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，才能完成细胞所需的任务。另一方面，当蛋白质折叠成错误的形状时，它们可能会扰乱细胞的正常功能，并与包括阿尔茨海默氏症和帕金森氏症在内的一系列疾病有关。蛋白质是如何“知道”如何折叠成正确的结构的(至少，大多数时候是这样)？简单蛋白质通常具有这样的特性，即在试管中分解后，它们可以自发地重新折叠成它们的天然结构。这意味着它们的本征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，到目前为止，大多数关于蛋白质折叠的研究都集中在少数几个简单的模型系统上，而忽略了这样一个事实，即细胞有数千种不同的蛋白质，其中许多比通常适合生物物理表征的蛋白质更大、更复杂。这个项目的目的是应用现代质谱学(MS)蛋白质组学方法--它可以常规地表征复杂混合物中的数千种蛋白质--来解决蛋白质折叠的问题。这些工具将使人们能够探索许多类别的蛋白质，它们的折叠从未被询问过。此外，由于大多数关于蛋白质折叠的概念和理论都是基于到目前为止模型蛋白质的“特权”子集，人们期望这项研究将“离开折叠”，并改变目前关于蛋白质折叠的范式。该项目还将提供机会，让科学界和劳动力中代表性不足的个人参与进来。其中一项活动针对的是第一代本科生，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介绍更多STEM职业发展资源的迫切需要。将开发一个网络数据库，目的是增加生物物理界对这一研究项目产生的蛋白质组数据的可及性。随着时间的推移，这个数据库可能会成为蛋白质折叠的权威资源，因为蛋白质组学实验能够量化大量蛋白质在统一条件下的折叠。具体地说，该项目利用并扩展了结构蛋白质组学中的两种新兴方法，并试图解决关于蛋白质折叠的两个影响深远的问题。在方法上，该项目采用有限的蛋白质分解和交联，以便将有关蛋白质的结构信息(以及它们的折叠中间体及其在复性尝试失败后错误折叠的形式)编码到裂解位点和交联物中，可以通过质谱仪对其进行集体测序。通过这些方法，这个项目将探索热力学折叠的极限。特别是，来自嗜热有机体和古代蛋白质的蛋白质可能比它们的中温和现存的同类蛋白质更具折叠能力。此外，伴侣有望帮助挽救那些不能自行折叠的蛋白质的重新折叠。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分解质谱学来检查各种蛋白质的可复性，并在一系列伴侣系统的帮助下，明确地检验这些假设。其次，本研究旨在揭示不可折叠的生物物理基础。当蛋白质不能重新折叠时，它采用了什么结构，什么中间体引导它走上了这条命运多舛的道路？实验将通过使用交联质谱来探索折叠蛋白质组的结构动力学来解决这些问题。这一结果将为蛋白质自由能景观的拓扑结构提供新的洞察力，并能够描述动力学陷阱错误折叠物种的特征。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.