Characterization of long and stable de novo single alpha-helix domains provides novel insight into their stability

Abstract

Naturally-occurring single α-helices (SAHs), are rich in Arg (R), Glu (E) and Lys (K) residues, and stabilized by multiple salt bridges. Understanding how salt bridges promote their stability is challenging as SAHs are long and their sequences highly variable. Thus, we designed and tested simple de novo 98-residue polypeptides containing 7-residue repeats (AEEEXXX, where X is K or R) expected to promote salt-bridge formation between Glu and Lys/Arg. Lys-rich sequences (EK3 (AEEEKKK) and EK2R1 (AEEEKRK)) both form SAHs, of which EK2R1 is more helical and thermo-stable suggesting Arg increases stability. Substituting Lys with Arg (or vice versa) in the naturally-occurring myosin-6 SAH similarly increased (or decreased) its stability. However, Arg-rich de novo sequences (ER3 (AEEERRR) and EK1R2 (AEEEKRR)) aggregated. Combining a PDB analysis with molecular modelling provides a rational explanation, demonstrating that Glu and Arg form salt bridges more commonly, utilize a wider range of rotamer conformations, and are more dynamic than Glu–Lys. This promiscuous nature of Arg helps explain the increased propensity of de novo Arg-rich SAHs to aggregate. Importantly, the specific K:R ratio is likely to be important in determining helical stability in de novo and naturally-occurring polypeptides, giving new insight into how single α-helices are stabilized.

Beyond function: Engineering improved peptides for therapeutic applications

“…This effect is not limited to small peptides. Substitution of Lys with Arg in the single α‐helix of the naturally occurring protein myosin‐6 increased its stability, whereas the substitution of Arg with Lys decreased stability, indicating the results for the single‐helix peptides can also be observed in the context of more complicated proteins or peptides.…”

“…Chiti et al showed that salt bridges also contribute to the aggregation of leucine‐rich peptides and tau and mitigated the aggregation using a D24Q mutation in leucine‐rich peptides and an R5L mutation in tau to eliminate undesirable intermolecular salt bridges . Similarly, this was demonstrated in a de novo α‐helical peptide composed of (AEEEXXX) n repeats (X = K or R) . The peptide was designed for improved thermostability by careful pairing of either Lys or Arg with Glu to stabilize the helix; however, the increased prevalence of Arg residues produced undesirable salt bridges that instead promoted aggregation, likely into amorphous aggregates.…”

“…For example, the antimicrobial activity and immunogenicity of peptides can rely on their ability to form and retain their secondary structures, so engineering higher thermodynamic stability can be crucial for their in vivo efficacy. When engineering a peptide for thermodynamic stability, a key consideration is whether the peptide contains α‐helical or β‐sheet secondary structure, as knowledge of the intramolecular interactions in these structures assists in designing or improving peptides.…”

“…Forming an α‐helical structure can stabilize a peptide, and rational design can be used to improve the propensity to form α‐helices and the stability of the helices. Using rational design, α‐helicity can be engineered via intramolecular salt bridges using Glu, Asp, Arg, or Lys salt bridges to increase helicity .…”

“…Using rational design, α‐helicity can be engineered via intramolecular salt bridges using Glu, Asp, Arg, or Lys salt bridges to increase helicity . To better understand how salt‐bridges affect stability, Wolny et al designed de novo, single α‐helix peptides with the motif AEEEXXX (X = K or R) . Experimentally, they showed the sequence AEEEKRK is more helical and thermostable than AEEEKKK, suggesting that Arg increases stability compared to Lys.…”