CAREER: Leaving the Fold: Leveraging Mass Spectrometry Proteomics to Shift the Paradigm on Protein Folding

蛋白质必须将自己折叠成特定的三维形状，才能执行细胞所需的任务。另一方面，当蛋白质折叠成错误的形状时，它们会破坏细胞的正常功能，并与包括阿尔茨海默氏症和帕金森病在内的多种疾病有关。蛋白质是如何“知道”如何折叠成正确的结构的（至少在大多数情况下）？简单的蛋白质通常具有这样的特性，即在试管中被拆解后，它们可以自发地重新折叠回其原始结构。这意味着它们的原生状态也是它们最稳定的形式，因此热力学自然有利于它们的形成。然而，迄今为止，大多数关于蛋白质折叠的研究都集中在少数简单的模型系统上，而忽略了细胞中有数千种不同的蛋白质这一事实，其中许多蛋白质比那些通常适合生物物理表征的蛋白质更大、更复杂。该项目的目的是应用现代质谱（MS）蛋白质组学方法-可以常规表征复杂混合物中的数千种蛋白质-来解决蛋白质折叠问题。这些工具将使人们能够探索许多种类的蛋白质，这些蛋白质的折叠从未被研究过。此外，由于大多数关于蛋白质折叠的概念和理论都是基于迄今为止“特权”的模型蛋白质子集，因此期望这项研究将“离开折叠”并改变当前关于蛋白质折叠的范式。该项目还将为科学界和劳动力中代表性不足的个人提供参与的机会。其中一项活动针对第一代本科生，并与研究计划紧密结合。第二项活动旨在满足巴尔的摩地区向低收入社区引入更多STEM职业发展资源的迫切需求。将开发一个网络数据库，旨在增加生物物理学界对本研究项目产生的蛋白质组学数据的可访问性。随着时间的推移，由于蛋白质组学实验能够在统一的条件下量化大量蛋白质的折叠，该数据库可以承担蛋白质折叠的权威资源的作用。具体而言，本项目利用并扩展了结构蛋白质组学中的两种新兴方法，并寻求解决关于蛋白质折叠的两个深远问题。在方法上，该项目采用有限的蛋白质水解和交联，以便将蛋白质的结构信息（以及它们的折叠中间体和在重新折叠失败后的错误折叠形式）编码为裂解位点和交联，这些信息可以通过质谱法进行大规模测序。通过这些方法，本项目将探索热力学再折叠的极限。特别是，来自嗜热生物的蛋白质和古老的蛋白质可能比它们的中温生物和现存的同类更容易折叠。此外，伴侣蛋白有望帮助挽救无法自行折叠的蛋白质的再折叠。作为该项目的一部分进行的实验将通过使用有限的蛋白质水解质谱法来检查各种蛋白质的可折叠性，并在一系列伴侣系统的帮助下，明确地测试这些假设。其次，本研究旨在揭示不可折叠性的生物物理基础。当一个蛋白质无法再折叠时，它会呈现出什么样的结构，又是什么中间产物引导它走上这条不幸的道路？实验将通过使用交联质谱法来探测蛋白质组重折叠的结构动力学来解决这些问题。这些结果将为蛋白质自由能景观的拓扑结构提供新的见解，并使动力学捕获的错误折叠物种的表征成为可能。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Proteins must fold themselves up into specific three-dimensional shapes to perform tasks required by the cell. On the other hand, when proteins fold into the wrong shape, they can disrupt the normal functioning of the cell, and are associated with a wide range of maladies including Alzheimer's and Parkinson's diseases. How is it that proteins ‘know’ how to fold into the correct structures (at least, most of the time)? Simple proteins typically have the property that after being disassembled in a test tube, they can spontaneously refold back into their native structures. This implies that their native states are also their most stable forms, and therefore thermodynamics naturally favor their formation. However, most studies of protein folding to date focus on a small number of simple model systems and leave out the fact that cells have thousands of different proteins, many of which are bigger and more complicated than the ones that are typically amenable for biophysical characterization. The purpose of this project is to apply modern mass spectrometry (MS) proteomics methods – which can routinely characterize thousands of proteins in complex mixtures – to problems in protein folding. These tools will enable the exploration of many classes of proteins whose folding has never been interrogated. Moreover, since the majority of the concepts and theories about protein folding have been based on a heretofore “privileged” subset of model proteins, the expectation is that this research will “leave the fold” and shift current paradigms on protein folding. The project will also provide opportunities to engage individuals that are underrepresented in the scientific community and workforce. One of these activities targets first-generation undergraduates and integrates closely with the research plan. The second activity is designed to meet a pressing need in the Baltimore area to introduce more STEM career development resources to low-income communities. A web database aimed at increasing the accessibility of proteomic data generated by this research project to the biophysical community will be developed. Over time, this database could take on the role as the authoritative resource of protein folding due to the ability of proteomics experiments to quantify folding for large numbers of proteins under uniform conditions. Specifically, this project utilizes and expands two emerging approaches in structural proteomics and seeks to address two far-reaching questions about protein folding. Methodologically, the project employs limited proteolysis and crosslinking in order to encode structural information about proteins (as well as their folding intermediates and their misfolded forms following failed attempts at refolding) into cleavage sites and crosslinks, which can be sequenced en masse by mass spectrometry. With these methods, this project will explore the limits of thermodynamic refolding. In particular, proteins from thermophilic organisms and ancient proteins might be expected to be more refoldable than their mesophilic and extant peers. Moreover, chaperones are expected to help rescue the refolding of proteins that could not refold on their own. Experiments conducted as part of this project will explicitly test these hypotheses by using limited proteolysis mass spectrometry to examine the refoldability of a wide range of proteins and with the assistance of a range of several chaperone systems. Secondly, this research aims to uncover the biophysical basis of non-refoldability. When a protein fails to refold, what structure does it assume and what intermediates lead it down that ill-fated path? Experiments will address these questions by using crosslinking mass spectrometry to probe the structural dynamics of refolding proteomes. The results will provide new insight into the topologies of protein free energy landscapes and enable the characterization of kinetically-trapped misfolded species.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.