Crystal contact-free conformation of an intrinsically flexible loop in protein crystal: Tim21 as the case study
Crystal contact-free conformation of an intrinsically flexible loop in protein crystal: Tim21 as the case study
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DOI：
10.1016/j.bbagen.2019.129418
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发表时间：
2020-02-01
影响因子：
3
通讯作者：
Kohda, Daisuke
中科院分区：
文献类型：
--
作者：
Bala, Siqin;Shinya, Shoko;Kohda, Daisuke
背景：在蛋白质晶体中，柔性环经常因晶体接触而变形，而在溶液中，大的运动导致核磁共振结构测定中此类柔性环的收敛性较差。我们需要一种实验技术来表征蛋白质分子本质上柔性环的结构和动态特性。方法：我们在蛋白质晶体中设计了一个预期的晶体无接触空间（CCFS），并将感兴趣的柔性环布置在CCFS中。选择酵母 Tim 21 蛋白作为模型蛋白，因为其中一个环（环 2）因常规晶体中的晶体接触而变形。结果：酵母 Tim21 通过刚性 a 螺旋接头与 MBP 蛋白融合。两种蛋白质之间产生的空间被用作 CCFS。连接子长度提供了在 CCFS 中排列环 2 的可调节自由度。我们重新测定了酵母Tim21的NMR结构，并进行MD模拟进行比较。使用多维标度可视化环2的构象相似性。我们发现环2的晶体无接触构象位于NMR和MD结构中靠近环2构象整体的中心。结论：CCFS中酵母Tim21的环2在溶液中采用了具有代表性的主导构象。一般意义：没有单一强大的技术可用于 蛋白质分子柔性结构的表征。 NMR 分析和 MD 模拟提供了有用但不完整的信息。 CCFS 晶体学提供了实现这一目标的第三条途径。
Background: In protein crystals, flexible loops are frequently deformed by crystal contacts, whereas in solution, the large motions result in the poor convergence of such flexible loops in NMR structure determinations. We need an experimental technique to characterize the structural and dynamic properties of intrinsically flexible loops of protein molecules.Methods: We designed an intended crystal contact-free space (CCFS) in protein crystals, and arranged the flexible loop of interest in the CCFS. The yeast Tim 21 protein was chosen as the model protein, because one of the loops (loop 2) is distorted by crystal contacts in the conventional crystal.Results: Yeast Tim21 was fused to the MBP protein by a rigid a-helical linker. The space created between the two proteins was used as the CCFS. The linker length provides adjustable freedom to arrange loop 2 in the CCFS. We re-determined the NMR structure of yeast Tim21, and conducted MD simulations for comparison. Multidimensional scaling was used to visualize the conformational similarity of loop 2. We found that the crystal contact-free conformation of loop 2 is located close to the center of the ensembles of the loop 2 conformations in the NMR and MD structures.Conclusions: Loop 2 of yeast Tim21 in the CCFS adopts a representative, dominant conformation in solution.General significance: No single powerful technique is available for the characterization of flexible structures in protein molecules. NMR analyses and MD simulations provide useful, but incomplete information. CCFS crystallography offers a third route to this goal.