Quantitative Studies of Intrinsically Disordered Protein Structure and Function

在这项研究项目中，将开发广泛的原理来描述高度灵活的蛋白质的结构，这将提供对其功能的预测性洞察。PI将高度柔性蛋白质的分子结构和功能联系起来的新模型将导致这些分子系统的常规定量表征。将实施一种新的实验方法来表征高度灵活的蛋白质所具有的结构，以及这些结构经常伴随着与其他大分子结合而发生的变化。因此，这项研究和相关的培训活动将产生对基本生物过程的分子水平的基本见解，这将使生物技术行业受益。该计划将培训初级科学家，利用物理科学的原理和定量规律，从生物化学中寻求对系统和过程的基本见解。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，向他们展示科学和工程领域的高等教育带来的机会。此外，PI还被任命为宾夕法尼亚州立大学两个项目的导师，这两个项目旨在将未被充分代表的少数族裔本科生带入研究实验室。通过这些计划，PI每年将至少有一名注册的学生进入他的实验室。由该项目支持的学员将继续在PI的指导下，为所有参与这些外展项目的学生提供日常指导，PI坚定地致力于为来自不同背景的学生扩大科学机会。该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)集合和结构-功能关系。挑战是找到一个最好地描述高度柔性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，目标是开发新的蛋白质构象和动力学模型，导致将结构生物学工具应用于国内流离失所者，假设这些境内流离失所者具有直接负责赋予其特定功能的天然结构。为国内流离失所者确定结构-功能关系需要拓宽为合作折叠系统开发的传统上狭窄的范例。该项目继续努力开发定量和有效的工具，通过碳检测的核磁共振光谱学，通过实验限制IDP结构和与其他生物分子的特定位置相互作用。传统上获得的(即1H-检测的)IDPs的核磁共振谱存在化学位移色散差的问题，使得它们在大多数情况下不适合高分辨率应用。研究表明，~(13)C-直接检测光谱一般适用于国内流离失所者的定量分析。在这一项目期间，将继续开发用于研究国内流离失所者的碳检测核磁共振方法。最近，蛋白质FCP1在其非结合状态下的完全原子化的结构系综已经被解决。该项目将进一步努力，包括FCP1与其折叠伙伴Rap74之间形成的复合体的结构特征，同时还定义RNA聚合酶II C-末端结构域的构象集合，该结构域是FCP1磷酸酶活性的生物目标。最后，有必要将结构性发现与功能结果联系起来。对于本项目中研究的模型系统，可以通过定义研究中的国内流离失所者与其合作折叠的伙伴之间的结合相互作用来分析生物功能。功能假设检验将主要通过定量的量热结合研究来进行，确定内在无序的能量后果，并确定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.