Interplay among side chain sequence, backbone composition, and residue rigidification in polypeptide folding and assembly

Abstract

The extent to which polypeptide conformation depends on side-chain composition and sequence has been widely studied, but less is known about the importance of maintaining an α-amino acid backbone. Here, we examine a series of peptides with backbones that feature different repeating patterns of α- and β-amino acid residues but an invariant side-chain sequence. In the pure α-backbone, this sequence corresponds to the previously studied peptide GCN4-pLI, which forms a very stable four-helix bundle quaternary structure. Physical characterization in solution and crystallographic structure determination show that a variety of α/β-peptide backbones can adopt sequence-encoded quaternary structures similar to that of the α prototype. There is a loss in helix bundle stability upon β-residue incorporation; however, stability of the quaternary structure is not a simple function of β-residue content. We find that cyclically constrained β-amino acid residues can stabilize the folds of α/β-peptide GCN4-pLI analogues and restore quaternary structure formation to backbones that are predominantly unfolded in the absence of cyclic residues. Our results show a surprising degree of plasticity in terms of the backbone compositions that can manifest the structural information encoded in a sequence of amino acid side chains. These findings offer a framework for the design of nonnatural oligomers that mimic the structural and functional properties of proteins.

Structural and biological mimicry of protein surface recognition by α/β-peptide foldamers

“…The magnitude of the CD signatures among the well-folded mixtures is similar, but the ratio of intensities at 208 and 222 nm changes as a function of ␤-residue content. This trend is consistent with our previous studies on helical oligomers containing mixed ␣/␤ backbones (24,26). The complexes formed by 1ϩ3, 1ϩ5, 1ϩ8, and 1ϩ10 each showed highly cooperative thermal transitions (Fig.…”

“…␣/␤-Peptides 5 and 6 represent an improvement in gp41 mimicry relative to 4 (vide supra), but it would be desirable to place ␤-residues throughout an ␣/␤-peptide sequence to maximize resistance to proteolysis (30)(31)(32). Each ␣3␤ 3 replacement, however, adds a flexible bond to the backbone, which should increase the conformational entropy penalty associated with helix formation (26,33,34). The greater conformational entropy of the unfolded state of 4 relative to 3, arising from eleven ␣3␤ 3 replacements, may account for the large difference in binding affinity for gp41-5 between these two oligomers.…”

“…When the two bundles are aligned via the NHR trimer (Fig. 3B), the CHR helices track very closely in the C-terminal segment (0.84 Å C ␣ rmsd for residues [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33], but diverge near the N terminus (4.2 Å C ␣ rmsd for residues [2][3][4][5][6][7][8][9][10][11][12][13][14][15]. This divergence reflects a greater superhelical twist in ␣-peptide 3 relative to ␣/␤-peptide 10.…”

“…␣-Peptide 3 represents one of the most successful examples reported to date of improving the antiviral efficacy of gp41 CHR ␣-peptides via modification of the ␣-amino acid sequence. We began with the side chain sequence optimized in 3 and explored the orthogonal variable of backbone composition in the form of ␣3␤ residue substitution [a method we term ''sequence-based design'' (24)(25)(26)]. In ␣/␤-peptide 4, a subset of the ␣-residues in 3 has been replaced by corresponding ␤ 3 -residues ( Fig.…”

“…1 A). Preliminary findings indicated that this sequence-based design strategy generates ␣/␤-peptides that can form protein-like quaternary assemblies or mimic short ␣-helical domains involved in apoptotic signaling (24)(25)(26). The gp41 protein represents an attractive target for exploratory studies related to sequence-based backbone modification because of the extensive structural data available for this system and the prospect of evaluating the functional consequences of backbone alterations in biological assays.…”