Molecular Mechanisms of Spontaneous and Hsp 70-assisted Renaturation of Misfolded Proteins

蛋白质在活细胞中发挥着许多基本功能。它们是折叠成独特的3D结构以支持特定蛋白质功能的聚合物。在非理想的情况下，如热休克，结构可能会变性，在这种情况下，蛋白质不能发挥功能，甚至对细胞有毒。所有的细胞都进化出了一种机制，在称为伴侣的酶的帮助下纠正变性蛋白质的结构。一个重要的分子伴侣系统是Hsp 70，Hsp 70分子伴侣重折叠和拯救变性蛋白的机制仍不清楚。了解Hsp 70机制具有根本意义，因为伴侣蛋白的失效使细胞容易受到环境变化的影响，并与老年痴呆症或帕金森氏症等与年龄相关的疾病有关。该项目的目标是通过涉及原子力显微镜和低温电子显微镜的新研究策略来解决Hsp 70机制，这些研究策略允许检查单个蛋白质和伴侣分子的行为。此外，计算机建模将用于检查变性蛋白质和它们的救援分子伴侣之间的相互作用，以确定分子伴侣的关键机制作用。该项目将促进对负责保护蛋白质结构和功能的机制的理解，并将有助于培养研究生和本科生，培养一支接受分子生物物理学教育的新的科学家队伍。该项目还将拓宽公众对负责生命自我保存的分子机制的基本知识。该项目将利用蛋白质工程与基于原子力显微镜（AFM）的单分子力谱（SMFS）、单粒子低温电子显微镜（SP-cryo-EM）和计算建模的组合。重点将是阐明错误折叠的蛋白质的自发重折叠途径和重折叠途径操纵机械力和分子伴侣之间的差异。SMFS测量将检查蛋白质的错误折叠倾向，并将捕获自发重折叠中间体，其将通过SP-cryo-EM进一步表征。通过AFM模拟热休克蛋白70的“解折叠酶作用”来验证这一假说。这些测量将确定解折叠酶的作用是否是自发重折叠所必需和充分的。目前未知的Hsp 70和其底物之间的力将被直接测量，并且它们的大小将与计算预测进行比较，以评估Hsp 70的当前“动力冲程”模型，其中蛋白质机械地夹在其底物上以去除非天然接触。一个新的替代模型提出热休克蛋白70行动重置错误折叠的蛋白质的结构，以他们的“新生链状形式”将被测试。这个奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

Proteins play numerous fundamental functions within a living cell. They are polymers that fold into unique 3D structures to support specific protein functions. Under non-ideal circumstances such as heat shock, the structures may become denatured, in which case proteins cannot function or even become toxic to cells. All cells have evolved mechanisms to correct the structures of denatured proteins with the help of enzymes termed chaperones. One important chaperone system is Hsp70, and the mechanism by which Hsp70 chaperones refold and rescue denatured proteins remains unclear. Understanding the Hsp70 mechanism is of fundamental significance, as failure of chaperones makes cells vulnerable to changes in the environment and is associated with age-related diseases such as Alzheimer's or Parkinson's. The goal of this project is to resolve the Hsp70 mechanism by novel research strategies involving Atomic Force Microscopy and cryogenic electron microscopy that allow examination of the behavior of individual protein and chaperone molecules. In addition, computer modeling will be used to examine interactions between denatured proteins and their rescue chaperones to identify key mechanistic actions of chaperones. This project will advance understanding of the mechanisms responsible for preserving protein structure and function, and will contribute to training graduate and undergraduate students, developing a new cadre of scientists educated in molecular biophysics. The project will also broaden basic knowledge among the general public about molecular mechanisms responsible for life self-preservation.The project will exploit a combination of protein engineering with Atomic Force Microscopy (AFM)-based single molecule force spectroscopy (SMFS), single-particle cryogenic electron microscopy (SP-cryo-EM) and computational modeling. The focus will be on elucidating the differences between spontaneous refolding pathways of misfolded proteins and refolding pathways steered by mechanical forces and by chaperones. SMFS measurements will examine proteins' propensity to misfold and will capture spontaneous refolding intermediates, which will be further characterized by SP-cryo-EM. The hypothesis about the "unfoldase action" of Hsp70 will be tested by emulating this action with AFM. These measurements will determine whether the unfoldase action is necessary and sufficient for spontaneous refolding. The forces between Hsp70 and its substrates, which are currently unknown, will be directly measured and their magnitude will be compared with computational predictions in order to evaluate the current "power stroke" model of Hsp70, in which the protein mechanically clamps on its substrate to remove non-native contacts. A new alternative model proposing that Hsp70 action resets the structure of misfolded proteins to their "nascent chain-like form" will be tested.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.