Quantitative Studies of Intrinsically Disordered Protein Structure and Function

在这个研究项目中，将开发广泛的原则来描述高度灵活的蛋白质的结构，这将为它们的功能提供预测性的见解。PI的新模型连接分子结构和功能的高度灵活的蛋白质将导致这些分子系统的常规定量表征。一种新的实验方法将实施原子分辨率表征的结构所拥有的高度灵活的蛋白质和变化，这些结构往往伴随着结合到其他大分子。因此，这项研究和相关的培训活动将产生基本的，分子水平的见解，将有利于生物技术产业的基本生物过程。该计划将培养初级科学家，以寻求从生物化学系统和过程的基本见解，使用物理科学的原理和定量定律。在该项目的每一年，PI将把来自弱势背景的中学生带到宾夕法尼亚州立大学，以向他们说明科学和工程领域的高等教育所带来的机会。此外，PI还被任命为宾夕法尼亚州立大学两个项目的导师，这些项目旨在将代表性不足的少数民族本科生带入研究实验室。通过这些项目，PI每年将至少有一名注册学生进入他的实验室。本项目的研究目标是在原子和分子尺度上定量描述内在无序蛋白质（IDP）集合体和结构-功能关系。本项目的研究目标是在原子和分子尺度上定量描述内在无序蛋白质（IDP）集合体和结构-功能关系。挑战在于找到一种最好地描述高度灵活的生物分子的构象特征的模型，因为这种结构描述与细胞功能密切相关。从更广泛的角度来看，其目标是开发新的蛋白质构象和动力学模型，导致结构生物学工具的应用程序，以国内流离失所者，这是假设拥有天然结构，直接负责赋予其特定的功能。定义结构-功能关系的IDP需要扩大传统的狭隘的合作折叠系统开发的范例。该项目继续努力开发定量和有效的工具，通过碳检测核磁共振光谱实验限制IDP结构和与其他生物分子的位点特异性相互作用。传统上获得的（即，1H-检测的）NMR光谱的IDP遭受差的化学位移分散，使他们不适合在大多数情况下的高分辨率应用。已经表明，13 C-直接检测光谱法通常产生适合于IDP定量分析的光谱。在本项目执行过程中，将继续开发用于研究国内流离失所者的碳探测核磁共振方法。最近，一个完全原子结构合奏的蛋白质FCP 1在其未结合状态已被解决。该项目将扩大努力，包括FCP 1及其折叠伴侣Rap 74之间形成的复合物的结构表征，同时还定义了RNA聚合酶II C-末端结构域的一个片段的构象集合，这是FCP 1磷酸酶活性的生物靶标。最后，有必要将结构调查结果与功能成果联系起来。对于本项目中研究的模型系统，生物功能可以通过定义所研究的IDP与其合作折叠伙伴之间的结合相互作用来测定。功能假设检验将主要通过定量量热结合研究进行，建立内在无序的能量后果，并定义无序蛋白质相互作用的驱动力。

In this research project broad principles wil be developed to describe the structure of highly flexible proteins, which will provide predictive insight into their function. The PI's new models connecting molecular structure and function for highly flexible proteins will lead to the routine quantitative characterization of these molecular systems. A new experimental methodology will be implemented for atomic resolution characterization of the structures possessed by highly flexible proteins and the changes to those structures that often accompany binding to other macromolecules. As a result, this research and associated training activities will yield fundamental, molecular level insights into essential biological processes that will benefit the biotechnology industry. This program will train junior scientists to seek fundamental insight into the systems and processes from biochemistry, using the principles and quantitative laws from the physical sciences. During each year of the project, the PI will bring middle school students from disadvantaged backgrounds to Penn State University in order to illustrate for them the opportunities that arise from higher education in science and engineering fields. Additionally, the PI has been named a mentor for two Penn State programs that aim to bring under-represented minority undergraduate students into the research laboratory. Through these programs, the PI will bring a minimum of one enrolled student per year of the project into his laboratory. Trainees supported by this project will continue to provide the daily mentorship of all students engaging in these outreach programs, with guidance from the PI, who is firmly committed to expanding opportunities in science for students from diverse backgrounds.The research objective of this project is to quantitatively describe intrinsically disordered protein (IDP) ensembles and structure-function relationships on the atomic and molecular scale. The challenge is to find a model that best describes the conformational features of highly flexible biomolecules, as this structural description is intimately connected with cellular function. From a broader point of view, the objective is to develop new models for protein conformation and dynamics that lead to the application of structural biological tools to IDPs, which are hypothesized to possess native structure that is directly responsible for imparting their specific functions. Defining structure-function relationships for IDPs requires broadening the traditionally narrow paradigm developed for cooperatively folding systems. This project continues efforts to develop quantitative and efficient tools for experimentally constraining IDP structure and site-specific interactions with other biomolecules through carbon-detected NMR spectroscopy. Traditionally acquired (i.e., 1H-detected) NMR spectra of IDPs suffer from poor chemical shift dispersion, rendering them unsuitable to high resolution applications in most cases. It has been shown that 13C-direct detection spectroscopy generally produces spectra suitable for quantitative analysis of IDPs. The development of carbon-detected NMR methods for the study of IDPs will continue to be developed during the course of this project. Recently a fully atomistic structural ensemble of the protein FCP1 in its unbound state has been resolved. This project will expand efforts to include structural characterization of the complex formed between FCP1 and its folded partner Rap74, while also defining the conformational ensemble of a segment from the RNA Polymerase II C-terminal domain, which is the biological target of the FCP1 phosphatase activity. Finally, it is necessary to connect structural findings to functional outcomes. For the model systems investigated in this project, biological function can be assayed by defining binding interactions between the IDPs under study and their cooperatively folded partners. Functional hypothesis testing will be primarily carried out through quantitative calorimetric binding studies, establishing the energetic consequences of intrinsic disorder and defining the driving forces for disordered protein interactions.