Xu Yingqi, Zheng Yu, Fan Jing-Song, Yang Daiwen
Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543.
Nat Methods. 2006 Nov;3(11):931-7. doi: 10.1038/nmeth938.
So far high-resolution structure determination by nuclear magnetic resonance (NMR) spectroscopy has been limited to proteins <30 kDa, although global fold determination is possible for substantially larger proteins. Here we present a strategy for assigning backbone and side-chain resonances of large proteins without deuteration, with which one can obtain high-resolution structures from (1)H-(1)H distance restraints. The strategy uses information from through-bond correlation experiments to filter intraresidue and sequential correlations from through-space correlation experiments, and then matches the filtered correlations to obtain sequential assignment. We demonstrate this strategy on three proteins ranging from 24 to 65 kDa for resonance assignment and on maltose binding protein (42 kDa) and hemoglobin (65 kDa) for high-resolution structure determination. The strategy extends the size limit for structure determination by NMR spectroscopy to 42 kDa for monomeric proteins and to 65 kDa for differentially labeled multimeric proteins without the need for deuteration or selective labeling.
到目前为止，通过核磁共振（NMR）光谱法进行高分辨率结构测定仅限于分子量小于30 kDa的蛋白质，尽管对于大得多的蛋白质也可以确定其整体折叠。本文我们提出了一种无需氘代即可对大蛋白质的主链和侧链共振进行归属的策略，利用该策略可以从1H-1H距离约束获得高分辨率结构。该策略利用通过键相关实验的信息来过滤通过空间相关实验中的残基内和序列相关性，然后匹配过滤后的相关性以获得序列归属。我们在三种分子量范围为24至65 kDa的蛋白质上展示了该策略用于共振归属，并在麦芽糖结合蛋白（42 kDa）和血红蛋白（65 kDa）上展示了该策略用于高分辨率结构测定。该策略将通过NMR光谱法进行结构测定的分子量限制扩展到单体蛋白质为42 kDa，差异标记的多聚体蛋白质为65 kDa，而无需氘代或选择性标记。
