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

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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.