Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California, USA.
Biophys J. 2012 Jul 3;103(1):79-88. doi: 10.1016/j.bpj.2012.05.047.
The backbone dynamics of human α-thrombin inhibited at the active site serine were analyzed using R(1), R(2), and heteronuclear NOE experiments, variable temperature TROSY 2D [(1)H-(15)N] correlation spectra, and R(ex) measurements. The N-terminus of the heavy chain, which is formed upon zymogen activation and inserts into the protein core, is highly ordered, as is much of the double beta-barrel core. Some of the surface loops, by contrast, remain very dynamic with order parameters as low as 0.5 indicating significant motions on the ps-ns timescale. Regions of the protein that were thought to be dynamic in the zymogen and to become rigid upon activation, in particular the γ-loop, the 180s loop, and the Na(+) binding site have order parameters below 0.8. Significant R(ex) was observed in most of the γ-loop, in regions proximal to the light chain, and in the β-sheet core. Accelerated molecular dynamics simulations yielded a molecular ensemble consistent with measured residual dipolar couplings that revealed dynamic motions up to milliseconds. Several regions, including the light chain and two proximal loops, did not appear highly dynamic on the ps-ns timescale, but had significant motions on slower timescales.
采用 R(1)、R(2)和异核 NOE 实验、变温 TROSY 2D [(1)H-(15)N]相关谱和 R(ex)测量法，分析了人凝血酶活性部位丝氨酸抑制后的骨干动力学。重链的 N 端在酶原激活时形成并插入蛋白核心，高度有序，双β-桶核心的大部分也是如此。相比之下，一些表面环仍然非常动态，其顺序参数低至 0.5，表明在 ps-ns 时间尺度上存在显著的运动。在酶原中被认为是动态的并在激活时变得僵硬的蛋白质区域，特别是γ-环、180s 环和 Na(+)结合位点，其顺序参数低于 0.8。在大多数γ-环、靠近轻链的区域和β-片核心中都观察到了显著的 R(ex)。加速分子动力学模拟产生了一个与测量的残差偶极耦合一致的分子集合，该集合揭示了高达毫秒的动态运动。包括轻链和两个邻近环在内的几个区域在 ps-ns 时间尺度上似乎没有高度动态，但在较慢的时间尺度上有显著的运动。
