NMR and CD analysis of an intermediate state in the thermal unfolding process of mouse lipocalin-type prostaglandin D synthase
NMR and CD analysis of an intermediate state in the thermal unfolding process of mouse lipocalin-type prostaglandin D synthase
复制标题
小鼠脂质运载蛋白型前列腺素 D 合酶热解折叠过程中间态的 NMR 和 CD 分析
DOI：
10.1093/jb/mvr140
复制
发表时间：
2012
期刊：
影响因子：
--
通讯作者：
T.
中科院分区：
文献类型：
--
作者：
Miyamoto;Y.;Noda;Y.;Iida;T.;Yamaguchi;K.;Nishimura;S.;Tanaka;A.;Segawa;S.;Inui;T.
我们以前报道过小鼠脂质运载蛋白型前列腺素D合酶（L-PGDS）在酸性条件下的热去折叠是一个完全可逆的过程，遵循三态途径，包括天然状态（N）和去折叠状态之间的中间状态（I）。在本研究中，我们研究了小鼠C65 A L-PGDS的中间状态，并澄清了局部构象变化的上部和底部区域，通过使用NMR和CD光谱。1H-15 N HSQC测量表明，在45°C时，β-桶上部区域的骨架构象被破坏，这与N ParticipI跃迁的Tm值接近，但位于L-PGDS底部区域的残基的信号在54°C时保持不变，其中发现了中间态的最大积累。1H-NMR和CD测量表明，通过监测β-桶上部区域的Trp 54和底部区域的Trp 43获得的Tm值分别为41.4和47.5°C，表明上部区域的构象变化发生在比底部区域低的温度下。这些发现表明，在中间状态下，底部区域的骨架构象仍然保持。
We previously reported that the thermal unfolding of mouse lipocalin-type prostaglandin D synthase (L-PGDS) is a completely reversible process under acidic conditions and follows a three-state pathway, including an intermediate state (I) between native state (N) and unfolded state. In the present study, we investigated the intermediate state of mouse C65A L-PGDS and clarified the local conformational changes in the upper and bottom regions by using NMR and CD spectroscopy. The1H-15N HSQC measurements revealed that the backbone conformation was disrupted in the upper region of the β-barrel at 45°C, which is around theTmvalue for the N ↔ I transition, but that the signals of the residues located at the bottom region of L-PGDS remained at 54°C, where the maximum accumulation of the intermediate state was found.1H-NMR and CD measurements showed that theTmvalues obtained by monitoring Trp54 at the upper region and Trp43 at the bottom region of the β-barrel were 41.4 and 47.5°C, respectively, suggesting that the conformational change in the upper region occurred at a lower temperature than that in the bottom region. These findings demonstrate that the backbone conformation of the bottom region is still maintained in the intermediate state.