Helical stability of de novo designed alpha-aminoisobutyric acid-rich peptides at high temperatures.

1D and 2D NMR spectroscopy is used to determine the helical stability of two Aib-rich peptides, iBoc-(Aib)3-DkNap-Leu-Aib-Ala-(Aib)2-NH(CH2)2OCH3 (Dk4[7/9]) and Ac-(Aib)2-beta-(1'-naphthyl)Ala-(Aib)2-Phe-(Aib)2-NHMe (Nap3Phe6[6/8]), where the bracket notation indicates the number of Aib-class residues/total number of residues. 2D ROESY experiments, carried out previously on Nap3Phe6[6/8] in DMSO (Basu & Kuki, 1993), showed that this compound adopts the 3(10)-helical conformation at 20 degrees C. The first step in the present work is to apply this technique to the peptide Dk4[7/9], demonstrating that it likewise adopts the 3(10)-helical conformation in chloroform at 20 degrees C. The amide proton shifts of Nap3-Phe6[6/8] in DMSO and Dk4[7/9] in C2D2Cl4 were then monitored by means of 1D NMR over a large temperature range, up to 150 and 120 degrees C, respectively. The nonamer Dk4[7/9] exhibits no evidence of any conformational or unfolding transition as the temperature is raised. The nearly temperature independent amide proton chemical shifts of this nonamer are an indication of retention of the intrahelical hydrogen bonding, which was then verified directly by solvent perturbation with DMSO at 120 degrees C. The resulting hydrogen-bonding pattern confirms that Dk4[7/9] retains its 3(10)-helical conformation in C2D2Cl4 over the entire temperature range. This conformational quietness is exploited to examine the intrinsic temperature dependence of free versus intrahelically hydrogen bonded amide proton shifts within the same peptide structure. It is also shown that Nap3Phe6[6/8] retains its 3(10)-helical conformation over the entire temperature range in the stronger hydrogen-bonding solvent DMSO. The extreme thermal stability of these octameric and nonameric Aib-rich peptides in both solvents is contrasted with that of much longer alanine-rich peptides in water.