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Thermodynamics of the reconstitution of tuna cytochrome c from two peptide fragments
Author(s) -
Yokota Atsushi,
Takenaka Hidekazu,
Oh Tomohiro,
Noda Yasuo,
Segawa ShinIchi
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560070806
Subject(s) - chemistry , isothermal titration calorimetry , enthalpy , calorimetry , heat capacity , van der waals force , crystallography , standard enthalpy of formation , heme , hydrogen bond , circular dichroism , stereochemistry , thermodynamics , molecule , organic chemistry , enzyme , physics
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 spectrometry. It was found from CD and NMR spectra that the structure of the fragment complex formed is similar to that of an intact cyt c, although each isolated fragment itself is unstructured. Binding constants and enthalpies upon the complex formation were directly observed by isothermal titration calorimetry. Thermodynamic parameters (ΔG° b , Δ H b , Δ S° b , and Δ C b p ) associated with the complex formation were determined at various pHs and temperatures. Δ Hb was found to be almost independent of pH values. The change in heat capacity accompanying the complex formation (ΔC b p ) was directly determined from the temperature dependence of Δ H b . In addition, the change in heat capacity and enthalpy upon tuna cyt c unfolding were determined by differential scanning calorimetry. Thermodynamic parameters for the unfolding/ dissociation process of the fragment complex were compared with those for cyt c unfolding at pH 3.9 and 303 K. In a comparison of two unfolding processes, the heat capacity change 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 . These thermodynamic data suggest that the internal interactions between polar groups (hydrogen bonding) and nonpolar groups (van der Waals interactions) are preserved in the fragment complex as well as in the native state of cyt c .