在蛋白质晶体中,柔性环经常因晶体接触而变形,而在解决方案中,大的运动导致在NMR结构确定中这种柔性回路的收敛性差。我们需要一种实验技术来表征蛋白质分子固有柔性环的结构和动态特性。
我们在蛋白质晶体中设计了一个预期的晶体接触自由空间(CCFS),并在CCFS中安排了灵活的兴趣循环。选择酵母Tim21蛋白作为模型蛋白,因为其中一个环(环2)被常规晶体中的晶体接触扭曲。
酵母Tim21通过刚性α-螺旋接头与MBP蛋白融合。在两种蛋白质之间产生的空间用作CCFS。链接器长度为在CCFS中布置环2提供了可调节的自由度。我们重新确定了酵母Tim21的NMR结构,并进行了MD模拟进行比较。使用多维缩放来可视化环2的构象相似性。我们发现,在NMR和MD结构中,环2的晶体无接触构象位于环2构象集合的中心附近。
CCFS中的酵母Tim21的Loop2采用代表,溶液中的显性构象。
没有一种强大的技术可用于表征蛋白质分子中的柔性结构。NMR分析和MD模拟提供了有用的,但信息不完整。CCFS晶体学为实现这一目标提供了第三条途径。
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.
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.
Yeast Tim21 was fused to the MBP protein by a rigid α-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.
Loop 2 of yeast Tim21 in the CCFS adopts a representative, dominant conformation in solution.
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.