Fukagawa Akihiro, Hiroshima Michio, Sakane Isao, Tokunaga Makio
Structural Biology Center, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan; Research Center for Allergy and Immunology, RIKEN, Yokohama, Kanagawa 230-0045, Japan.
Structural Biology Center, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan.
Biophysics (Nagoya-shi). 2009 Sep 4;5:25-35. doi: 10.2142/biophysics.5.25. eCollection 2009.
Experimental probing of a protein-folding energy landscape can be challenging, and energy landscapes comprising multiple intermediates have not yet been defined. Here, we quasi-statically unfolded single molecules of staphylococcal nuclease by constant-rate mechanical stretching with a feedback positioning system. Multiple discrete transition states were detected as force peaks, and only some of the multiple transition states emerged stochastically in each trial. This finding was confirmed by molecular dynamics simulations, and agreed with another result of the simulations which showed that individual trajectories took highly heterogeneous pathways. The presence of Ca did not change the location of the transition states, but changed the frequency of the emergence. Transition states emerged more frequently in stabilized domains. The simulations also confirmed this feature, and showed that the stabilized domains had rugged energy surfaces. The mean energy required per residue to disrupt secondary structures was a few times the thermal energy (1-3 ), which agreed with the stochastic feature. Thus, single-molecule quasi-static measurement has achieved notable success in detecting stochastic features of a huge number of possible conformations of a protein.
对蛋白质折叠能量景观进行实验探测可能具有挑战性，并且尚未定义包含多个中间体的能量景观。在这里，我们使用反馈定位系统通过恒速机械拉伸准静态展开金黄色葡萄球菌核酸酶的单分子。多个离散的过渡态被检测为力峰，并且在每次试验中只有一些多个过渡态随机出现。这一发现通过分子动力学模拟得到证实，并且与模拟的另一个结果一致，该结果表明各个轨迹采取高度异质的途径。Ca的存在没有改变过渡态的位置，但改变了出现的频率。过渡态在稳定结构域中出现得更频繁。模拟也证实了这一特征，并表明稳定结构域具有崎岖的能量表面。破坏二级结构每个残基所需的平均能量是热能（1-3 ）的几倍，这与随机特征一致。因此，单分子准静态测量在检测蛋白质大量可能构象的随机特征方面取得了显著成功。
