Calorimetric Evidence for a Two-State Unfolding of the β-Hairpin Peptide Trpzip4

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Abstract
β-Sheets are a common secondary structural element found in proteins. The difficulty in studying β-sheet folding and stability is that their formation is often dependent on the tertiary structural environment within the protein. However, the discovery of water-soluble β-hairpin peptides has allowed them to be used as model systems because they represent the smallest units of β-sheet structure independent of tertiary structural context. Trpzip4 has been used as a model β-hairpin peptide to study β-hairpin folding and stability because it is highly soluble in aqueous solutions, maintains its monomeric state, and shows reversible cooperative thermal unfolding. The previously determined thermodynamic parameters for trpzip4 thermal unfolding vary depending on the spectroscopic probe used, which questions the assumption that trpzip4 unfolds in a two-state manner. Here we provide direct calorimetric evidence that the unfolding of trpzip4 follows a two-state unfolding mode. Furthermore, the thermal unfolding of trpzip4 monitored using near- and far-UV−CD yielded thermodynamic parameters similar to those determined calorimetrically, providing additional evidence for a two-state unfolding mode.