The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel.
J Phys Chem B. 2021 Jan 28;125(3):805-816. doi: 10.1021/acs.jpcb.0c10124. Epub 2021 Jan 15.
We report on amide (N-H) NMR relaxation from the protein S100A1 analyzed with the slowly relaxing local structure (SRLS) approach. S100A1 comprises two calcium-binding "EF-hands" (helix-loop-helix motifs) connected by a linker. The dynamic structure of this protein, in both calcium-free and calcium-bound form, is described as the restricted local N-H motion coupled to isotropic protein tumbling. The restrictions are given by a rhombic potential, (∼10 ), the local motion by a diffusion tensor with rate constant (∼10 s), and principal axis tilted from the N-H bond at angle β (10-20°). This parameter combination provides a physically insightful picture of the dynamic structure of S100A1 from the N-H bond perspective. Calcium binding primarily affects the C-terminal EF-hand, among others slowing down the motion of helices III and IV approximately 10-fold. Overall, it brings about significant changes in the shape of the local potential, , and the orientation of the local diffusion axis, β. Conformational entropy derived from makes an unfavorable entropic contribution to the free energy of calcium binding estimated at 8.6 ± 0.5 kJ/mol. The N-terminal EF-hand undergoes moderate changes. These findings provide new insights into the calcium-binding process. The same data were analyzed previously with the extended model-free (EMF) method, which is a simple limit of SRLS. In that interpretation, the protein tumbles anisotropically. Locally, calcium binding increases ordering in the loops of S100A1 and conformational exchange () in the helices of its N-terminal EF-hand. These are very unusual features. We show that they most likely stem from problematic data-fitting, oversimplifications inherent in EMF, and experimental imperfections. is shown to be mainly a fit parameter. By reanalyzing the experimental data with SRLS, which is largely free of these deficiencies, we obtain-as delineated above-physically-relevant structural, kinetic, geometric, and binding information.
我们报告了通过缓慢弛豫局部结构（SRLS）方法分析的蛋白质 S100A1 的酰胺（N-H）NMR 弛豫。S100A1 由两个钙结合“EF 手”（螺旋-环-螺旋基序）通过连接体连接而成。该蛋白质的动态结构，无论是在无钙还是钙结合形式下，都被描述为限制局部 N-H 运动与各向同性蛋白质翻滚的耦合。限制由菱形势，（10），局部运动由扩散张量给出，速率常数为（10 s），主轴相对于 N-H 键倾斜β角（10-20°）。这种参数组合从 N-H 键的角度提供了 S100A1 动态结构的物理洞察力。钙结合主要影响 C 端 EF 手，除其他外，使螺旋 III 和 IV 的运动大约减慢 10 倍。总体而言，它导致局部势，β和局部扩散轴的取向发生重大变化。从得出的构象熵对钙结合自由能做出不利的熵贡献，估计为 8.6±0.5 kJ/mol。N 端 EF 手发生适度变化。这些发现为钙结合过程提供了新的见解。相同的数据以前也使用扩展无模型（EMF）方法进行了分析，这是 SRLS 的简单极限。在该解释中，蛋白质各向异性地翻滚。局部地，钙结合增加了 S100A1 环中的有序性和其 N 端 EF 手螺旋中的构象交换（）。这些都是非常不寻常的特征。我们表明，它们很可能源自有问题的数据拟合、EMF 固有的过度简化以及实验缺陷。被证明主要是一个拟合参数。通过 SRLS 重新分析实验数据，该方法在很大程度上没有这些缺陷，我们获得了如上所述的物理相关的结构、动力学、几何和结合信息。
