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

<jats:title>Abstract</jats:title><jats:p>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 <jats:italic>de novo</jats:italic> 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 <jats:italic>vice versa)</jats:italic> in the naturally-occurring myosin-6 SAH similarly increased (or decreased) its stability. However, Arg-rich <jats:italic>de novo</jats:italic> 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 <jats:italic>de novo</jats:italic> Arg-rich SAHs to aggregate. Importantly, the specific K:R ratio is likely to be important in determining helical stability in <jats:italic>de novo</jats:italic> and naturally-occurring polypeptides, giving new insight into how single α-helices are stabilized.</jats:p>