Analysis on the early events of protein folding at subzero temperatures

零下温度下蛋白质折叠的早期事件分析

• 批准号: 12680663
• 负责人: KIHARA Hiroshi
• 金额: $ 2.24万
• 依托单位: Kansai Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680663/
• 关键词: Protein folding; Compaction of protein; Formation rate of alpha-helix; Subzero temperature; Stopped-flow; x-ray solution scattering; フォールディング; ビータラクトグロブリン; アポミオグロビン; ユビキティン; アルフアービータ変換; 円偏光二色性

Protein takes (a) definite pathway(s) when it folds into its native form. It is so-called folding problem. One of the most important subject in the folding process is when and how the three major conformational formation takes place ; i.e. secondary structure formation, compaction and formation of the folding core.Protein folding starts at the time range of sub-micro-seconds. We reduced this rate to millisecond region, by decreasing temperature so as to slow down the folding process. With the use of stopped-flow technique combined with circular dichroism, fluorescence and X-ray scattering, we could monitor formation of alpha-helix, folding core formation and compaction. Results so far obtained are ; (1) formation rate of alpha-helix is too fast to be monitored even at -38C. (2) In case of bovine beta-lactogloblulin, we monitored a phase of alpha-helix increase at -28C. (3) In case of ubiquitin, the initial burs phase of alpha-helical formation was observed at -20C.From the findings above, it would be suggested that formation of alpha-helix is commonly observed at the early stage of the folding even in the case of beta-rich proteins.Results above are also very consistent with findings observed by temperature-jump method, and thus demonstrate the importance of our methodology ; cooling down the folding process.

蛋白质折叠成其天然形式时有一定的途径。这就是所谓的折叠问题。蛋白质折叠过程中最重要的问题之一是蛋白质折叠过程中的三个主要构象形成过程，即二级结构形成、压缩和折叠核心的形成。我们通过降低温度来减缓折叠过程，从而将该速率降低到毫秒区域。利用停流技术结合圆二色谱、荧光和X射线散射，我们可以监测α-螺旋的形成、折叠核心的形成和压实。结果表明：（1）α-螺旋的形成速度太快，即使在-38 ℃下也无法监测。(2)在牛β-乳球蛋白的情况下，我们在-28 ℃监测到α-螺旋增加的阶段。(3)在泛素的情况下，α-螺旋形成的初始布尔斯相在-20 ℃下观察到，从以上结果可以看出，即使在富含β的蛋白质中，α-螺旋的形成也通常在折叠的早期阶段观察到，以上结果也与温度跳跃法观察到的结果非常一致，从而证明了我们方法的重要性;冷却折叠过程。

项目成果

Zhi-jie Qin, Dong-mei Hu, Lui Shimada, Tatsuo Nakagawa, Munehito Arai, Jun-Mei Zhou and Hiroshi Kihara.: "Refolding of β-Lactoglobulin Studied by Stopped-Flow Circular Dichroism at Subzero Temperatures"FEBS lett.. 507. 299-302 (2001)

秦志杰、胡冬梅、Lui Shimada、Tatsuo Nakakawa、Munehito Arai、Jun-Mei Zhou 和 Hiroshi Kihara.：“在零下温度下通过停流圆二色性研究 β-乳球蛋白的重折叠”FEBS lett.. 507 .299-302 (2001)

Qin et al.: "Refolding of β-lactoglobulin studied by stopped-flow ciecular dichroism at subzero temperatures"FEBS Lett.. 507. 299-302 (2001)

秦等人：“在零度以下温度下通过停流环二色性研究β-乳球蛋白的重折叠”FEBS Lett.. 507. 299-302 (2001)

Oin et al.: "Refolding of β-lactoglobulin studied by stopped-flow ciecular dichroism at subzero temperatures"FEBS Lett.. 507. 299-302 (2001)

Oin 等人：“在零下温度下通过停流环二色性研究 β-乳球蛋白的重折叠” FEBS Lett.. 507. 299-302 (2001)

A.A.Timchenko et al.: "GroES co-chaperonin small-angle X-ray scattering study shows ring orifice increase in solution"FEBS Lett.. 471. 211-214 (2000)

A.A.Timchenko 等人：“GroES 辅助伴侣小角 X 射线散射研究显示溶液中环形孔口增加”FEBS Lett.. 471. 211-214 (2000)