THERMODYNAMIC CHARACTERIZATION OF AN ARTIFICIALLY DESIGNED AMPHIPHILIC ALPHA-HELICAL PEPTIDE-CONTAINING PERIODIC PROLINES - OBSERVATIONS OFHIGH THERMAL-STABILITY AND COLD DENATURATION

To investigate the structural stability of proteins, we analyzed the t hermodynamics of an artificially designed 30-residue peptide. The desi gned peptide, NH2-EELLPLAEALAPLLEALLPLAEALAPLLKK-COOH (PERI COIL-1), w ith prolines at i + 7 positions, forms a pentameric alpha-helical stru cture in aqueous solution. The thermal denaturation curves of the CD a t 222 nm (pH 7.5) show an unusual cold denaturation occurring well abo ve 0 degrees C and no thermal denaturation is observable under 90 degr ees 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 (H-5) was sufficient to analyze 5 thermal denaturation curves of PERI COIL-1 with concentrations betwe en 23 and 286 mu M. The analysis was carried out by a nonlinear least- squares method using 3 fitting parameters: the midpoint temperature, T -m, the enthalpy change, Delta H(T-m), and the heat capacity change, D elta C-p. The association number (n = 5) was determined by sedimentati on equilibrium and was not used as a fitting parameter. The heat capac ity change suggests that the hydrophobic residues are buried in the he lical state and exposed in the denatured one, as it occurs normally fo r natural globular proteins. On the other hand, the enthalpy and the e ntropy changes have values close to those found for coiled-coils and a re quite distinct from typical values reported for natural globular pr oteins. In particular, the enthalpy change extrapolated at 110 degrees C is about 3 kJ/mol per amino acid residue, i.e., half of the value f ound for globular proteins. Thus, the helices of PERI COIL-1, observed by GD, would be stabilized by entropic effect rather than enthalpic e ffect. This might be a general feature for de novo designed proteins t hat lack the rigid tertiary structure, and are mainly stabilized by no nspecific hydrophobic interactions, as well as for some molten globule s of natural proteins.