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。它们是可以折叠成独特的3D结构的聚合物，以支持特定的蛋白质功能。在热休克等非理想环境下，这些结构可能会变性，在这种情况下，蛋白质无法发挥功能，甚至对细胞产生毒性。所有细胞都进化出了在称为伴侣的酶的帮助下纠正变性蛋白质结构的机制。Hsp70是一个重要的伴侣系统，其折叠和挽救变性蛋白的机制尚不清楚。了解Hsp70机制具有重要意义，因为伴侣蛋白的失效使细胞容易受到环境变化的影响，并与阿尔茨海默氏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原子力显微镜和低温电子显微镜等新的研究策略来解决Hsp70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，计算机模拟将被用来检查变性蛋白质和它们的救援伴侣之间的相互作用，以确定伴侣的关键机械作用。这个项目将促进对负责保存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，培养受过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关于生命自我保护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)和计算建模的结合。重点将集中在阐明错误折叠的蛋白质的自发折叠途径与机械力和伴侣引导的复性途径之间的差异。SMFS测量将检查蛋白质错误折叠的倾向，并将捕获自发折叠中间产物，这将由SP-CRYO-EM进一步表征。关于Hsp70“展开酶作用”的假说将通过用原子力显微镜模拟这一作用来检验。这些测量将确定去折叠作用是否对自发折叠是必要的和充分的。Hsp70和其底物之间的力目前尚不清楚，将直接测量其大小，并将其大小与计算预测进行比较，以评估目前Hsp70的“动力冲程”模型，在该模型中，蛋白质机械地夹在其底物上，以消除非天然接触。将测试一种新的替代模型，该模型提出Hsp70行动将错误折叠的蛋白质的结构重置为其“新生的链状形式”。这一裁决反映了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 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.