Measuring Molecular Electric Fields at the Active Site of a Protein using Single Molecule and Hole-Burning Techniques

在这个由实验物理化学计划资助的项目中，威斯康星大学密尔沃基分校的Geissinger和Woehl教授将开发方法，以分子和原子分辨率定量测定蛋白质活性部位的内部电场。 测量这些由蛋白质电荷分布本身产生的场的方法是基于高分辨率光谱技术（特别是在低温下的单分子光谱和烧孔光谱），结合量子力学数据分析和蛋白质的静电模型计算。 实验工作将集中在两个密切相关的模型系统，肌红蛋白和血红蛋白。 预计在分子甚至原子水平上的内部电场的大小和方向的可用性将在这些系统中的配体歧视的基本问题上揭示新的光，因为连续介质方法无法最终建立静电结构和功能之间的联系。 促进对肌红蛋白和血红蛋白的生理功能负责的因素的理解和发现对于许多生物技术应用是重要的，例如设计有效的血液替代品或有效地从食物或其他氧敏感产品中去除氧的物质。 更一般地，将开发的用于从光谱数据提取内部电场值的方法预期容易适用于含有一个或多个卟啉分子的任何生物系统。 此外，提供实验访问内部电场将允许调查的问题，是否生物系统是由自然设计的，以优化这些系统的某些功能部位的内部电场的目标，烧孔和单分子光谱从相同的系统的可用性将构成教学和学习的宝贵资源。 特别是单分子研究提供了优秀和独特的教育资源，用于展示个体分子参数如何在宏观尺度上导致物质的某些行为。 教学上的好处是，抽象的数学公式，如统计热力学的分布函数，可以引入作为一个非常具体和详细的实验知识的个别分子的属性的直接后果，从而提高学生的学习。 因此，这个项目的结果将成为物理研究所在本科和研究生阶段物理化学课程教学的一个组成部分。 此外，这两个PI将为本科生和访问高中生提供机会，让他们参与这些领域的研究活动，例如，通过国家支持的UROP（本科生研究机会）和向上拓展计划。这些课程为本科生和高中生提供研究机会。

In this project funded by the Experimental Physical Chemistry Program, Professors Geissinger and Woehl of the University of Wisconsin-Milwaukee will develop methods to determine quantitatively internal electric fields at the active sites of proteins at molecular and atomic resolution. The approach to measuring these fields, which are generated by the protein charge distributions themselves, is based on high-resolution spectroscopic techniques (in particular single-molecule spectroscopy and hole-burning spectroscopy at cryogenic temperatures) combined with quantum-mechanical data analysis and electrostatic model calculations of proteins. Experimental work will focus on two closely related model systems, myoglobin and hemoglobin. It is expected that the availability of magnitude and direction of internal electric fields at a molecular or even at an atomic level will shed new light on the fundamental issue of ligand discrimination in these systems, because continuum dielectric approaches are unable to conclusively establish the link between electrostatic structure and function. Advancing understanding and discovery of the factors that are responsible for the physiological functions of myoglobin and hemoglobin is important for a number of biotechnological applications, such as the design of efficient blood substitutes or of substances that efficiently remove oxygen from foods or other oxygen-sensitive products. More generally, the methods that will be developed for the extraction of internal electric field values from spectroscopic data are expected to be readily adaptable to any biological system that contains one or more porphyrin molecules. In addition, providing experimental access to internal electric fields will allow for investigating the question whether biological systems were designed by nature with the goal of optimizing internal electric fields at certain functional sites of these systems.The availability of hole-burning and single-molecule spectra from the same systems will constitute a valuable resource for teaching and learning. Single molecule studies in particular provide excellent and unique educational resources for demonstrating how individual, molecular parameters lead to certain behavior of matter on the macroscopic scale. The pedagogical benefit is that abstract mathematical formulas such as distribution functions of statistical thermodynamics can be introduced as a direct consequence of very concrete and detailed experimental knowledge about properties of individual molecules, thereby improving student learning. Thus, the results of this project will form an integral part for the PIs' teaching of Physical Chemistry courses at both the undergraduate and graduate levels. Moreover, both PIs will provide opportunities for undergraduate and visiting high school students to participate in research activities in these areas, for example, through the state-supported UROP (Undergraduate Research Opportunities) and the Upward Bound program. These programs provide research opportunities for undergraduate and high-school students.