Thermodynamics of the reconstitution of tuna cytochrome c from two peptidefragments

Two peptide fragments from tuna cytochrome c (cyt c), N-fragment (residues 1-44 containing the heme) and C-fragment (residues 45-103), combine to form a 1:1 fragment complex. This was clearly proved by ion-spray mass spectrom etry. It was found from CD and NMR spectra that the structure of the fragme nt complex formed is similar to that of an intact cyt c, although each isol ated fragment itself is unstructured. Binding constants and enthalpies upon the complex formation were directly observed by isothermal titration calor imetry. Thermodynamic parameters (Delta G degrees(b), Delta H-b, Delta S de grees(b). and Delta C-p(b)) associated with the complex formation were dete rmined at various pHs and temperatures. Delta H-b was found to be almost in dependent of pH values. The change in heat capacity accompanying the comple x formation (Delta C-p(b)) was directly determined from the temperature dep endence of Delta H-b. In addition, the change in heat capacity and enthalpy upon tuna cyt c unfolding were determined by differential scanning calorim etry. Thermodynamic parameters for the unfolding/dissociation process of th e fragment complex were compared with those for cyt c unfolding at pH 3.9 a nd 303 K. In a comparison of two unfolding processes, the heat capacity cha nge of each was very close to the other, while both the unfolding enthalpy and entropy of the fragment complex were larger than those of tuna cyt c. T hese thermodynamic data suggest that the internal interactions between pola r groups (hydrogen bonding) and nonpolar groups (van der Waals interactions ) are preserved in the fragment complex as well as in the native state of c yt c.