Real-time monitoring of protein conformational changes using a nano-mechanical sensor.
Real-time monitoring of protein conformational changes using a nano-mechanical sensor.
复制标题
DOI：
10.1371/journal.pone.0103674
复制
发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Longo G
中科院分区：
文献类型：
--
作者：
Alonso-Sarduy L;De Los Rios P;Benedetti F;Vobornik D;Dietler G;Kasas S;Longo G
蛋白质可以在不同的构象之间转换，以响应刺激，如pH或温度变化，或配体的结合。这种可塑性及其动力学可能具有关键的功能作用，它们的表征已经成为蛋白质研究的中心。作为一个例子，拓扑异构酶是特别有趣的酶，能够管理缠结和超螺旋的双链DNA，从而促进许多生理过程。在这项工作中，我们描述了使用的一个基于纳米运动传感器来表征人类拓扑异构酶II（拓扑II）酶的动力学和它们对不同种类的配体，如ATP，这增强了构象动力学的响应。该传感器的灵敏度和时间分辨率允许定量确定ATP浓度和拓扑II构象变化速率之间的相关性。此外，我们展示了如何合理化的实验结果，在一个全面的模型，考虑到物理的悬臂梁和酶的ATP酶循环的动力学，揭示了动力学的过程。最后，我们研究了一种抗癌药物aclaravine的作用，表明它直接影响Topo II分子，抑制其构象变化。这些结果为研究蛋白质和蛋白质复合物内在动力学的新方法铺平了道路，从而实现了从基础蛋白质组学到药物发现和开发以及可能的临床实践的新应用。
Proteins can switch between different conformations in response to stimuli, such as pH or temperature variations, or to the binding of ligands. Such plasticity and its kinetics can have a crucial functional role, and their characterization has taken center stage in protein research. As an example, Topoisomerases are particularly interesting enzymes capable of managing tangled and supercoiled double-stranded DNA, thus facilitating many physiological processes. In this work, we describe the use of a cantilever-based nanomotion sensor to characterize the dynamics of human topoisomerase II (Topo II) enzymes and their response to different kinds of ligands, such as ATP, which enhance the conformational dynamics. The sensitivity and time resolution of this sensor allow determining quantitatively the correlation between the ATP concentration and the rate of Topo II conformational changes. Furthermore, we show how to rationalize the experimental results in a comprehensive model that takes into account both the physics of the cantilever and the dynamics of the ATPase cycle of the enzyme, shedding light on the kinetics of the process. Finally, we study the effect of aclarubicin, an anticancer drug, demonstrating that it affects directly the Topo II molecule inhibiting its conformational changes. These results pave the way to a new way of studying the intrinsic dynamics of proteins and of protein complexes allowing new applications ranging from fundamental proteomics to drug discovery and development and possibly to clinical practice.
登录
查看更多内容
影响因子：
4.3
作者：
Ho BK;Agard DA
通讯作者：
Agard DA
影响因子：
4.8
作者：
Hammonds, TR;Maxwell, A
通讯作者：
Maxwell, A
影响因子：
64.8
作者：
BARNES, JR;STEPHENSON, RJ;GIMZEWSKI, JK
通讯作者：
GIMZEWSKI, JK
影响因子：
5.8
作者：
JENSEN, PB;SORENSEN, BS;HANSEN, HH
通讯作者：
HANSEN, HH
影响因子：
15
作者：
MacBeath, G;Koehler, AN;Schreiber, SL
通讯作者：
Schreiber, SL