Department of Chemistry, University of Zürich, Winterthurerstrasse 190, 8057, Zürich, Switzerland.
Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Max-von-Laue-Straße 9, 60438, Frankfurt am Main, Germany.
J Biomol NMR. 2021 Sep;75(8-9):319-334. doi: 10.1007/s10858-021-00376-8. Epub 2021 Aug 2.
NMR structure calculation using NOE-derived distance restraints requires a considerable number of assignments of both backbone and sidechains resonances, often difficult or impossible to get for large or complex proteins. Pseudocontact shifts (PCSs) also play a well-established role in NMR protein structure calculation, usually to augment existing structural, mostly NOE-derived, information. Existing refinement protocols using PCSs usually either require a sizeable number of sidechain assignments or are complemented by other experimental restraints. Here, we present an automated iterative procedure to perform backbone protein structure refinements requiring only a limited amount of backbone amide PCSs. Already known structural features from a starting homology model, in this case modules of repeat proteins, are framed into a scaffold that is subsequently refined by experimental PCSs. The method produces reliable indicators that can be monitored to judge about the performance. We applied it to a system in which sidechain assignments are hardly possible, designed Armadillo repeat proteins (dArmRPs), and we calculated the solution NMR structure of YMA, a dArmRP containing four sequence-identical internal modules, obtaining high convergence to a single structure. We suggest that this approach is particularly useful when approximate folds are known from other techniques, such as X-ray crystallography, while avoiding inherent artefacts due to, for instance, crystal packing.
使用基于 NOE 的距离约束进行 NMR 结构计算需要对大量的骨架和侧链共振进行分配，这对于大型或复杂的蛋白质来说通常是困难或不可能的。伪接触位移（PCSs）在 NMR 蛋白质结构计算中也起着既定的作用，通常用于补充现有的结构信息，这些信息主要来自 NOE。使用 PCSs 的现有精修协议通常需要相当数量的侧链分配，或者由其他实验约束来补充。在这里，我们提出了一种自动迭代过程，用于执行仅需要有限数量的骨架酰胺 PCSs 的骨架蛋白质结构精修。从起始同源模型（在这种情况下是重复蛋白的模块）中已经知道的结构特征被框定为支架，然后通过实验 PCS 对其进行精修。该方法产生了可靠的指标，可以用来监测性能。我们将其应用于一个几乎不可能进行侧链分配的系统，设计了 Armadillo 重复蛋白（dArmRPs），并计算了包含四个序列相同内部模块的 dArmRP YMA 的溶液 NMR 结构，得到了高收敛到单个结构。我们认为，当从其他技术（例如 X 射线晶体学）中已知近似折叠时，这种方法特别有用，同时避免了由于例如晶体堆积而产生的固有伪影。
