Modulation of Calmodulin Plasticity by the Effect of Macromolecular Crowding
Modulation of Calmodulin Plasticity by the Effect of Macromolecular Crowding
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DOI：
10.1016/j.jmb.2009.06.073
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发表时间：
2009-09-04
影响因子：
5.6
通讯作者：
Cheung, Margaret S.
中科院分区：
文献类型：
--
作者：
Homouz, Dirar;Sanabria, Hugo;Cheung, Margaret S.
体外生物化学反应最常在稀溶液中研究，稀溶液是真核细胞的细胞内空间的较差模拟物，其挤满了移动的和非移动的大分子。这种拥挤的条件产生体积排斥和其他熵力，这些熵力有可能影响化学平衡和反应速率。在这篇文章中，我们使用的特点和无处不在的分子钙调素（CaM）和理论和实验相结合的方法来解决拥挤如何影响钙调素的构象可塑性。CaM是一种哑铃形分子，含有四个EF手（两个在N叶，两个在C叶），每个EF手都可以结合Ca2+，导致某些亚基的稳定，有利于与其他靶蛋白的相互作用。使用粗粒度的分子模拟，我们探讨了拥挤剂的存在下，钙调素构象的分布。这些预测，其中拥挤效应增强人口的紧凑结构，然后证实了在实验测量中使用荧光共振能量转移技术的供体和受体标记的钙调素在正常和拥挤的条件下。使用蛋白质重建方法，我们进一步探索了折叠能量景观，并研究了CaM在自由能盆地的结构特征。我们发现，拥挤稳定了几种不同的紧凑的构象，这反映了固有的可塑性钙调素的结构。根据这些结果，我们认为C瓣中的EF指针是灵活的，可以被认为是开关，而N瓣中的EF指针是僵硬的，类似于变阻器。新的组合信号的性质可能会出现从产品的微分可塑性的两个不同的叶钙调素在拥挤的存在下。我们讨论了这些结果对调节钙调素结合钙离子和靶蛋白的能力的影响。(C)2009爱思唯尔有限公司保留所有权利。
In vitro biochemical reactions are most often studied in dilute solution, a poor mimic of the intracellular space of eukaryotic cells, which are crowded with mobile and immobile macromolecules. Such crowded conditions exert volume exclusion and other entropic forces that have the potential to impact chemical equilibria and reaction rates. In this article, we use characterized and ubiquitous molecule calmodulin (CaM) and a combination of theoretical and experimental approaches to address how crowding impacts CaM's conformational plasticity. CaM is a dumbbell-shaped molecule that contains four EF hands (two in the N-lobe and two in the C-lobe) that each could bind Ca2+, leading to stabilization of certain substates that favor interactions with other target proteins. Using coarse-grained molecular simulations, we explored the distribution of CaM conformations in the presence of crowding agents. These predictions, in which crowding effects enhance the population of compact structures, were then confirmed in experimental measurements using fluorescence resonance energy transfer techniques of donor- and acceptor-labeled CaM under normal and crowded conditions. Using protein reconstruction methods, we further explored the folding-energy landscape and examined the structural characteristics of CaM at free-energy basins. We discovered that crowding stabilizes several different compact conformations, which reflects the inherent plasticity in CaM's structure. From these results, we suggest that the EF hands in the C-lobe are flexible and can be thought of as a switch, while those in the N-lobe are stiff, analogous to a rheostat. New combinatorial signaling properties may arise from the product of the differential plasticity of the two distinct lobes of CaM in the presence of crowding. We discuss the implications of these results for modulating CaM's ability to bind Ca2+ and target proteins. (C) 2009 Elsevier Ltd. All rights reserved.