Nishiguchi Shingo, Goto Yuji, Takahashi Satoshi
Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.
J Mol Biol. 2007 Oct 19;373(2):491-502. doi: 10.1016/j.jmb.2007.08.003. Epub 2007 Aug 19.
Solvation and desolvation dynamics around helices during the kinetic folding process of apomyoglobin (apoMb) were investigated by using time-resolved infrared (IR) spectroscopy based on continuous-flow rapid mixing devices and an IR microscope. The folding of apoMb can be described by the collapse and search mechanism, in which the initial collapse occurring within several hundreds of microseconds is followed by the search for the correct secondary and tertiary structures. The time-resolved IR measurements showed a significant increase in solvated helix possessing a component of amide I' at 1633 cm(-1) within 100 mus after initiating the folding by a pD jump from pD2.2 to 6.0. In contrast, there was a minor increase in buried helices having amide I' at 1652 cm(-1) in this time domain. The observations demonstrate that the initially collapsed conformation of apoMb possesses a large amount of solvated helices, and suggest that much water is retained inside the collapsed domain. The contents of solvated and buried helices decrease and increase, respectively, in the time domain after the collapse, showing that the stepwise desolvation around helices is associated with the conformational search process. Interestingly, the largest changes in solvated and buried helices were observed at the final rate-limiting step of the apoMb folding. The persistence of the solvated helix until the final stage of apoMb folding suggests that the dissociation of hydrogen bonds between water and main-chain amides contributes to the energy barrier in the rate-determining step of the folding.
利用基于连续流动快速混合装置和红外显微镜的时间分辨红外（IR）光谱，研究了脱辅基肌红蛋白（apoMb）动力学折叠过程中螺旋周围的溶剂化和去溶剂化动力学。apoMb的折叠可以用塌缩和搜索机制来描述，即在几百微秒内发生初始塌缩，随后搜索正确的二级和三级结构。时间分辨红外测量结果表明，通过从pD2.2到6.0的pD跃迁引发折叠后，在100微秒内，具有1633 cm-1处酰胺I'成分的溶剂化螺旋显著增加。相比之下，在该时域内，具有1652 cm-1处酰胺I'的埋藏螺旋略有增加。这些观察结果表明，apoMb最初的塌缩构象具有大量的溶剂化螺旋，并表明在塌缩区域内保留了大量的水。在塌缩后的时域内，溶剂化螺旋和埋藏螺旋的含量分别减少和增加，这表明螺旋周围的逐步去溶剂化与构象搜索过程有关。有趣的是，在apoMb折叠的最终限速步骤中观察到溶剂化螺旋和埋藏螺旋的最大变化。溶剂化螺旋一直持续到apoMb折叠的最后阶段，这表明水与主链酰胺之间氢键的解离在折叠的限速步骤中对能量屏障有贡献。
