Hu Kaifeng, Eletsky Alexander, Pervushin Konstantin
Laboratorium für Physikalische Chemie, Eidgenössische Technische Hochschule Hönggerberg, CH-8093 Zürich, Switzerland.
J Biomol NMR. 2003 May;26(1):69-77. doi: 10.1023/a:1023008719248.
A new method for backbone resonance assignment suitable for large proteins with the natural (1)H isotope content is proposed based on a combination of the most sensitive TROSY-type triple-resonance experiments. These techniques include TROSY-HNCO, (13)C'-detected 3D multiple-quantum HACACO and the newly developed 3D TROSY multiple-quantum-HN(CA)HA and 4D TROSY multiple-quantum-HACANH experiments. The favorable relaxation properties of the multiple-quantum coherences, signal detection using the (13)C' antiphase coherences, and the use of TROSY optimize the performance of the proposed set of experiments for application to large protonated proteins. The method is demonstrated with the 44 kDa uniformly (15)N,(13)C-labeled and fractionally (35%) deuterated trimeric B. Subtilis Chorismate Mutase and is suitable for proteins with large correlation times but a relatively small number of residues, such as membrane proteins embedded in micelles or oligomeric proteins.
基于最灵敏的TROSY型三重共振实验的组合，提出了一种适用于具有天然（1）H同位素含量的大蛋白的骨架共振归属新方法。这些技术包括TROSY-HNCO、（13）C'检测的3D多量子HACACO以及新开发的3D TROSY多量子-HN（CA）HA和4D TROSY多量子-HACANH实验。多量子相干的良好弛豫特性、使用（13）C'反相相干进行信号检测以及TROSY的使用优化了所提出的一组实验在大质子化蛋白应用中的性能。该方法通过44 kDa均匀（15）N、（13）C标记且部分（35%）氘代的枯草芽孢杆菌分支酸变位酶三聚体进行了验证，适用于具有长相关时间但残基数量相对较少的蛋白，如嵌入胶束中的膜蛋白或寡聚蛋白。
