Water-Solubilized, Cap-Stabilized, Helical Polyalanines:  Calibration Standards for NMR and CD Analyses

NMR and CD studies are reported for two length series of solubilized, spaced, highly helical polyalanines that are N-capped by the optimal helix stabilizer <sup>β</sup>Asp-Hel and C-capped by β-aminoalanine <i>beta</i> and that are studied in water at 2 °C, pH 1−8. NMR analysis yields a structural characterization of the peptide Ac<sup>β</sup>AspHelAla<sub>8</sub><i>beta</i>NH<sub>2</sub> and selected members of one <sup>β</sup>AspHelAla<i><sub>n</sub></i><i>beta</i> series. At pH > 4.5 the <sup>β</sup>AspHel cap provides a preorganized triad of carboxylate anion and two amide residues that is complementary to the helical polyalanine N-terminus. The C-terminal β-aminoalanine assumes a helix-stabilizing conformation consistent with literature precedents. H(N)CO NMR experiments applied to capped, uniformly <sup>13</sup>C- and <sup>15</sup>N-labeled Ala<sub>8</sub> and Ala<sub>12</sub> peptides define Ala<i><sub>n</sub></i> hydrogen bonding signatures as α-helical without detectable 3<sub>10</sub> character. Relative NH→ND exchange rates yield site protection factors PF<i><sub>i</sub></i> that define uniquely high fractional helicities FH for the peptide Ala<i><sub>n</sub></i> regions. These Ala<i><sub>n</sub></i> calibration series, studied in water and lacking helix-stabilizing tertiary structure, yield the first <sup>13</sup>C NMR chemical shifts, <sup>3</sup><i>J</i><sub>HNH</sub><sub>α</sub> coupling constants, and CD ellipticities [θ<sub>Molar</sub>]<sub>λ</sub><sub>,</sub><i><sub>n</sub></i> characteristic of a fully helical alanine within an Ala<i><sub>n</sub></i> context. CD data are used to assign parameters <i>X</i> and [θ]<sub>λ</sub><sub>,</sub><sub>∞</sub>, required for rigorous calculation of FH values from CD ellipticities.