p53PRISM: Regulation of life-and-death decisions by conformational switches

P53蛋白通过协调癌前细胞的自我破坏来预防癌症。因此，在大多数癌症中，P53功能失常，允许细胞不受控制地生长，这是疾病的核心。因此，了解P53‘S功能的分子机制是至关重要的，研究人员现在知道，P53在一定程度上受到过多的翻译后修饰(PTM)的调节。在这个项目中，我将研究非典型PTM在P53中的功能后果：通过顺式和反式立体异构体(Pro-Iso)之间的Pro-Iso异构化来重新定位蛋白质的骨架。具体地说，我假设Pro-Iso起着“构象开关”的作用，驱动p53进入不同的功能状态。由于很难获得化学上定义明确的P53变异体，以及P53‘S对结构生物学技术的抵抗，这一假说尚未得到验证。为了克服这些挑战，我将通过跨学科的化学生物学和生物物理方法来探索P53’S功能中前Iso的作用。首先，我将使用我的导师曼努埃尔·穆勒博士(伦敦国王学院)开创的蛋白质半合成技术来生产带有精确化学修饰的全长p53变体，这些变体探测Pro-Iso的不同方面。其次，我将对这些变体进行一系列生化分析，以确定关键特性和功能的差异。最后，我将使用单分子Förster共振能谱来研究Pro-Iso对潜在的P53‘S分子相互作用的构象动力学的影响。这个项目将为我提供作为独立研究小组组长的职业生涯所需的可移植技能、培训和记录。更重要的是，这项研究将有助于进一步破译p53如何做出细胞生死决定，从而扩大全球利益相关者寻求解决老龄化世界中不断上升的癌症发病率的知识库。

The protein p53 guards against cancer by orchestrating the self-destruction of precancerous cells. Accordingly, p53 in most cancers ismalfunctioning and permits the unchecked cellular growth central to the disease. Understanding the molecular mechanismsunderlying p53's function is thus crucial, and researchers now know that p53 is regulated in part by a plethora of post-translationalmodifications (PTMs).In this project, I will investigate the functional consequences in p53 of an atypical PTM: the reorientation of a protein's backbone bythe isomerisation of a proline between cis and trans stereoisomers (Pro-Iso). Specifically, I hypothesise Pro-Iso acts as a"conformational switch" which drives p53 into distinct functional states. This hypothesis is untested due to the difficulty of obtainingchemically well-defined p53 variants and p53's recalcitrance towards structural biology techniques.To overcome these challenges, I will explore the role of Pro-Iso in p53's function via an interdisciplinary chemical biology andbiophysical approach. First, I will use the protein semisynthesis techniques pioneered by my supervisor Dr. Manuel Müller (King'sCollege London) to produce full-length p53 variants bearing precise chemical modifications that probe different aspects of Pro-Iso.Second, I will subject these variants to a host of biochemical assays to ascertain differences in key properties and functions. Finally, Iwill use single-molecule Förster resonance energy spectroscopy to investigate the effect of Pro-Iso on the conformational dynamicsunderlying p53's molecular interactions.This project will provide me with the transferable skills, training and track record needed to pursue a career as an independentresearch group leader. More importantly, this research will help further decode how p53 makes cellular life-and-death decisions andthus expand the knowledgebase from which global stakeholders seek to tackle growing cancer rates in an aging world.