The work contained in this thesis is focused on the structural characterization through 2D-NMR and the thermodynamic analysis based on optical spectroscopic studies of a series of related β-hairpin peptides. Studies of small peptide systems provide valuable insight toward understanding β-sheet folding and stability. A 12-residue β-hairpin peptide model based on a design from Cochran, which was stabilized by four tryptophans forming a strong hydrophobic core and a common Asn-Gly β-turn, was studied here to understand the stability contributions from cross-strand hydrophobic interactions and turn constraints.
The primary optical spectroscopic methods utilized in this thesis are infrared (IR) absorption and electronic circular dichroism (ECD). FTIR was used to characterize the secondary structure of these peptides, because carbonyl coupling provides the fundamental interaction that leads to the spectral variations characteristic of different backbone structures. ECD was used to monitor the local tertiary structure, because the pairwise Trp-Trp interactions in Trpzip peptides result in a strong exciton CD signal that indicates a distinctive cross-strand contact, effectively a tertiary structure. Furthermore, NMR spectroscopy, particular TOCSY and NOESY 2D-NMR were used to study the geometry of interacting aromatic sidechain pairs and thus derive the whole peptide structure. By combining the optical spectroscopic measurements with NMR spectroscopy, the particular local structure can be correlated with analysis of the spectral variation.
First, three sets of Trp-Trp interactions in Trpzip hairpin were pairwise substituted by Val residues, on the outer cross-strand pair, the inner pair and the diagonal-pair. The solution-phase structures were determined based on NMR analysis, and the thermal unfolding transition of the Trpzip peptides monitored with FTIR and ECD were analyzed to yield Tm values and a full set of thermal parameters assuming a two-state transition model. Variation of the Trp residues to Val, maintaining hydrophobic but not aromatic interaction, has an impact on both the structure and the thermodynamics. Aromatic interaction is more effective in stabilizing β-hairpin formation than hydrophobic interaction, and an edge-to-face interact geometry for the aromatic sidechain is more effective than a face-to-face geometry. Also the position of interacting aromatic pairs also affects hairpin backbone formation. In other words, when the Trp-Trp interacting pair is located at the termini of two strands, the hairpin tends to form flatter β-sheets than when it’s located near the turn.
Since the Trp-Trp interacting pair is an important means of creating stable hairpin designs, this leads to further comparison of pairwise Trp-Trp interactions with other aromatic interactions, such as Trp-Tyr and Tyr-Tyr, and their relative impact on hairpin stability. Structures from NMR spectroscopy suggest aromatic interactions are excellent stabilizing agents of the secondary structure scaffold, and Tryptophan tends to contribute more to peptide stability than Tyrosine. The thermodynamic data indicates that specific Trp/Trp edge-to-face interactions could be reduced by effects of electronic coupling to other neighboring aromatic (Tyr) residues or by steric interferences. In other words, all adjacent aromatic pairs appear to interact together and result in a coupled unfolding mechanism.
And further study of the impact of turn stability on hairpins has also been carried out emphasizing a thermodynamic approach. I substituted the β-turn residues in the Trpzip2 hairpin peptide with five different β-turn sequences, Asn-Gly, Gly-Asn, Thr-Gly, Aib-Gly and DPro-Gly, which represent either flexible or constrained turn types. Among these, the most common tight β-turns in protein structures, NG turn, is very stable. Reversing the sequence of the NG turn into GN turn (Trpzip1) destabilized both cross-strand coupling and Trp-Trp interaction. The TG turn presents no conformational constraint, and offers no added stability to hairpin structure. And the DPG turn is the most different one; it stabilizes the turn locally but distorts the Trp-Trp interaction and reduces the exciton effect. DPG segment has a high loop-forming propensity and thus decreases the entropic cost of β-hairpin formation as compared to the more flexible NG segment. DPro-Gly turn is proposed to adopt a unique rigid local conformation to promote β-hairpin formation, but its rigidity does not allow the strands to achieve more energetically favorable contacts with one another.