Thermodynamic characterization of an artificially designed amphiphilic α‐helical peptide containing periodic prolines: Observations of high thermal stability and cold denaturation

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Abstract
To investigate the structural stability of proteins, we analyzed the thermodynamics of an artificially designed 30-residue peptide. The designed peptide, NH2-EELLPLAEALAPLLEALLPLAEALAPLLKK-COOH (PERI COIL-l), with prolines at i + 7 positions, forms a pentameric α-helical structure in aqueous solution. The thermal denaturation curves of the CD at 222 nm (pH 7.5) show an unusual cold denaturation occurring well above 0 °C and no thermal denaturation is observable under 90 °C. This conformational change is reversible and depends on peptide concentration. A 2-state model between the monomeric denatured state (5D) and the pentameric helical state (H5) was sufficient to analyze 5 thermal denaturation curves of PERI COIL-1 with concentrations between 23 and 286 μM. The analysis was carried out by a nonlinear least-squares method using 3 fitting parameters: the midpoint temperature, Tm, the enthalpy change, ΔH(Tm), and the heat capacity change, Δ Cp. The association number (n= 5) was determined by sedimentation equilibrium and was not used as a fitting parameter. The heat capacity change suggests that the hydrophobic residues are buried in the helical state and exposed in the denatured one, as it occurs normally for natural globular proteins. On the other hand, the enthalpy and the entropy changes have values close to those found for coiled-coils and are quite distinct from typical values reported for natural globular proteins. In particular, the enthalpy change extrapolated at 110 °C is about 3 kJ/mol per amino acid residue, i.e., half of the value found for globular proteins. Thus, the helices of PERI COIL-1, observed by CD, would be stabilized by entropic effect rather than enthalpic effect. This might be a general feature for de novo designed proteins that lack the rigid tertiary structure, and are mainly stabilized by nonspecific hydrophobic interactions, as well as for some molten globules of natural proteins.