Thermodynamic of the reconstitution of protein structure from peptide fragments.

金枪鱼细胞色素c的两个多肽片段(1-44)H[H表示该片段含有血红素]和(45-103)结合形成1：1片段复合体。离子喷雾质谱仪清楚地证明了这一点。圆二色谱和核磁共振谱表明，所形成的片段复合体的结构与完整的细胞色素c相似，尽管每个分离的片段本身都是非结构化的。用等温滴定量热法直接观察了络合物形成时的结合常数和结合热焓。由于(45-103)端的氨基与丙酸亚铁血红素之间的静电吸引作用，络合物形成时的热变略大于蛋白质折叠时的变化。当KCI的加入降低了静电作用力时，热焓的变化趋于一致。此外，还制备了较小的多肽片段，以考察蛋白质整个氨基酸序列中失去某些多肽片段的影响。制备的多肽片段为(1-21)H、(22-44)、(45-66)和(67-103)。量热和光谱实验表明，多肽片段(45-66)对于N-末端和C-末端螺旋之间形成能量稳定的结构是重要的。

Two peptide fragments from tuna cytochrome c, (1-44)H [H means that the fragment contains heme] and (45-103), combine to from a 1 : 1 fragment complex. This was clearly proved by ion-spray mass spectrometry. CD and NMR spectra showed that the structure of the fragment complex formed is similar to that of on intact cytochrome c, although each isolated fragment itself is unstructured. Binding constants and enthalpies upon the complex formation were directly observed by isothermal titration calorimetry. The change in enthalpy upon the complex formation was somewhat larger than that upon protein folding, because of the electrostatic attractive interactions between the amino group of N-terminus of (45-103) and heme propionates. When the electrostatic force was reduced by the addition of KCI, the enthalpy change become to coincide with each other.In addition, smaller peptide fragments were prepared to investigate the effect of the losing some peptide segment from the whole amino acid sequence of protein. Prepared peptide fragments were (1-21)H, (22-44), (45-66) and (67-103). Calorimetric and spectroscopic experiments showed that the peptide segment (45-66) is important for the formation of energetically stable structure between N- and C-terminal helices.