Internal dynamics in the enzyme barnase

芽孢杆菌RNA酶的内部动力学

• 批准号: BB/J014966/1
• 负责人: Michael Williamson
• 金额: $ 51.57万
• 依托单位: University of Sheffield
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FJ014966%2F1
• 关键词: Internal; dynamics; enzyme; barnase

Enzymes are the catalysts that carry out all of the reactions in nature. We have known for over 100 years that the structure of an enzyme has to be matched closely to the structures of the molecules that are reacting ('substrates'), and X-ray and NMR structures have shown how this is achieved in detail for many enzymes (the lock and key hypothesis). However, our attempts to design new enzymes have so far been rather pathetic in comparison with the impressive catalytic ability of real enzymes. The best rationally designed enzymes are at least a million times slower than the real thing. Partly this is because the structure has to be very accurately correct. However, another reason, which we are only just beginning to come to grips with, is that an enzyme is not just a static framework, but it moves constantly, mainly as a result of continual bombardment by solvent molecules. This provides it with a lot of kinetic energy, and it appears that somehow this random thermal kinetic energy is channeled into a few very specific motions in order to help the enzyme perform its catalysis. One of the main ways in which this is achieved is that the 'normal' or resting state of an enzyme is an 'open' state, in which the active site (where the reaction occurs) is not in its optimum configuration. Motion within the enzyme very specifically closes the active site, and is precisely tuned so that only a few percent of enzyme molecules are in this active or 'closed' state at any one time. The substrates bind more tightly to the closed state than the open one, and therefore the presence of substrate pulls almost all of the enzyme molecules over into the more active closed state. This model is a refinement of the induced fit hypothesis, and is called conformational selection. It is not clear why enzymes need to do this. In some cases it is because the substrate cannot get into the closed state, but the more general reason may be that evolution does not want the enzyme to be active unless there are substrates bound, to avoid unwanted reactions.This proposal aims to understand these motions for a model enzyme called barnase, which digests RNA. We have shown that in barnase there are two different motions required to make the closed state. One of these is a simple bending of the enzyme, like a hinge closing, and is a low-energy and common motion. The other requires several loops around the active site to close up together, rather like the fingers of a hand closing, and cannot occur efficiently unless the hinge is closed already. We have good evidence that this happens, but we need more details in order to understand it properly: we need to know rates, energies and structures, and how these motions are determined by the structure of barnase. Exactly what does it do and how does it do it? The first motion is easy to understand, but the second is not. Once we have understood it, we also want to explain it in ways that everyone can understand.This is important, because until we understand how enzymes really work, we are to a large extent groping around in the dark, and we are unlikely to be able to build an enzyme that works well. Many scientists think that because we know the structural details of enzymes, we understand them already. This is sadly not true. Science has shown that real progress comes from a proper understanding of the problem, which is what this research aims to produce.

酶是进行自然界所有反应的催化剂。 100 多年来，我们就知道酶的结构必须与正在反应的分子（“底物”）的结构紧密匹配，X 射线和 NMR 结构已经详细展示了如何实现这一点酶（锁和钥匙假说）。然而，与真正酶令人印象深刻的催化能力相比，我们设计新酶的尝试迄今为止是相当可悲的。最合理设计的酶至少比真正的酶慢一百万倍。部分原因是结构必须非常准确。然而，我们才刚刚开始认识到的另一个原因是，酶不仅仅是一个静态框架，而且它不断移动，这主要是溶剂分子持续轰击的结果。这为其提供了大量的动能，并且似乎这种随机的热动能以某种方式被引导到一些非常特定的运动中，以帮助酶进行催化。实现这一目标的主要方式之一是酶的“正常”或静止状态是“开放”状态，其中活性位点（反应发生的地方）不处于其最佳配置。酶内的运动非常具体地关闭活性位点，并且经过精确调整，以便在任何时候只有百分之几的酶分子处于这种活性或“关闭”状态。底物与封闭状态的结合比开放状态更紧密，因此底物的存在将几乎所有酶分子拉入更活跃的封闭状态。该模型是诱导拟合假设的改进，称为构象选择。目前尚不清楚为什么酶需要这样做。在某些情况下，这是因为底物无法进入封闭状态，但更普遍的原因可能是进化不希望酶具有活性，除非有底物结合，以避免不必要的反应。该提案旨在了解这些运动一种名为 Barnase 的模型酶，它可以消化 RNA。我们已经证明，在 barnase 中需要两种不同的运动才能达到关闭状态。其中之一是酶的简单弯曲，就像铰链关闭一样，是一种低能量且常见的运动。另一种需要围绕活性位点的几个环才能闭合在一起，就像手指闭合一样，并且除非铰链已经闭合，否则无法有效地发生。我们有充分的证据证明这种情况发生，但我们需要更多细节才能正确理解它：我们需要知道速率、能量和结构，以及这些运动是如何由芽孢杆菌RNA酶的结构决定的。它到底有什么作用以及如何做到这一点？第一个动议很容易理解，但第二个动议则不然。一旦我们理解了它，我们也想用每个人都能理解的方式来解释它。这一点很重要，因为在我们理解酶的真正工作原理之前，我们很大程度上是在黑暗中摸索，而且我们不太可能能够构建一种效果良好的酶。许多科学家认为，因为我们知道酶的结构细节，所以我们已经了解它们了。遗憾的是，事实并非如此。科学表明，真正的进步来自于对问题的正确理解，这正是本研究的目的。

项目成果

Close identity between alternatively folded state N2 of ubiquitin and the conformation of the protein bound to the ubiquitin-activating enzyme.

泛素的交替折叠状态 N2 和与泛素激活酶结合的蛋白质构象之间的密切一致性。

• DOI: 10.1021/bi401617n
• 发表时间: 2014-01-10
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Soichiro Kitazawa;T. Kameda;A. Kumo;M. Yagi;N. J. Baxter;Koichi Kato;M. Williamson;R. Kitahara
• 通讯作者: R. Kitahara

Why the Energy Landscape of Barnase Is Hierarchical.

为什么 Barnase 的能源格局是分层的。

• DOI: http://dx.10.3389/fmolb.2018.00115
• 发表时间: 2018
• 期刊: Frontiers in molecular biosciences
• 作者: Pandya MJ

Solution structure of the Q41N variant of ubiquitin as a model for the alternatively folded N2 state of ubiquitin.

泛素 Q41N 变体的溶液结构，作为泛素交替折叠 N2 态的模型。

• DOI: http://dx.10.1021/bi301420m
• 发表时间: 2013
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Kitazawa S

Conformational exchange in the potassium channel blocker ShK.

钾通道阻滞剂 ShK 中的构象交换。

• DOI: http://dx.10.1038/s41598-019-55806-3
• 发表时间: 2019
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Iwakawa N

Pressure-Dependent Chemical Shifts in the R3 Domain of Talin Show that It Is Thermodynamically Poised for Binding to Either Vinculin or RIAM

Talin R3 结构域中压力依赖性化学变化表明，它在热力学上已做好与 Vinculin 或 RIAM 结合的准备

• DOI: http://dx.10.1016/j.str.2017.10.008
• 发表时间: 2017
• 期刊: Structure
• 影响因子: 5.7
• 作者: Baxter N