DISSECTING AND EXPLOITING MOLECULAR RECOGNITION AT PROTEIN-PROTEIN INTERFACES

蛋白质调节许多对活细胞活动至关重要的过程。为了成功地实现其生物学功能，蛋白质经常与其他蛋白质以复合物的形式相互作用。拟议研究的主要主题是发展新方法，以促进我们对蛋白质-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，我主要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（或缺乏活性）？我们能否修改蛋白质界面，使小分子能越来越好地“粘”在表面？我们能从这些新的界面中学到什么？我们如何利用这些信息来发现可以通过与这些位点紧密结合而在细胞中起作用的新化合物？为了研究蛋白质界面，我将首先使用蛋白质工程，这是一种通过改变氨基酸（蛋白质的组成部分）从一种类型到另一种类型来产生蛋白质突变的技术。我将以一种确定的方式进行突变，用越来越小的氨基酸取代蛋白质界面上的大氨基酸，从而产生越来越大的空腔。结合在这些工程口袋上的小分子的位置和强度将被确定。这将为寻找其他可能在生物体中起作用的蛋白质界面提供有用的信息，这些界面可能有可能被小分子破坏。其次，我将进一步利用这些知识来促进通过破坏蛋白质界面起作用的小分子的鉴定。我将开发新的方法来检测结合在界面相邻位置的小分子。为此，我将使用核磁共振（NMR）光谱学，这种技术可以监测小分子的氢原子，并可以报告这些氢原子是否与彼此靠近的蛋白质结合。我还将制作蛋白质的晶体，其中的界面可以被小分子接触到，并让这些分子在蛋白质表面紧密结合时一起反应。由于与蛋白质结合的小分子的结构可以直接通过向蛋白质晶体发射x射线来确定，这是一种称为x射线晶体学的技术，这是一种快速识别任何成功组装在蛋白质界面上的化合物的方法。这项研究的重要性和令人兴奋的原因如下：1。蛋白质的界面往往相对平坦，没有特征，因为它们不是自然地“进化”来结合小分子的。因此，利用小分子调节蛋白质-蛋白质相互作用是一项具有挑战性的任务，并且处于分子识别的前沿。2. 一个新的科学视野是通过在细胞内以选择性的方式破坏通路和网络来提高我们对生物系统的理解。由于蛋白质-蛋白质相互作用在细胞内广泛发生，使用小分子对它们进行调节提供了一个询问和发现新生物学的机会。3. 蛋白质-蛋白质复合物的破坏为开发新药提供了一种新颖而通用的机制。总之，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利益，并在与疾病的斗争中带来令人兴奋的机会。

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.