Single Molecule Studies of Tau Conformations and Dynamics

随着越来越多的蛋白质被确定为本质上无序的--完全由或包含不存在明确定义的二级或三级结构的长条区域--对这些蛋白质的兴趣相应地增长。它们挑战了现代结构生物学的核心教条，即功能源于结构。在无序蛋白质中，了解生物功能是如何实现的是具有挑战性的，部分原因是它们具有内在的异质性，同时具有多个构象和物种，并且具有不同的寿命。与传统的系综方法相比，本研究中使用的单分子生物物理方法非常适合在广泛的溶液条件下表征非均相分子物种，并与相关的结合伙伴相关联。这个项目的重点是tau蛋白，这是一种微管结合蛋白，在溶液中完全无序，当功能结合到微管时只有部分结构。在使用单分子实验和计算方法的系统研究中，tau的功能片段--例如微管结合域--将独立于整个蛋白质链并在整个蛋白质链的背景下进行表征。这将允许精确地确定特定区域的构象和动态是如何协调的，因为对蛋白质一个区域的工程扰动将被分析其对远处区域功能的影响。此外，这里描述的研究可以作为设计其他内在无序蛋白质的研究的模型，并有助于阐明瞬时结构形成和长程相互作用如何在这组有趣的蛋白质中产生功能。PI积极致力于让本科生参与研究。这些研究项目是本科生研究的理想之选；几名本科生已经积极参与其中，其中一名学生来自一个针对少数族裔的暑期项目，另一名学生将在即将到来的一年里度过暑假，在PI的实验室完成她的高级论文。另一个组成部分是国际学生联合会为耶鲁大学理科研究生招收人数不足的学生所做的努力。除了在部门多样性委员会任职外，PI还出席了针对这些少数族裔学生的会议并在会上发言，包括年度少数族裔学生生物医学研究会议(ABRCMS)和耶鲁大学本科生物理女性会议。最后是一个多方面的计划，以改善对生物物理科学感兴趣的学生的跨学科培训机会。这包括开发和扩展一门课程，旨在向生物学本科生和研究生教授数学方法。本课程涵盖分析生物数据所需的分析和数值方法，重点是学习如何使用MatLab软件来解决和模拟复杂的生物学问题。随着这门课程的发展，PI参与了一个新的生物物理培训计划，该计划招募对跨学科研究感兴趣但缺乏生物学或物理学术培训的有才华的研究生。

As a growing number of proteins are identified as intrinsically disordered--consisting entirely of or containing long stretches of regions that do not populate well-defined secondary or tertiary structures--there is a corresponding growth in interest in these proteins. They challenge the central dogma in modern structural biology that function follows from structure. Gaining an understanding of how biological function is achieved is challenging in disordered proteins, in part due to the fact that they are inherently heterogeneous with multiple conformations and species populated simultaneously and with different lifetimes. In contrast to traditional ensemble methods, the single molecule biophysical approaches used in this research are uniquely suited to characterize heterogeneous molecular species in a wide range of solution conditions and associated with relevant binding partners. This project focuses on the protein tau, a microtubule binding protein that is entirely disordered in solution and only partly structured when functionally bound to microtubules. In a systematic study using single molecule experimental and computational approaches, functional fragments of tau--such as the microtubule-binding domain--will be characterized both independently of and in the context of the entire protein chain. This will allow for a precise determination how the conformations and dynamics of specific regions are coordinated, as engineered perturbations to one region of the protein will be assayed for their effects on the functionality of distant regions. Furthermore, the studies described here may serve as a model for designing studies of other intrinsically disordered proteins, and help elucidate how transient structure formation and long range interactions generate function in this intriguing group of proteins.There are a number of educational and outreach activities that are integral to the PI's research goals. The PI actively works to involve undergraduate students in research. The research projects are ideal for undergraduate research; several undergraduate students are already actively involved, including one from a summer program for underrepresented minorities, and another who will spend the summer and complete her senior thesis in the PI's lab in the upcoming year. Another component is the PI's effort in the recruitment of underrepresented students to graduate study in the sciences at Yale. In addition to serving on the departmental diversity committee, the PI has attended and spoken at conferences aimed at these minority students, including the Annual Biomedical Research Conference for Minority Students (ABRCMS) and the Conference for Undergraduate Women in Physics at Yale. Lastly is a multifaceted plan to improve interdisciplinary training opportunities for students interested in biophysical science. This includes the development and expansion of a course designed to teach mathematical methods to biology undergraduate and graduate students. The course covers analytical and numerical methods needed for analyzing biological data, with a strongemphasis on learning to use MATLAB software for solving and modeling complex biological problems. Synergistic with this course development, the PI is involved with a new training program in Biological Physics that recruits talented graduate students with an interest in interdisciplinary research, but lacking the academic training in biology or physics.