title: 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.
authors.affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Marcin
authors.initials: M
authors.lastname: Wolny
authors.affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Matthew
authors.initials: M
authors.lastname: Batchelor
authors.affiliation: School of Chemistry, University of Bristol, Cantock's Close, Bristol, BS8 1TS, UK.
authors.forename: Gail J
authors.initials: GJ
authors.lastname: Bartlett
authors.affiliation: School of Chemistry, University of Bristol, Cantock's Close, Bristol, BS8 1TS, UK.
authors.forename: Emily G
authors.initials: EG
authors.lastname: Baker
authors.affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Marta
authors.initials: M
authors.lastname: Kurzawa
authors.affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Knight
authors.affiliation: Astbury Centre for Structural Molecular Biology and School of Physics and Astronomy, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Lorna
authors.initials: L
authors.lastname: Dougan
authors.affiliation: School of Chemistry, University of Bristol, Cantock's Close, Bristol, BS8 1TS, UK.|School of Biochemistry, University of Bristol, Biomedical Sciences Building, Bristol, BS8 1TD, UK.|BrisSynBio, University of Bristol, Life Sciences Building, Bristol, BS8 1TQ, UK.
authors.forename: Derek N
authors.initials: DN
authors.lastname: Woolfson
authors.affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Emanuele
authors.initials: E
authors.lastname: Paci
authors.affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Michelle
authors.initials: M
authors.lastname: Peckham
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry*; D006860:Hydrogen Bonding; D008239:Lysine / Q000737:chemistry*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D010455:Peptides / Q000737:chemistry*; D000072756:Protein Conformation, alpha-Helical; D017510:Protein Folding; D055550:Protein Stability; D013816:Thermodynamics
chemical_list: D010455:Peptides; D018698:Glutamic Acid; D001120:Arginine; D008239:Lysine