Thermodynamic of the reconstitution of protein structure from peptide fragments.

从肽片段重建蛋白质结构的热力学。

• 批准号: 09680660
• 负责人: SEGAWA Shin-ichi
• 金额: $ 2.11万
• 依托单位: Kwansei Gakuin University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680660/
• 关键词: Cytochrome c; Peptide fragment-complex; Thermodynamics; Protein folding; Calorimetry; チトクロムC; ペプチド断片複合体

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.

金枪鱼细胞色素c的两个肽片段（1-44）H [H表示片段含有血红素]和（45-103）结合形成1:1的片段复合物。离子喷雾质谱法清楚地证明了这一点。CD和NMR表明，形成的片段复合物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焓。复合物形成时的焓变略大于蛋白质折叠时的焓变，这是由于（45-103）的n端氨基与丙酸血红素之间的静电吸引相互作用。当静电力因KCI的加入而减小时，焓变趋于一致。此外，制备了更小的肽片段，以研究从蛋白质的整个氨基酸序列中丢失一些肽段的影响。制备的肽片段分别为（1-21）H、（22-44）、（45-66）和（67-103）。量热和光谱实验表明，肽段（45-66）对N端和c端螺旋之间能量稳定结构的形成至关重要。

项目成果

Noda Yasuo: "A Two-Dimensional NMR Study of Exchange Behavior of Amide Hydrogens in a Lysozyme Denvative with an Extra Cross-Link Between Glu35 and Trp108" Biopolymers. 41. 131-143 (1997)

Noda Yasuo：“溶菌酶衍生物中酰胺氢交换行为的二维核磁共振研究，在 Glu35 和 Trp108 之间具有额外的交联”生物聚合物。

Yokota Atsushi: "The transition state in the folding-unfolding reaction of four Species of three-disulfide variant of hen lysozyme"J. Mol. Biol.. 295. 1275-1288 (2000)

横田敦：“四种母鸡溶菌酶三二硫键变体折叠-展开反应中的过渡态”J.

Kubo Shigeru: "Improvement in the oxidative folding of endothelin-1 by a Lys-Arg extension at the amino terminus" Letters in Peptide Science. 4. 185-192 (1997)

Kubo Shigeru：“通过氨基末端的赖氨酸-精氨酸延伸改善内皮素-1 的氧化折叠”肽科学快报。

Noda, Y. , Fukuda, Y. & Segawa, S.: ""A Two-Dimensional NMR Study of Exchange Behavior of Amide Hydrogens in a Lysozyme Derivative with an Extra Cross-Link Between Glu35 and Trp 108""Biopolymers. 41. 131-143 (1997)

野田 Y.、福田 Y.

Yokota, A., Takenaka, H., Oh, T., Noda, Y. & Segawa, S.: ""Thermodynamics of the reconstitution of tuna cytochrome c from two peptide fragments""Prptein Science. 7. 1717-1727 (1998)

横田，A.，竹中，H.，哦，T.，野田，Y.