Integrative Approaches for Characterising Small-Molecule Binding to Disordered Proteins

生物学中的许多过程（如信号、刺激）以及我们如何操纵它们（如杀虫剂、药物）在分子水平上取决于被称为蛋白质的大分子，以及它们如何与较小的化学物质（或“小分子”）相互作用。大多数蛋白质（称为“结构蛋白质”）采用与特定功能相关的明确的3D形状。一般来说，小分子将自己插入结构蛋白质表面的凹槽中，进而改变蛋白质的运动和功能。通常，这被称为“锁与钥匙”结合机制，因为小分子能钻进蛋白质的凹槽，就像钥匙能钻进锁一样。然而，许多蛋白质（包括来自人类、病毒和植物的蛋白质）从不采用单一的形状，而是在多种形状之间快速转换。这些被称为“无序蛋白质”的蛋白质缺乏小分子（“钥匙”）可以结合的长寿命凹槽（“锁”）。几十年来，人们一直认为小分子不会与无序蛋白质相互作用，因为人们不清楚这些相互作用是如何发生的。然而，最近的研究表明，无序蛋白质确实可以结合小分子，但其机制不同于“锁与键”结合。相反，无序蛋白质与小分子“共舞”，这样每一种蛋白质形状都以独特的方式与小分子相互作用。我是最早描述这些新机制的人之一（针对与阿尔茨海默病有关的一种无序蛋白质），但关于结合的分子细节，还有很多需要了解的。解决这一差距的最大瓶颈是缺乏可用的工具来研究这些过程。作为BBSRC研究员，我将结合两种方法，一种是实验方法，一种是计算方法，以开发新的跨学科工具。这种实验技术被称为核磁共振波谱（NMR），它利用强磁场来研究蛋白质和小分子的物理和化学性质。大多数核磁共振实验是针对结构蛋白开发的。因此，我将专门为小分子结合在无序蛋白质上的应用建立新的方法。我还将采用一种称为分子动力学（MD）模拟的计算方法，在这种方法中，我将使用超级计算机模拟无序蛋白质和小分子的运动。MD模拟将允许我创建“电影”来“看到”小分子和无序蛋白质如何相互作用，这是我无法从核磁共振单独获得的宝贵见解。然而，在我的模型中必须进行某些近似，以使计算负担得起，而这些可能导致不准确。因此，我将开发新的工具，使我能够将实验核磁共振数据纳入模拟以提高准确性。通过结合NMR和MD模拟，我可以克服每种技术单独的局限性，并为无序蛋白质如何与小分子相互作用的分子机制提供新的见解。我将把这些工具应用于特定的无序蛋白质，以发现新的小分子结合机制。例如，来自丙型肝炎病毒的非结构蛋白5A （NS5A）与几种小分子相互作用，包括抗病毒药物，但这些分子如何结合（以及抗病毒药物如何起作用）的机制仍不清楚。我还将运用这些工具来研究一种叫做FUS的人类紊乱蛋白。FUS经历了一种称为“液-液相分离”的现象，这与水中油滴的形成非常相似。在某些被称为“核苷酸”的高浓度小分子存在的情况下，FUS不会发生相分离，但原因尚不清楚。我的新工具将使我能够“看到”和理解这些结合机制，发现新的基础生物学，并利用它来开发新的生物技术。

Many processes in biology (e.g. signalling, stimulation) and how we manipulate them (e.g. pesticides, drugs) depend, at the molecular level, on large biomolecules called proteins, and how they interact with smaller chemicals (or 'small molecules').Most proteins (termed 'structured proteins') adopt well-defined 3D shapes which are associated with a specific function. Generally, small molecules insert themselves into grooves on the surfaces of structured proteins, which in turn, alter protein movement and function. Often, this is referred to as the 'lock-and-key' binding mechanism because the small molecule fits into the protein's grooves much like a key fits into a lock.Nevertheless, many proteins (including those from humans, viruses, & plants) never adopt a single shape and instead rapidly interconvert between many shapes. These proteins, called 'disordered proteins' lack long-lived grooves ('locks') to which small molecules ('keys') can bind. For many decades it was believed that small molecules do not interact with disordered proteins, because it was unclear how these interactions could take place. Nevertheless, recent work suggests that disordered proteins can indeed bind small molecules, but the mechanisms differ from 'lock-and-key' binding. Instead, disordered proteins 'dance' with small molecules, such that each protein shape interacts with the small molecule in a unique way. I was one of the first to describe these new mechanisms (for a disordered protein involved in Alzheimer's disease), but there is still much to be understood about the molecular details of binding. The greatest bottleneck to addressing this gap is the lack of available tools to study these processes. As a BBSRC Fellow, I will combine two approaches, one experimental and one computational, towards the development of new interdisciplinary tools. The experimental technique is called Nuclear Magnetic Resonance spectroscopy (NMR), in which strong magnetic fields are used to study the physical and chemical properties of proteins and small molecules. Most NMR experiments were developed for structured proteins. Thus, I will establish new methods specifically for the application of small-molecule binding to disordered proteins. I will also employ a computational approach called molecular dynamics (MD) simulations, in which I will model the movement of disordered proteins and small molecules using supercomputers. The MD simulations will allow me to create 'movies' to 'see' how small molecules and disordered proteins interact with one-another, which is valuable insight that I cannot get from NMR alone. Nevertheless, certain approximations must be made in my models to make the calculations affordable, and these can lead to inaccuracies. Thus, I will develop new tools that allow me to incorporate experimental NMR data into the simulations to improve accuracy. By combining both NMR and MD simulations, I can overcome the limitations of each technique alone and provide new insight into the molecular mechanisms of how disordered proteins interact with small molecules. I will apply these tools to specific disordered proteins to discover new small-molecule binding mechanisms. For example, the Non-Structural protein 5A (NS5A) from hepatitis C virus interacts with several small molecules, including an antiviral, but the mechanisms of how these molecules bind (and how the antiviral works) remain unclear. I will also apply these tools to study a human disordered protein called FUsed in Sarcoma (FUS). FUS undergoes a phenomenon termed 'liquid-liquid phase separation' which is very similar to the formation of oil droplets in water. In the presence of high concentrations of certain small molecules, called 'nucleotides', FUS does not undergo phase separation, but it is not clear why. My new tools will allow me to 'see' and understand these binding mechanisms, discover new fundamental biology, and exploit it towards the development of novel biotechnology.