Molecular Mechanisms in Photolyase and Cryptochrome

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。智力优势：光裂合酶/隐花色素家族中的蓝光光感受器都结合黄素腺嘌呤二核苷酸（FAD），需要吸收蓝光光子，这是启动其生理功能所需的一个步骤。 光解酶修复通过暴露于紫外线在DNA中形成的环丁烷嘧啶二聚体（CPD），隐花色素参与调节动物的昼夜循环沿着植物的幼苗发育和茎生长。 隐花色素DASH（cryDASH）亚家族的成员能够修复CPD损伤，但与光解酶不同，它们仅修复单链DNA。 所有的光解酶和隐花色素都含有三个高度保守的FAD辅因子光还原所必需的FAD蛋白。 虽然光还原在DNA修复中可能不起直接作用，但它被认为是隐花色素信号功能的关键步骤，本研究将表征蛋白质-FAD和蛋白质-DNA相互作用对光裂合酶和cryDASH的物理和化学性质的影响，并阐明它们在CPD结合和修复能力方面的差异。 它将揭示这些酶中新的结构-功能关系，从而详细了解它们利用光能转移电子来修复DNA的分子机制。 该项目还将确定在光解酶和cryDASH中色氨酸光还原FAD后蛋白质电子转移（PET）的机制，并测试拟议的质子耦合电子转移（PCET）模型。 阐明色氨酸自由基在PET和PCET中的光解酶和cryDASH中的作用将有助于更深入地了解它们在隐花色素和PET和PCET中的信号传导中的作用和功能。 PCET是非常及时的兴趣，和光解酶和cryDASH是两个自然系统，允许详细研究这一重要的生物过程。 稳态和时间分辨吸收和共振拉曼光谱的组合将被使用，和黄素和色氨酸自由基的振动正常模式将获得同位素标记和计算化学。 这些任务是必要的，与所观察到的分子振动的结构变化，因为没有任务是目前可用的，将是重要的贡献，永久的文献。 更广泛的影响：该项目提供时间分辨激光光谱学的研究培训和博士后助理和研究生的职业指导。 本科生研究对下一代科学家的发展至关重要，该项目为本科生提供了有意义的研究机会。 当地的高中生也将有机会通过大学的几个推广项目参与研究。 研究成果将通过研讨会、会议上的演讲和同行评审期刊上的出版物传播，而一些研究成果将在课堂讲座中用作例子。 更好地了解决定蓝光感光器中光能转化为电子转移的因素也将加深太阳能转化为化学能的知识基础。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Intellectual Merit: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. Broader Impact: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.