Investigation of alternative states of barnase

酶是催化生物反应的分子。几乎所有的酶都是蛋白质，它们是大而复杂的分子。这是不可避免的，因为他们有工作要做。所有的酶都是通过稳定“过渡态”来工作的，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物质（“底物”）、过渡态和反应产物。这意味着酶必须是灵活的，以适应反应的这三个阶段，这三个阶段总是有不同的形状和（通常）不同的电荷分布。这是已知的，但令人惊讶的是，人们对其他很多东西知之甚少。例如，很久以前就有人提出，酶会经历“诱导契合”，在这种情况下，底物的结合会导致酶的结构发生变化，以便更好地匹配过渡状态。然而，最近已经很清楚，即使在没有底物的情况下，酶也可能经历诱导的拟合运动。在这种情况下，问题出现了；底物到底做了什么？例如，它是改变酶的运动，还是通过将酶冻结在活性状态来阻止酶的运动？或者它重新定向运动，使酶以适当的方式“推动”底物，以帮助反应发生？这些都是基础问题，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。这里研究的酶的功能是消化RNA，被称为藤蔓酶。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：一种是底物的模拟物，另一种是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），它提供了关于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不同状态，这些状态只占蛋白质的百分之几：这些就是这些诱导契合运动所涉及的状态类型。这项研究的目的是将我们的技术与其他核磁共振技术进行详细的比较，以探测不同的状态，特别是一种称为弛豫色散的技术。弛豫色散是一种令人兴奋的测量方法，因为它提供了时间尺度和可选状态的总体。然而，它只对相对有限的时间尺度范围敏感，大约在10-3秒到10-6秒之间。还有其他的核磁共振技术可以观察10-9秒甚至更快的范围，但到目前为止，还没有一种技术可以观察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。我们的研究将提供一个完整的画面，运动发生了什么，蛋白质在哪里，有多快。我们还将测量不同原子的运动是否相关，也就是说，它们是相同运动的一部分还是独立的。这些都是详细的测量，但它们将第一次使我们有把握地说蛋白质是如何运动的，因此运动是如何与它的功能联系起来的。

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.