Folding mechanism of a protein from a hyperthermophile with unusually slow folding rates

来自超嗜热生物的蛋白质的折叠机制，其折叠速率异常缓慢

• 批准号: 17570102
• 负责人: YUTANI Katsuhide
• 金额: $ 2.18万
• 依托单位: RIKEN
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17570102/
• 关键词: Protein Folding; Denatured Structure of a Protein; Hyperthermophile; NMR; Protein Stability; α-Helix; Pyrrolidone Carboxyl Peptidase; Mutant Protein; 蛋白質の変性; CD; 蛋白質の変異型

We have hound that the refolding reaction of pyrrolidone carboxyl peptidase (PCP) from a hyperthermophile, Pyrococcus furiosus, is unusually slow at acidic pH and can be controlled by regulating the incubation temperature ; the refolding reaction significantly stops at pH 2.3 and 4 ℃. In this period, in order to elucidate the folding mechanism of a protein from a hyperthermophile with unusually slow folding rates, we have completed two papers using cysteine-free PCP (PCP-OSH) with unusually slow refolding rates. PCP-OSH was used in place of PCP to avoid troubles due to the formation of disulfide bonds. (1) PCPs from hyperthermophiles have a structurally conserved and completely buried Glu192 in the hydrophobic core ; in contrast, the corresponding residue in mesophile protein is a hydrophobic residue, He. To elucidate the role of the buried Glu in stability and folding rates of PCP from hyperthermophiles, we examined changes in stability and structure due to mutations at Glu192. The results indicated that completely buried Glu192 contributes to stabilization of PCP-OSH due to the formation of strong intramolecular hydrogen bonds, and the hydrogen bonds by the non-ionized and buried Glu can contribute more than the burial of hydrophobic groups to the conformational stability of proteins (Kausahik et al., 2006). (2) The above denatured state (D_1 state) of PCP corresponds to the denatured structure that exists in equilibrium with the native state under physiological conditions. To elucidate the structural basis of the D_1 state, H/D exchange experiments with PCP-OSH were performed at pD 3.4 and 4 ℃. The results indicated that amide protons in the C-terminal a6-helix region hardly exchanged in the D_1 state with deuterium even after 7 days, suggesting that the a6-helix (from Ser188 to Glu205) of PCP-OSH was stably formed in the D_1 state (Iimura et al., 2007).

我们发现，一株嗜热菌的吡咯烷酮羧基肽酶的复性反应在酸性pH下异常缓慢，可通过调节孵育温度来控制，在pH2.3和4℃时，复性反应显著停止。在此期间，为了阐明来自极端嗜热者的蛋白质折叠速度异常缓慢的折叠机制，我们使用不含半胱氨酸的PCP(PCP-OSH)完成了两篇论文，折叠速度异常缓慢。用PCP-OSH代替PCP，避免了二硫键的形成带来的麻烦。(1)来自高温菌的PCP在疏水核心有一个结构保守且完全埋藏的Glu192，而在中温蛋白中对应的残基是一个疏水残基He。为了阐明埋藏的Glu在超嗜热菌PCP的稳定性和折叠率中的作用，我们检测了Glu192位突变引起的稳定性和结构的变化。结果表明，完全掩埋的Glu192通过形成较强的分子内氢键有助于PCP-OSH的稳定，而非电离和掩埋的Glu的氢键对蛋白质构象稳定性的贡献大于疏水基团的掩埋(Kausahik等人，2006)。(2)PCP的上述变性状态(D_1状态)对应于生理条件下与天然状态平衡存在的变性结构。为了阐明D_1态的结构基础，分别在PD3.4和4℃进行了与PCP-OSH的H/D交换实验。结果表明，即使在7天后，PCP-OSH的C-末端a6-螺旋区的酰胺质子也几乎不能在D_1状态与氘交换，这表明PCP-OSH的a6-螺旋(从Ser188到Glu205)在D_1状态下稳定形成(Iimura等人，2007)。

项目成果

Hyper-thermostability of CutAl protein, with a denaturation temperature of nearly 150℃

CutAl蛋白具有超热稳定性，变性温度接近150℃

• 发表时间: 2006
• 期刊: FEBS Letters, 580
• 作者: Tanaka;T. et al.
• 通讯作者: T. et al.

Hyper-thermostability of CutA1 protein, with a denaturation temperature of nearly 150℃

CutA1蛋白具有超热稳定性，变性温度接近150℃

• 发表时间: 2006
• 期刊: FEBS Letters 580
• 作者: Tanaka;T. et al.
• 通讯作者: T. et al.

Characterization of the Denatured Structure of Pyrrolidone Carboxyl Peptidase from a Hyperthermophile under Non-denaturing Conditions : Role of the C-terminal a-helix of the Protein in Folding and Stability.

非变性条件下超嗜热菌吡咯烷酮羧基肽酶变性结构的表征：蛋白质 C 端 α 螺旋在折叠和稳定性中的作用。

• 发表时间: 2007
• 期刊: Biochemistry 46(In press)
• 作者: Iimura;S. et al.
• 通讯作者: S. et al.

蛋白質立体構造から安定化のメカニズムを定量的に理解する方法

一种从蛋白质3D结构定量理解稳定机制的方法

• 发表时间: 2005
• 期刊: 日本結晶学会誌 47
• 作者: 舩橋順;油谷克英
• 通讯作者: 油谷克英

生物工学ハンドブック(日本生物工学会編)(コロナ社)

生物工学手册（日本生物工学学会编）（Corona出版社）

• 发表时间: 2005
• 作者: Soo Jae Lee;Kyoko Ogasahara;Jichun Ma;Kazuya Nishio;Masami Ishida;Yuriko Yamagata;Tomitake Tsukihara;Katsuhide Yutani;黒木良太など
• 通讯作者: 黒木良太など