Investigation of alternative states of barnase

芽孢杆菌RNA酶替代状态的研究

• 批准号: BB/D015308/1
• 负责人: Michael Williamson
• 金额: $ 42.96万
• 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: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FD015308%2F1
• 关键词: Investigation; alternative; states; barnase

Enzymes are the molecules that catalyse biological reactions. Almost all enzymes are proteins, and they are large and complicated molecules. This is inevitable, because of the job they have to do. All enzymes work by being able to stabilise the 'transition state', which is the highest energy part of the reaction pathway. They therefore have to be able to recognise, and bind to, the starting materials of the reaction ('substrates'), the transition state, and the products of the reaction. This means that an enzyme has to be flexible, to accommodate these three stages of the reaction, which always have different shapes and (usually) a different distribution of electric charges. This much is known, but much else is surprisingly poorly understood. It was for example suggested a long time ago that enzymes undergo 'induced fit', in which binding of the substrate causes the structure of the enzyme to change, in order to better match the transition state. However, more recently it has become clear that enzymes may undergo induced fit motions even in the absence of substrates. In which case the question arises; what exactly does the substrate do? For example, does it change the motions of the enzyme, or does it stop them, by freezing the enzyme in an active state? Or does it redirect the motion so that the enzyme 'pushes' the substrate in the appropriate way to help the reaction to happen? These are fundamental questions, which are important to answer because they will enable us to engineer better enzymes in future. The enzyme being studied here functions to digest RNA, and is called barnase. Rather than study binding of substrate, we will study inhibitors, which are more stable. We will study two different types of inhibitor: a mimic of the substrate, plus a naturally occurring protein inhibitor. The technique we will use is nuclear magnetic resonance (NMR), which provides detailed information about motional states of individual atoms in the enzyme. We have been developing a novel technique to characterise alternative states of proteins, that are only populated a few percent: these are the types of states involved in these induced fit motions. The purpose of this research is to make detailed comparisons of our technique to other NMR techniques for probing alternative states, in particular a technique called relaxation dispersion. Relaxation dispersion is an exciting measurement, because it provides timescales and populations of alternative states. However, it is only sensitive to a relatively limited range of timescales, between about 10-3 s and 10-6 s. There are other NMR techniques that can look at the range from 10-9 s and faster, but so far nothing that can look in the intermediate range, between 10-6 and 10-9 s. This is a big gap, and one that includes many of the motions that are suspected to be important for enzyme function. Our research will provide a complete picture of what motion is happening, where in the protein, and how fast. We will also measure whether motions of different atoms are correlated, that is, whether they are part of the same movements or are independent. These are detailed measurements, but they will for the first time enable us to say with confidence how the protein moves, and therefore how the motions relate to its function.

酶是催化生物反应的分子。几乎所有的酶都是蛋白质，而且它们是大而复杂的分子。这是不可避免的，因为他们必须做的工作。所有酶的工作原理都是能够稳定“过渡态”，这是反应途径中能量最高的部分。因此，它们必须能够识别并结合反应的起始材料（“底物”）、过渡态和反应产物。这意味着酶必须具有灵活性，以适应反应的这三个阶段，这三个阶段始终具有不同的形状和（通常）不同的电荷分布。已知的事情就这么多，但令人惊讶的是，还有很多事情我们却知之甚少。例如，很久以前就有人提出，酶会经历“诱导配合”，即底物的结合导致酶的结构发生变化，以便更好地匹配过渡态。然而，最近人们已经清楚，即使在没有底物的情况下，酶也可能发生诱导拟合运动。在这种情况下就会出现问题；基材到底有什么作用？例如，它是否通过将酶冻结在活性状态来改变或阻止酶的运动？或者它是否会改变运动方向，以便酶以适当的方式“推动”底物以帮助反应发生？这些都是基本问题，回答这些问题很重要，因为它们将使我们能够在未来设计出更好的酶。这里研究的酶具有消化 RNA 的功能，被称为芽孢杆菌RNA酶。我们将研究更稳定的抑制剂，而不是研究底物的结合。我们将研究两种不同类型的抑制剂：底物的模拟物，加上天然存在的蛋白质抑制剂。我们将使用的技术是核磁共振 (NMR)，它提供有关酶中单个原子运动状态的详细信息。我们一直在开发一种新技术来表征蛋白质的替代状态，这些状态只占百分之几：这些是参与这些诱导拟合运动的状态类型。这项研究的目的是将我们的技术与其他用于探测替代状态的核磁共振技术进行详细比较，特别是一种称为弛豫色散的技术。松弛离散度是一个令人兴奋的测量方法，因为它提供了替代状态的时间尺度和群体。然而，它仅对相对有限的时间尺度范围敏感，大约在 10-3 s 到 10-6 s 之间。还有其他 NMR 技术可以查看 10-9 秒及更快的范围，但到目前为止还没有任何技术可以查看 10-6 到 10-9 秒之间的中间范围。这是一个很大的差距，其中包括许多被怀疑对酶功能很重要的运动。我们的研究将提供关于运动正在发生的情况、蛋白质中的位置以及运动速度的完整图片。我们还将测量不同原子的运动是否相关，即它们是同一运动的一部分还是独立的。这些是详细的测量，但它们将第一次使我们能够自信地说出蛋白质如何运动，以及运动与其功能的关系。

项目成果

Use of quantitative (1)H NMR chemical shift changes for ligand docking into barnase.

使用定量 (1)H NMR 化学位移变化将配体对接至芽孢杆菌RNA酶中。

• DOI: http://dx.10.1007/s10858-008-9286-7
• 发表时间: 2009
• 期刊: Journal of biomolecular NMR
• 影响因子: 2.7
• 作者: Cioffi M

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

Pressure-dependent structure changes in barnase on ligand binding reveal intermediate rate fluctuations.

配体结合时 Barnase 的压力依赖性结构变化揭示了中等速率波动。

• DOI: http://dx.10.1016/j.bpj.2009.06.022
• 发表时间: 2009
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Wilton DJ

Dimerisation of the UBA domain of p62 inhibits ubiquitin binding and regulates NF-kappaB signalling.

p62 UBA 结构域的二聚化可抑制泛素结合并调节 NF-kappaB 信号传导。

• DOI: 10.1016/j.jmb.2009.11.032
• 发表时间: 2010-02-12
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: J. Long;Thomas P. Garner;M. P;ya;ya;C. Craven;Ping;B. Shaw;M. Williamson;R. Layfield;M. Searle
• 通讯作者: M. Searle