This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
The blue-light photoreceptors in the photolyase/cryptochrome family all bind a flavin adenine dinucleotide (FAD) and need to absorb a blue-light photon, a step required to initiate their physiological function. Photolyases repair cyclobutane pyrimidine dimers (CPD) that form in DNA through exposure to ultraviolet light, and cryptochromes are involved in regulation of the day-night cycle in animals along with seedling development and stem growth in plants. Members of the cryptochrome-DASH (cryDASH) subfamily are capable of repairing CPD lesions, but unlike photolyases, they only repair single-stranded DNA. All photolyases and cryptochromes contain three highly conserved tryptophans that are essential for photoreduction of the FAD cofactor. Although photoreduction may not play a direct role in DNA repair, it is considered a critical step in the signaling function of cryptochromes.
This research will characterize the effect of protein-FAD and protein-DNA interactions on the physical and chemical properties of photolyase and cryDASH and elucidate the differences in their CPD binding and repair capabilities. It will reveal new structure-function relationships in these enzymes that will provide detailed insight into the molecular mechanism by which they use light energy to transfer an electron to repair DNA. This project will also determine the mechanism of protein electron transfer (PET) following photoreduction of FAD by tryptophan in photolyase and cryDASH and test a proposed proton-coupled electron-transfer (PCET) model. The elucidation of the role of tryptophan radicals in PET and PCET in photolyase and cryDASH will contribute to a deeper understanding of their role and function in signaling in cryptochromes and in PET and PCET, in general. PCET is of great timely interest, and photolyase and cryDASH are two natural systems that allow for a detailed study of this important biological process. A combination of steady-state and time-resolved absorption and resonance Raman spectroscopy will be used, and the vibrational normal modes of flavin and tryptophan radicals will be obtained from isotopic labeling and computational chemistry. These assignments are necessary to relate the observed molecular vibrations to structural changes and, since no assignments are currently available, will be important contributions to the permanent literature.
The project provides research training in time-resolved laser spectroscopy and career mentoring for a postdoctoral associate and a graduate student. Undergraduate research is of great importance for the development of the next generation of scientists, and the project provides meaningful research opportunities for undergraduate students. Local high-school students will also have the opportunity to participate in the research through several outreach programs at the university. The outcome of the research will be disseminated through seminars, presentations at meetings, and publications in peer-reviewed journals, while some of the research will be used as examples during lectures in the classroom. A better understanding of the factors that determine the conversion of light energy into electron transfer in blue-light photoreceptors will also deepen the knowledge base for the conversion of solar energy into chemical energy.
|Effective start/end date||1/08/09 → 31/07/13|
- National Science Foundation: $419,453.00