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.
Intellectual Merit: Proteins are highly organized three-dimensional structures that perform specific biological functions such as photosynthesis, enzymatic activity, protein folding and assembly, or intra- and intercellular transport of molecules. Each atom of the constituting amino acids contains a positively charged nucleus (at a well-defined position within the protein) surrounded by a negatively charged electron cloud. The presence of these positive and negative charges leads to an innate electric field at the active site of a protein, which can be quite strong due to the small distances involved. In recent years, a new picture of protein function has emerged, pointing to such molecular electric fields as a significant contributor to their biological function. It appears sensible to postulate that proteins, which participate in virtually every process within living cells, evolved over billions of years to perfect their three-dimensional structure not only to enhance their chemical and mechanical properties, but also to generate electric fields at active sites, which assist with or enable specific biological functions. Despite their importance for understanding protein function, obtaining detailed information about molecular electric fields from experiments has remained challenging. In this project, we have used high-resolution laser spectroscopy techniques to study the effects of (small) externally applied electric fields on the model protein myoglobin (responsible for oxygen transport to and stroage in the muscles) and developed a general approach for analyzing the experimental data in order to extract quantitative information about the electric field generated by myoglobin itself. The effect of the (molecular and applied) electric field was observed by substituting the heme cofactor in myoglobin with its fluorescent, iron-free analog protoporphyrin IX, and monitoring the energy shift of the lowest electronic transition as the externally applied field was increased. The resulting experimental spectra were analyzed using a rigorous, quantum-mechanical approach that is easily generalized for other heme- and non-heme containing proteins. We have shown that the magnitude and (in-plane) orientation of the molecular electric field can be determined using protoporphyrin IX as a "molecular antenna", and that the exact numerical values remain relatively stable once a sufficient number of electronic states are included in the quantum-mechanical analysis. A general protocol for determining the number and order of important electronic states for inclusion in the analysis has been developed and tested, and is easily transferable to other protein systems of interest. Broader Impacts: The three graduate students and multiple undergraduate and high school students involved in this research project have gained a breadth of experience in experimental and theoretical biophysical chemistry, learning techniques that include fluorescence excitation laser spectroscopy, high-resolution optical microscopy, cryogenic techniques, molecular biology, modern quantum chemistry, data acquisition programming, and the building and interfacing of scientific instrumentation (such as a confocal laser scanning optical microscope operating at 1.4 degrees Kelvin above absolute zero). It is the experience of the PIs that research in biological physics greatly stimulates interest for science in students, and provides a perfect vehicle for sharing the excitement of scientific discovery with undergraduate and high school students. Both PIs have provided opportunities for undergraduate and visiting high school students to participate in research activities related to these areas, thereby stimulating scientific curiosity and fostering excitement about next-generation scientific techniques. In addition, one of the PIs and a graduate student have mentored a team of high school students who have created a 3D printed myoglobin model to investigate the effect of molecular electric fields in the framework of the NSF-funded SMART Team program.
