Folding of the KIX domain:: Characterization of the equilibrium analog of a folding intermediate using 15N/13C relaxation dispersion and fast 1H/2H amide exchange NMR spectroscopy
Folding of the KIX domain:: Characterization of the equilibrium analog of a folding intermediate using 15N/13C relaxation dispersion and fast 1H/2H amide exchange NMR spectroscopy
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DOI：
10.1016/j.jmb.2008.05.040
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发表时间：
2008-07-18
影响因子：
5.6
通讯作者：
Tollinger, Martin
中科院分区：
文献类型：
--
作者：
Schanda, Paul;Brutscher, Bernhard;Tollinger, Martin
转录共激活因子CBP的KIX结构域是一种三螺旋束蛋白，其通过中间状态的快速积累折叠，然后是较慢的折叠阶段。最近的NMR弛豫色散研究揭示了KIX的低填充（激发）状态的存在，其在非变性条件下与天然折叠形式平衡存在，并且可能代表折叠中间体的平衡类似物。在这里，我们结合联合收割机酰胺氢/氘交换测量使用快速NMR数据采集技术与骨干（15）N和（13）C弛豫分散实验，以进一步研究平衡折叠的KIX域。通过这两种方法检测折叠中间体内的残留结构，并且它们的组合使得能够可靠地定量持久残留结构的量。三个定义明确的折叠亚基被发现，显示可变的稳定性，并密切对应于在自然状态下的个别螺旋。虽然三个螺旋中的两个α（2）和α（3）部分地形成在折叠中间体中（在20 ℃下分别为50%和80%），但第三个螺旋是无序的。所观察到的激发态内的螺旋内容超过了相应的肽区域的螺旋倾向预测，这表明这两个螺旋弱相互稳定，而甲基（13）C弛豫分散数据表明，一个定义的包装安排是不可能的。温度依赖性的实验表明，最大的焓和熵的变化沿着折叠反应发生在从中间体到天然状态的最后过渡。我们的实验数据与螺旋α（2）和α（3）快速形成的折叠机制一致，尽管程度不同，而螺旋α（1）仅在折叠过程中合并以完成天然状态结构。(C)2008爱思唯尔有限公司版权所有。
The KIX domain of the transcription co-activator CBP is a three-helix bundle protein that folds via rapid accumulation of an intermediate state, followed by a slower folding phase. Recent NMR relaxation dispersion studies revealed the presence of a low-populated (excited) state of KIX that exists in equilibrium with the natively folded form under non-denaturing conditions, and likely represents the equilibrium analog of the folding intermediate. Here, we combine amide hydrogen/deuterium exchange measurements using rapid NMR data acquisition techniques with backbone (15)N and (13)C relaxation dispersion experiments to further investigate the equilibrium folding of the KIX domain. Residual structure within the folding intermediate is detected by both methods, and their combination enables reliable quantification of the amount of persistent residual structure. Three well-defined folding subunits are found, which display variable stability and correspond closely to the individual helices in the native state. While two of the three helices alpha(2) and alpha(3) are partially formed in the folding intermediate (to similar to 50% and similar to 80%, respectively, at 20 degrees C), the third helix is disordered. The observed helical content within the excited state exceeds the helical propensities predicted for the corresponding peptide regions, suggesting that the two helices are weakly mutually stabilized, while methyl (13)C relaxation dispersion data indicate that a defined packing arrangement is unlikely. Temperature-dependent experiments reveal that the largest enthalpy and entropy changes along the folding reaction occur during the final transition from the intermediate to the native state. Our experimental data are consistent with a folding mechanism where helices alpha(2) and alpha(3) form rapidly, although to different extents, while helix alpha(1) consolidates only as folding proceeds to complete the native state-structure. (C) 2008 Elsevier Ltd. All rights reserved.