Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC, 27695-8204, USA.
J Biomol NMR. 2019 May;73(5):229-244. doi: 10.1007/s10858-019-00251-7. Epub 2019 May 10.
Multidimensional solid-state NMR spectra of oriented membrane proteins can be used to infer the backbone torsion angles and hence the overall protein fold by measuring dipolar couplings and chemical shift anisotropies, which depend on the orientation of each peptide plane with respect to the external magnetic field. However, multiple peptide plane orientations can be consistent with a given set of angular restraints. This ambiguity is further exacerbated by experimental uncertainty in obtaining and interpreting such restraints. The previously developed algorithms for structure calculations using angular restraints typically involve a sequential walkthrough along the backbone to find the torsion angles between the consecutive peptide plane orientations that are consistent with the experimental data. This method is sensitive to experimental uncertainty in interpreting the peak positions of as low as ± 10 Hz, often yielding high structural RMSDs for the calculated structures. Here we present a significantly improved version of the algorithm which includes the fitting of several peptide planes at once in order to prevent propagation of error along the backbone. In addition, a protocol has been devised for filtering the structural solutions using Rosetta scoring functions in order to find the structures that both fit the spectrum and satisfy bioinformatics restraints. The robustness of the new algorithm has been tested using synthetic angular restraints generated from the known structures for two proteins: a soluble protein 2gb1 (56 residues), chosen for its diverse secondary structure elements, i.e. an alpha-helix and two beta-sheets, and a membrane protein 4a2n, from which the first two transmembrane helices (having a total of 64 residues) have been used. Extensive simulations have been performed by varying the number of fitted planes, experimental error, and the number of NMR dimensions. It has been found that simultaneously fitting two peptide planes always shifted the distribution of the calculated structures toward lower structural RMSD values as compared to fitting a single torsion-angle pair. For each protein, irrespective of the simulation parameters, Rosetta was able to distinguish the most plausible structures, often having structural RMSDs lower than 2 Å with respect to the original structure. This study establishes a framework for de-novo protein structure prediction using a combination of solid-state NMR angular restraints and bioinformatics.
多维固态 NMR 谱可用于推断取向膜蛋白的骨架扭转角，从而推断整个蛋白质构象，方法是测量偶极耦合和化学位移各向异性，这些依赖于每个肽平面相对于外磁场的取向。然而，多个肽平面取向可能与给定的角度约束集一致。这种歧义进一步因获得和解释这些约束的实验不确定性而加剧。以前使用角度约束进行结构计算的算法通常涉及沿着骨架进行顺序遍历，以找到与实验数据一致的连续肽平面取向之间的扭转角。这种方法对解释峰位置的实验不确定性很敏感，即使低至±10 Hz 的不确定性也经常导致计算结构的高结构 RMSD。在这里，我们提出了该算法的一个显著改进版本，该版本包括同时拟合多个肽平面，以防止沿骨架传播误差。此外，还设计了一种使用 Rosetta 评分函数过滤结构解决方案的方案，以找到既适合光谱又满足生物信息学约束的结构。新算法的稳健性已通过使用已知结构为两种蛋白质生成的合成角度约束进行测试：一种可溶性蛋白 2gb1（56 个残基），选择它是因为它具有不同的二级结构元素，即一个α-螺旋和两个β-折叠，以及一种膜蛋白 4a2n，其中前两个跨膜螺旋（总共 64 个残基）已被使用。通过改变拟合的平面数量、实验误差和 NMR 维度数量，进行了广泛的模拟。已经发现，与拟合单个扭转角对相比，同时拟合两个肽平面总是将计算结构的分布移向较低的结构 RMSD 值。对于每种蛋白质，无论模拟参数如何，Rosetta 都能够区分最合理的结构，通常相对于原始结构的结构 RMSD 低于 2 Å。这项研究为使用固态 NMR 角度约束和生物信息学进行从头蛋白质结构预测建立了一个框架。
