Department of Chemistry, Nanoscience Center, University of Jyväskylä, Jyväskylä, Finland.
Department of Biochemistry, University of Turku, Turku, Finland.
J Biomol NMR. 2020 Mar;74(2-3):147-159. doi: 10.1007/s10858-020-00299-w. Epub 2020 Jan 13.
Resonance assignment of intrinsically disordered proteins is remarkably challenging due to scant chemical shift dispersion arising from conformational heterogeneity. The challenge is even greater if repeating segments are present in the amino acid sequence. To forward unambiguous resonance assignment of intrinsically disordered proteins, we present iHACANCO, HACACON and (HACA)CONCAHA, three H-detected 4D experiments with C as an additional dimension. In addition, we present (HACA)CON(CA)NH and (HACA)N(CA)CONH, new 4D H-start, H-detect experiments which have two N dimensions to enhance peak dispersion in a sequential walk through C', N and H, and provide more accurate N/H chemical shifts than those that can be obtained from a crowded H, N-HSQC spectrum. Application of these 4D experiments is demonstrated using BilRI (165 aa), an outer-membrane intrinsically disordered protein from the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans. BilRI amino acid sequence encompasses three very similar repeats with a 13-residue identical stretch in two of them.
由于构象异质性导致化学位移分散度较小，因此对无规卷曲蛋白质进行共振分配是一项极具挑战性的任务。如果氨基酸序列中存在重复片段，则挑战更大。为了对无规卷曲蛋白质进行明确的共振分配，我们提出了 iHACANCO、HACACON 和 (HACA)CONCAHA，这三个 H 检测的 4D 实验，其中 C 作为附加维度。此外，我们还提出了 (HACA)CON(CA)NH 和 (HACA)N(CA)CONH，这两个新的 4D H 起始、H 检测实验具有两个 N 维度，可以增强在 C'、N 和 H 中连续行走时的峰分散，并提供比拥挤的 H、N-HSQC 光谱中获得的更准确的 N/H 化学位移。使用机会性病原体 Aggregatibacter actinomycetemcomitans 的外膜无规卷曲蛋白 BilRI（165 个氨基酸）演示了这些 4D 实验的应用。BilRI 的氨基酸序列包含三个非常相似的重复，其中两个重复中有 13 个相同的残基。
