We additionally performed molecular dynamics (MD) simulations to investigate salt-bridge formation and the dynamic behaviour of salt bridges formed by E–K and E–R parings in the SAHs. To interpret our experimental findings, we analysed side chain interactions in α-helices from the Protein Data Bank (PDB) 21 for E–R pairs and compared these to E–K pairs. Their length was specifically chosen to be similar to that of many natural SAHs 2 as a better test of the properties of a SAH compared to short peptides. These long (98-residue) polypeptides contain 7-residue repeats, AEEEXXX, where X is K or R, and were designed to emulate the properties of SAHs using simpler constructs. To determine how Lys and Arg contribute to the stability of long SAHs, we first designed, expressed and characterized de novo SAHs. Although it was recently reported that E–R pairs may be more stabilizing than E–K in very short (<12 residue) peptides 20, it is unclear which pairings are utilized, or why E–R pairs are more stabilizing. However, the equivalent study for E–R pairs is lacking. Studies on short peptides have additionally suggested that K → E(+4) pairs are more helix-stabilizing than the reversed E → K(+4) orientation 17, resolving previous conflicting reports 16, 18, 19. ![]() Single E–K or E–R pairs of this type are known to promote the folding of monomeric α-helices in short (<20 residue) alanine-based peptides 15, 16, and thus are assumed to stabilize long SAHs. However, understanding how such salt bridges promote the highly helical states of natural sequences is challenging, as SAHs have a range of different potential salt bridges in terms of sequence separation and residue type. “E → K(+3)” or “K → E(+3)” where the K residue is 3 residues downstream or upstream from E respectively) 2, 4. These pairings are spaced three or four residues apart ( e.g. SAHs are likely to form when a sequence is rich in acidic and basic residues (Glu, E Arg, R and Lys, K), lacks a hydrophobic seam, and contains many potential intrahelical interactions (salt bridges) between either E and K (E–K), or E and R (E–R). Thus it is important to understand the underlying stability of these intriguing domains, in order to understand their contribution to protein function. Most recently, it has been shown that in some cases, a region of the SAH can mediate binding to other proteins 13, 14. However, only a few of these have been characterized experimentally and in detail 4, 5, 6, 9, 10, 11, 12. Many putative SAHs have now been identified in a wide range of proteins with diverse functions, typically ranging in length between 40 and ~200 residues 1, 2, 7, 8, 9. They are stiff enough to replace the canonical lever in myosin 6, can behave as a “constant force spring” when extended by small (<30 pN) forces 5, and have a persistence length of ~15 nm (equivalent to ~100 residues of α-helix) 7. SAHs are not stabilized by a tertiary fold, remain monomeric and highly helical over a broad range of salt concentrations and pH, and exhibit a thermal denaturation profile that is only weakly cooperative 2, 5. Naturally occurring single α-helices (SAHs) are found in ~4% of proteins 1, 2, 3, and have been commonly misidentified as coiled coils 4. 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. This promiscuous nature of Arg helps explain the increased propensity of de novo Arg-rich SAHs to aggregate. 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. However, Arg-rich de novo sequences (ER3 (AEEERRR) and EK1R2 (AEEEKRR)) aggregated. Substituting Lys with Arg (or vice versa) in the naturally-occurring myosin-6 SAH similarly increased (or decreased) its stability. Lys-rich sequences (EK3 (AEEEKKK) and EK2R1 (AEEEKRK)) both form SAHs, of which EK2R1 is more helical and thermo-stable suggesting Arg increases stability. 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. Understanding how salt bridges promote their stability is challenging as SAHs are long and their sequences highly variable. Naturally-occurring single α-helices (SAHs), are rich in Arg (R), Glu (E) and Lys (K) residues, and stabilized by multiple salt bridges.
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