O signifies β³-homo-ornithine.

Figure 2

Isothermal (A,B) or temperature-dependent (C) CD analysis of Acid-1F, Base-1F, or a 1:1 mixture thereof. Sedimentation equilibrium analysis of (D) Acid-1F fit to a monomer model, (E) Base-1F fit to a monomer model, or (F) a 1:1 mixture thereof fit to a monomer–octamer model acquired at 42 000 (black), 50 000 (blue), and 60 000 (red) rpm. All experiments were performed at a β³-peptide concentration of 25 μM in PBC buffer (1 mM phosphate, 1 mM borate, and 1 mM citrate (pH 7.1)).

Figure 3

(A) Equilibrium between Base-1F^SSBase-1F and Acid-1F^SS Acid-1F homodimers and Base-1F^SS Acid-1F heterodimer under redox conditions (PBC buffer (pH 7.1) containing 500 μM reduced glutathione and 125 μM oxidized glutathione). (B) HPLC analysis of species observed upon incubation of 25 μM Base-1F^SS Acid-1F in redox buffer for 5 min and 6 and 24 h. G indicates glutathione.