Foldamer Tertiary Structure through Sequence-Guided Protein Backbone Alteration

Abstract

The prospect of recreating the complex structural hierarchy of protein folding in synthetic oligomers with backbones that are artificial in covalent structure ("foldamers") has long fascinated chemists. Foldamers offer complex functions from biostable scaffolds and have found widespread applications in fields from biomedical to materials science. Most precedent has focused on isolated secondary structures or their assemblies. In considering the goal of complex protein-like tertiary folding patterns, a key barrier became apparent. How does one design a backbone with covalent connectivity and a sequence of side-chain functional groups that will support defined intramolecular packing of multiple artificial secondary structures? Two developments were key to overcoming this challenge. First was the recognition of the power of blending α-amino acid residues with monomers differing in backbone connectivity to create "heterogeneous-backbone" foldamers. Second was the finding that replacing some of the natural α-residues in a biological sequence with artificial-backbone variants can result in a mimic that retains both the fold and function of the native sequence and, in some cases, gains advantageous characteristics. Taken together, these precedents lead to a view of a protein as chemical entity having two orthogonal sequences: a sequence of side-chain functional groups and a separate sequence of backbone units displaying those functional groups. In this Account, we describe our lab's work over the last ∼10 years to leverage the above concept of protein sequence duality in order to develop design principles for constructing heterogeneous-backbone foldamers that adopt complex protein-like tertiary folds. Fundamental to the approach is the utilization of a variety of artificial building blocks (e.g., d-α-residues, C_α-Me-α-residues, N-Me-α-residues, β-residues, γ-residues, δ-residues, polymer segments) in concert, replacing a fraction of α-residues in a given prototype sequence. We provide an overview of the state-of-the-art in terms of design principles for choosing substitutions based on consideration of local secondary structure and retention of key side-chain functional groups. We survey high-resolution structures of backbone-modified proteins to illustrate how diverse artificial moieties are accommodated in tertiary fold contexts. We detail efforts to elucidate how backbone alteration impacts folding thermodynamics and describe how such data informs the development of improved design rules. Collectively, a growing body of results by our lab and others spanning multiple protein systems suggests there is a great deal of plasticity with respect to the backbone chemical structures upon which sequence-encoded tertiary folds can manifest. Moreover, these efforts suggest sequence-guided backbone alteration as a broadly applicable strategy for generating foldamers with complex tertiary folding patterns. We conclude by offering some perspective regarding the near future of this field, in terms of unanswered questions, technological needs, and opportunities for new areas of inquiry.

Figure 1

Heterogeneous backbones that blend natural α-amino acid residues with various artificial building blocks can adopt protein-like tertiary folds when backbone displayed side-chain sequence encodes such behavior.

Figure 2

Key to nomenclature for commonly used building blocks in heterogeneous-backbone peptide and protein mimics.

Figure 3

Overview of design principles for sequence-guided protein backbone alteration. Each panel presents a canonical secondary structure (from PDB 2QMT) alongside select replacement strategies shown viable in tertiary fold contexts.

Figure 4

Mimicry of GB1 by heterogeneous backbones. (A) Sequence and crystal structure (PDB 2QMT) of wild-type GB1. (B) Some backbone modifications made to GB1, grouped by secondary structure context. All panels are from published crystal structures of GB1 variants (PDB: 5HFY, 5HI1, 4OZB, 4OZC, 4KGR, 4KGS, 4KGT), except data for the PEG-modified loop (from a molecular dynamics simulation) and the γ^cyc-modified sheet (from the NMR structure of a hairpin peptide).

Figure 5

Mimicry of Sp1-3 by heterogeneous backbones. (A) Sequence of Sp1-3 and two variants (R groups, when present, are defined by the single letter code of the corresponding α-residue). (B) NMR structures of Sp1-3 (PDB 1SP1) and one variant (PDB 5US3). (C) Retention of a native-like metal-coordination environment (inset) in the modified backbones is supported by visible absorption spectra with bound Co²⁺. (D) Comparison of folding thermodynamics shows that the modified backbones have stabilities superior to native Sp1-3.