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Understanding the interactions that govern turn formationin theunfolded state of proteins is necessary for a complete picture ofthe role that these turns play in both normal protein folding andfunctionally relevant yet disordered linear motifs. It is still unclear,however, whether short peptides can adopt stable turn structures inaqueous environments in the absence of any nonlocal interactions.To explore the effect that nearest-neighbor interactions and the localpeptide environment have on the turn-forming capability of individualamino acid residues in short peptides, we combined vibrational (IR,Raman, and VCD), UV-CD, and 1H NMR spectroscopies in orderto probe the conformational ensemble of the central aspartic acidresidue of the triaspartate peptide (DDD). The study was motivatedby the recently discovered turn propensities of aspartic acid in GDG(Hagarman; et al. Chem.Eur. J.2011, 17, 6789). We investigated the DDD peptideunder both acidic and neutral conditions in order to elucidate theeffect that side-chain protonation has on the conformational propensityof the central aspartic acid residue. Amide I′ profiles wereanalyzed in terms of two-dimensional Gaussian distributions representingconformational subdistributions in Ramachandran space. Interestingly,our results show that while the protonated form of the DDD peptidesamples various turn-like conformations similar to GDG, deprotonationof the peptide eliminates this propensity for turns, causing the fullyionized peptide to exclusively sample pPII and β-strand-likestructures. To further explore the factors stabilizing these moreextended conformations in fully ionized DDD, we analyzed the temperaturedependence of both the UV-CD spectrum and the 3J(HN,Hα) coupling constantsof the two amide protons (N- and C-terminal) in terms of a simpletwo-state (pPII−β) thermodynamic model. Thus, we wereable to obtain the enthalpic and entropic differences between thepPII and β-strand conformations of the central and C-terminalresidue. For the central residue, we obtained ΔH3= −12.0 kJ/mol and ΔS3= −73.8 J/mol·K, resultingin a much larger room-temperature Gibbs free energy of 10.0 kJ/mol,which effectively locks the C-terminal in a β-like conformation.A comparison of the temperature dependence of the chemical shiftsreveals that there is indeed some type of protection of the amideprotons from solvent in ionized DDD. This finding and several otherlines of evidence suggest that both conformations of ionized DDD arestabilized by hydrogen bonding between the carboxylate groups of thecentral and C-terminal residue and the respective amide protons. Thesehydrogen bonds can be expected to be eliminated by side-chain protonationand substituted by hydrogen bonds between the N-terminal amide protonand the C-terminal carbonyl group as well as between the central aspartateside chain and the N-terminal amide proton. Hence, our results areindicative of a pH-induced switch in hydrogen-bonding patterns ofaspartic acid motifs. [ABSTRACT FROM AUTHOR]