This project will probe the linkage between molecular structure, molecular motions and function in proteins that recognize and associate specific sites on DNA, in order to deduce general principles of site-specific protein-DNA recognition. Past research using EcoRI endonuclease as a model has suggested that when a protein binds to its "correct" site on DNA, both the protein and the DNA suffer losses of conformational-vibrational mobility, but that these changes are much less pronounced when the protein binds at an incorrect DNA site. If verified, this would provide deeper insight into how a protein chooses among DNA sites. This hypothesis cannot be readily tested since the incorrect protein-DNA complexes are presently inaccessible to crystallography or nuclear magnetic resonance methods in solution. Electron spin resonance (ESR) spectroscopy provides an exciting alternative route to assessing structural and dynamic differences between the various classes of complexes.
Intellectual Merit: Early results on a DNA-protein complex by pulsed ESR establish an entirely new methodology that can measure the solution structure and range of conformational states for complexes with different classes of DNA sites. Such comparative measurements can provide information on how microscopic differences in molecular motions and structure contribute to the macroscopic differences in affinity and catalytic rates. In order to better quantify differences among the complexes, ESR methods that exploit bound copper(II) ions and site-directed nitroxide spin labeling will be developed, and applied to EcoRI-DNA complexes. Preliminary evidence shows that copper(II) coordinates exclusively between His114 and a DNA phosphate in the EcoRI-DNA complex, thus providing a unique reference point. Copper(II) coordination prevents significant motion of the ion, so that the resulting ESR signals aid in deconvoluting protein flexibility from motions of the spin label. The specific scientific aims are to: (1) Establish a pulsed electron spin resonance (ESR) method to measure large metal-metal and metal-nitroxide distances in proteins. (2) Leverage metal-spin label measurements to determine point-to-point distances and the range of conformational states of the endonuclease in specific, miscognate and nonspecific DNA complexes. (3) Combine research with outreach to 4-year colleges, local high schools and undergraduates.
Broader Impact: Two to three undergraduate researchers and graduate students working on this project will be trained at the interdisciplinary interface between chemistry and biophysics. The PI will seek to recruit undergraduates from 4-year colleges by actively presenting research at those institutes. Research ideas and themes developed in the project will be used to modernize the undergraduate Physical Chemistry laboratory. This initiative is in line with the American Chemical Society's call for the incorporation of interdisciplinary and integrative trends of chemical research into undergraduate curriculum without the creation of new course work. An annual workshop will be organized for high-school teachers in the Pittsburgh area to orient them with this research. Software for the extraction of distances from metal-based ESR data will be disseminated. This grant supports ESR training for females.
We have developed experimental and analysis procedures to measure Cu2+-ion based interspin distances using Electron Spin Resonance spectroscopy. This is significant because the technology can be applied to probe structure-function relationships in many metalloproteins. The technique is applicable to large protein complexes and membrance proteins – the structural properties of many of these systems cannot be measured by traditional biophysical techniques like NMR or x-ray diffraction. We have applied this methodology to investigate the coordination of copper ions by a DNA-modifying enzyme, the restriction endonuclease EcoRI. These measurements, together with biochemical results, have revealed the presence of a previously unknown metal binding site in this protein. This novel binding site is distal from the active site which coordinates the catalytic cofactor, magnesium. We have uncovered atomic-level details of the mechanism by which the copper ion binding at the distal site inhibits DNA cleavage. These results lead to deeper insight into the relationship between DNA protein sequence and catalytic specificity for this class of enzymes. These fundamental results can ultimately inform us about basic processes in the cell. Five undergraduates (one woman) were trained in research over the last three years. These students went on to programs in Chemistry, M.D./Ph.D. and Education. The graduate education of four students (three women) was partly supported by funds from this NSF award. The graduate students and undergraduate students learned sophisticated spectroscopic techniques as well as analysis techniques. They have worked on the interdisciplinary interface between chemistry and biology. These students have acquired skillsets that range from programming to protein mutation and overexpression. The students and a postdoctoral fellow were successfully placed into leadership positions in academia and industry upon completion of their research. Workshops were organized to acquaint high school teachers in the tri-state area to the NSF supported research so as to provide them tools to motivate their students as well as generate modules for K-12 teaching. The research was also presented at several 4-year colleges to disseminate knowledge as well as to generate enthusiasm for graduate research. Programs for analyzing EPR data were created.
