Dissecting the Rate Theory for Protein Folding Dynamics via Advanced Single-Molecule Fluorescence Experiments

蛋白质是生物纳米机器，通过自组装成特定结构并根据提示改变形状的能力来执行大多数细胞功能。控制蛋白质运作的机制决定了分子和细胞生物学的各个方面，也是开发预测，工程和设计生物功能的工具所必需的。这些机制的关键在于蛋白质折叠和展开的速率。然而，仍然没有一个统一的速率处理，可以：1）整合分析理论和分子模拟; 2）合理化在传统实验中观察到的令人困惑的简单速率模式; 3）直接用于分析现代单分子实验。这个项目将展示一种新的方法来实证剖析折叠率。它还将为该领域的许多悬而未决的问题提供答案，包括速率如何取决于各种物理因素和蛋白质折叠/结构，速率和机制之间的联系，以及这些机制的异质性。该项目利用加州大学默塞德（一所拥有超过60%第一代学生的西班牙裔服务机构）的特点，将有助于为各级STEM劳动力中代表性不足的少数民族开发一个强大的管道，并培养能够在学术界、政府实验室或工业界从事跨学科STEM职业的毕业生。理论和模拟预测与蛋白质折叠实验分析之间的有效桥梁是速率处理，该速率处理将速率描述为通过将能量景观投影到几个阶参数中获得的自由能表面上的扩散。为了有效地测试这种处理，实验者必须解决微观行为的分布，这些行为反映了速率的属性，这一壮举尚未完全实现。这个项目的目标是实验性地剖析蛋白质折叠-展开转变的速率。这将使用一种方法来完成，该方法旨在跟踪单个蛋白质分子沿其反应性转变路径沿着经历的随机变化，从而解决关键速率项：屏障、扩散系数和威尔斯和屏障顶部的曲率。该方法结合了先进的单分子荧光实验，光子轨迹的最大似然分析和扩散动力学模型。这种方法将适用于快速折叠蛋白质的代表三个基本的原型折叠。该实验将直接测试蛋白质折叠速率理论，与分子模拟相结合，并促进如何解释实验速率的范式转变。该奖项反映了NSF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 are biological nanomachines that perform most cellular functions through their ability to self-assemble into specific structures and change shape on cue. The mechanisms that govern protein operation determine every aspect of molecular and cell biology and are also essential for developing tools to predict, engineer, and design biological function. The keys to these mechanisms are in the rates at which proteins fold and unfold. However, there still is not a unified rate treatment that can: 1) integrate analytical theory and molecular simulations; 2) rationalize the puzzlingly simple rate patterns observed in conventional experiments; and 3) be directly used to analyze modern single-molecule experiments. This project will demonstrate a novel approach to empirically dissect folding rates. It will also provide answers to many outstanding issues in the field, including how the rates depend on various physical factors and protein fold/structure, the connections between rate and mechanism, and how heterogeneous these mechanisms truly are. Capitalizing on the special characteristics of the University of California Merced, a Hispanic serving institution with over 60% of first-generation students, the project will help develop a robust pipeline for underrepresented minorities in the STEM workforce at all levels and produce graduates that can pursue cross-disciplinary STEM careers in academia, government laboratories or industry. The effective bridge between predictions of theory and simulations and the analysis of experiments in protein folding is a rate treatment that describes rates as diffusion on the free energy surface obtained by projecting the energy landscape into a few order parameters. To effectively test this treatment, experimentalists must resolve the distribution of microscopic behaviors that inform on the rate’s properties, a feat that has not been yet fully achieved. The goal of this project is to experimentally dissect the rates of protein folding-unfolding transitions. This will be done using an approach designed to track the stochastic changes that individual protein molecules undergo along their reactive transition paths, and hence resolve the key rate terms: barrier, diffusion coefficient, and curvature of wells and barrier top. The approach combines advanced single-molecule fluorescence experiments, maximum likelihood analysis of photon trajectories, and diffusive kinetic models. This approach will be applied to fast-folding proteins representatives of three basic archetypal folds. The experiments will directly test protein folding rate theory, connect with molecular simulations, and promote a paradigm shift in how experimental rates are interpreted.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.