Comparison of design strategies for α-helix backbone modification in a protein tertiary fold

Abstract

We report here the comparison of five classes of unnatural amino acid building blocks for their ability to be accommodated into an α-helix in a protein tertiary fold context. High-resolution structural characterization and analysis of folding thermodynamics yield new insights into the relationship between backbone composition and folding energetics in α-helix mimetics and suggest refined design rules for engineering the backbones of natural sequences.

Heterogeneous-Backbone Foldamer Mimics of Zinc Finger Tertiary Structure

“…Taking the energetic values for 1 as a baseline (Figure 7), the backbone modifications in 4 (2 α→N-Me-α; 4 α→β 3 ) have a moderately unfavorable effect on folding ΔH (~0.6 kcal mol −1 per substitution) that is more than compensated for by a favorable effect on folding TΔS (~0.7 kcal mol −1 per substitution). The above qualitative trend follows that seen upon α→β 3 modifications in the helix of GB1; 16a however, the quantitative balance between parameters differs between the two systems. 5d The magnitude of the unfavorable ΔΔH in 4 vs. 1 is comparable in GB1 and Sp1–3 yet the corresponding favorable ΔΔS is much greater in the zinc finger motif.…”

“…We recently reported a stabilizing effect from β 3 →C α -Me-α substitution in the helix of the GB1 tertiary fold and were motivated to see to what degree those observations might hold in a different structural context. 16b The data obtained in the Co 2+ binding assay showed that peptide 9 supports a metal coordination environment indistinguishable from both analogue 4 and the native backbone ( 1 ). Thus, we advanced peptides 1 , 4 , and 9 to experiments aimed at a more thorough characterization of folded structure and stability.…”

“…4c,5d,16 A single α→N-Me-α substitution was made in each strand of the hairpin segment at sites where the newly introduced methyl group should project toward solvent. α→β 3 substitutions were incorporated in each turn of the α-helix, such that added CH 2 groups in the backbone reside opposite the hydrophobic core of the tertiary fold.…”

“…Here, backbone modification has an opposite favorable effect on folding, with a magnitude as large as 0.5 kcal mol −1 in the case of peptide 4 . Because the folded state of each analogue remains identical to that of the native sequence ( vide infra ), we reason that the differences in fold stability originate elsewhere along the folding pathway, either in the denatured ensemble 16a or folding intermediates. 35 …”

Systematic Targeting of Protein–Protein Interactions

“…Significantly, many of these tertiary and quaternary protein structure scaffolds have been engineered for phage display, allowing rapid selection of potent sequences for desired targets. Synthetic protein tertiary structures with non-natural backbones have also been described[110-112]. …”