DISSECTING AND EXPLOITING MOLECULAR RECOGNITION AT PROTEIN-PROTEIN INTERFACES

蛋白质调节许多对活细胞的活动至关重要的过程。为了成功地发挥其生物学功能，蛋白质常常与其他蛋白质以复合体的形式相互作用。拟议研究的主要主题是开发新的方法来提高我们对蛋白质-蛋白质相互作用的理解，以及我们如何利用小分子的结合来破坏这些相互作用。在这个项目中，我专注于研究小分子与蛋白质界面的结合。我提出了以下问题：蛋白质界面的哪些特征决定了小分子的结合和活性(或缺乏)？我们能不能改变蛋白质的界面，让小分子越来越好地粘在表面上？我们可以从这些新界面中学到什么？我们如何利用这些信息来发现新的化合物，这些化合物可以通过与这些位点紧密结合而在细胞中发挥作用？为了研究蛋白质的相互作用，我将首先使用蛋白质工程，这是一种通过将蛋白质的组成成分氨基酸从一种类型改变为另一种类型来在蛋白质上产生突变的技术。我将以一种明确的方式进行突变，用越来越小的氨基酸取代蛋白质界面上的大氨基酸，从而创造出越来越大的空洞。结合在这些工程化口袋上的小分子的位置和强度将被确定。这将提供有用的信息，以寻找其他可能在活着的有机体中起作用的蛋白质界面，以及可能被小分子破坏的蛋白质界面。其次，我将进一步利用这一知识来帮助识别通过破坏蛋白质界面发挥功能的小分子。我将开发新的方法来检测与界面相邻位置结合在一起的小分子。为此，我将使用核磁共振光谱学，这是一种允许监测小分子氢原子的技术，如果这些小分子与蛋白质结合得很近，它可以报告。我还将制造蛋白质的晶体，在晶体中，小分子可以接触到界面，并让这些分子在蛋白质表面相互结合时发生反应。由于与蛋白质结合的小分子的结构可以通过向蛋白质晶体发射X射线辐射直接确定，这是一种被称为X射线结晶学的技术，这是一种快速识别任何成功组装在蛋白质界面的化合物的方法。这项研究是重要的，也是令人兴奋的，原因如下：1.蛋白质的界面往往相对平坦和无特征，因为它们不是天生的‘进化’来结合小分子的。因此，利用小分子调控蛋白质-蛋白质相互作用是一项具有挑战性的任务，也是分子识别的前沿。2.一个新的科学视野是通过以选择性的方式扰乱细胞内的通路和网络来促进我们对生物系统的理解。由于蛋白质-蛋白质相互作用广泛发生在细胞内，利用小分子对它们的调节提供了询问和发现新生物学的机会。3.蛋白质-蛋白质复合体的破坏为新药开发提供了一种新的通用机制。总而言之，这项研究有可能对未来发现新生物学和新药的方式产生重大影响，为社会带来更广泛的好处，并在抗击疾病方面提供令人兴奋的机会。

Proteins regulate many of the processes that are crucial for the activity of a living cell. In order to successfully carry out their biological function, proteins often interact in complex with other proteins. The principal subject of the proposed research concerns the development of new approaches to advance our understanding of protein-protein interactions and of how we could disrupt these interactions using the binding of a small molecule. In this project, I focus on studying the binding of small molecules to protein interfaces. I pose the following questions: what features of protein interfaces determine binding and activity (or lack thereof) of a small molecule? Can we modify protein interfaces so that a small molecule can 'stick' to the surface better and better? What can we learn from these new interfaces? How can we use such information to discover new compounds that could function in the cell by binding tightly to these sites? To interrogate protein interfaces, I will first use protein engineering, a technique to generate mutations on a protein by changing amino acids, the building blocks of proteins, from one type to another. I will make mutations in a defined manner, by replacing large amino acids at the protein interface with smaller and smaller ones, hence creating larger and larger cavities. The location and strength of small molecules bound to these engineered pockets will be determined. This will provide useful information to find other protein interfaces that may be functional in living organisms, and that may have the potential to be disrupted using small molecules. Secondly, I will exploit this knowledge further to facilitate identification of small molecules that function by disrupting a protein interface. I will develop new methods to detect small molecules that bind together to adjacent sites of the interface. For this purpose, I will use nuclear magnetic resonance (NMR) spectroscopy, a technique that allows monitoring the hydrogen atoms of small molecules and that can report if these are bound to a protein close to one another. I will also make crystals of the protein in which the interface is accessible to small molecules, and let these molecules react together as they are bound close to each other at the protein surface. Since the structure of a small molecule bound to the protein can be determined directly by shooting X-ray radiations at the protein crystal, a technique called X-ray crystallography, this is a rapid way of identifying any compound that has successfully assembled at the protein interface. The research is important and exciting for the following reasons: 1. Protein interfaces tend to be relatively flat and featureless, as they were not 'evolved' by nature to bind small molecules. The modulation of protein-protein interactions using small molecules is therefore a challenging task, and is at the forefront of molecular recognition. 2. A new scientific horizon is to advance our understanding of biological systems by disrupting pathways and networks in a selective fashion inside the cell. As protein-protein interactions occur widely within the cell, their modulation using small molecules offers an opportunity to interrogate and discover new biology. 3. The disruption of protein-protein complexes offers a novel and general mechanism to develop new medicines. In conclusion, this research has the potential to significantly impact on the way new biology and new drugs will be discovered in the future, with wider benefits to society and exciting opportunities in the fight against disease.