High Resolution Single Molecule Analysis of Fast Folding and its Coupling to Binding

蛋白质是细胞纳米机器，负责大多数生物功能，包括能量生产、DNA复制和转录、酶催化、信号传递、细胞支架和防御。但蛋白质也是一种天生的柔性聚合物，在由其氨基酸序列中编码的化学蓝图决定的自组装过程中，必须折叠成与其生物功能状态相对应的复杂的天然3D结构。因此，蛋白质折叠和功能的机制几乎是分子和细胞生物学各个方面的关键组成部分。了解折叠和功能的相互交织的机制也带来了预测、工程和设计生物功能的机会，从而向社会传达无与伦比的变革性影响。此外，由于蛋白质处于生物学复杂性的最低梯队，生物学有效地结合了物理、化学和工程，他们的研究构成了培养新一代多学科研究人员的理想舞台，为他们进入新兴的定量和合成生物学领域做好准备。活动围绕以团队为基础的结构设计，旨在促进各级教育成员的融合，从博士后研究员和研究生到本科生和高中生/教师。该项目的一个同样重要的因素是坚定地致力于代表人数不足的群体参与研究。国际蛋白质学会将从现有的几个针对STEM领域未被充分代表的少数民族的研究指导计划中积极招募项目成员，以参与该项目的研究活动。现代蛋白质研究的一个主要动力是开发实验方法，以解决先进理论和原子分子模拟预测的无数途径和复杂机制。在实验中检测这种固有的动力学复杂性仍然是难以捉摸的，即使用现代方法表现出更好的时间、结构或单分子分辨率。该项目的总体目标是通过实验监测单个蛋白质分子折叠时的过渡路径来弥合这一差距。先进的单分子荧光方法将结合理论和计算分析来测量快速折叠蛋白质结构域的过渡路径和折叠机制。快速折叠结构域是最理想的目标，因为它们的边际协作性确保了大量的“激发”态，而且有些违反直觉的是，在它们宽广而浅的折叠障碍上，较慢的过渡路径。此外，这些区域的微秒级折叠便于与现代原子模拟进行直接比较。为了达到所需的分辨率，我们依赖于我们最近开发的实现微秒分辨率单分子荧光检测的方法，例如更好的光保护系统和光子到达时间的最大似然分析程序，以及实施2色和3色FRET方案来测量多个距离。通过实现这些实验和对选定的快速折叠结构域的计算分析，我们将研究折叠机制的结构、顺序和环境决定因素。该项目由分子和细胞生物科学系的分子生物物理组和物理系的生命系统物理学项目共同资助。

Proteins are the cellular nanomachines in charge of most biological functions, including energy production, DNA replication and transcription, enzymatic catalysis, signaling, cellular scaffolding and defense. But proteins are also inherently flexible polymers that must fold into the complex native 3D structures corresponding to their biologically functional states in a self-assembly process determined by the chemical blueprints encoded in their amino acid sequence. Thus the mechanisms by which proteins fold and function are a critical component of almost every aspect of molecular and cell biology. Understanding the intertwined mechanisms of folding and function also brings about the opportunity to predict, engineer, and design biological function "a la carte", thus conveying unparalleled transformative impact to Society. Moreover, because proteins are at the lowest echelon of biological complexity where Biology effectively meets Physics, Chemistry, and Engineering, their study constitutes an ideal arena for training the new generations of multidisciplinary researchers, preparing them for the emerging fields of Quantitative and Synthetic Biology. Activities are designed around a team-based structure aimed at facilitating integration of members at various levels of education ranging from postdoctoral fellows and graduate students to undergraduate and high school students/teachers. An equally important element of this project is the strong commitment to participation of underrepresented groups in research. The PI will actively recruit project members from several existing research mentoring programs for underrepresented minorities in STEM fields to participate in the research activities of this project.A major drive for modern protein research has been to develop experimental methods to resolve the myriads of pathways and complex mechanisms that are predicted by advanced theory and atomistic molecular simulations. Detecting such inherent kinetic complexity in experiments has remained elusive, even with modern methods that exhibit improved time, structural, or single-molecule resolution. The overall objective of this project is to bridge this gap by experimentally monitoring the transition paths of individual protein molecules as they fold. Advanced single-molecule fluorescence methods will be used in conjunction with theoretical and computational analyses to measure transition paths and folding mechanisms of fast-folding protein domains. Fast-folding domains are optimal targets because their marginal cooperativity ensures significant populations of "excited" states and, somewhat counterintuitively, slower transition paths over their broad, shallow folding barriers. Moreover, the microsecond folding of these domains facilitates direct comparison with modern atomistic simulations. To reach the required resolution, we rely on approaches we recently developed for achieving microsecond resolution single-molecule fluorescence detection, such as better photoprotection systems and procedures for maximum likelihood analysis of photon arrival times, together with implementation of 2-color and 3-color FRET schemes to measure multiple distances. Through the realization of these experiments and computational analyses on select fast-folding domains we will investigate the structural, sequence, and environmental determinants of the mechanisms for folding. This project is jointly funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences and the Physics of Living Systems Program in the Division of Physics.