Photoinduced electron transfer in DNA photolyase

DNA光裂合酶中的光诱导电子转移

• 批准号: 0847855
• 负责人: Robert Stanley
• 金额: $ 46万
• 依托单位: Temple University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0847855&HistoricalAwards=false
• 关键词: Photoinduced; electron; transfer; DNA; photolyase

This award funded by the Experimental Physical Chemistry Program of the Chemistry Division supports research by Professor Robert Stanley from Temple University to study electron transfer-mediated repair of DNA. DNA is an extraordinarily stable molecule, a property required in its role as a genetic storage medium. In spite of this stability, DNA is constantly being damaged by a variety of environmental agents. Of these, ultraviolet (UV) light is among the most mutagenic, leading to a crosslinking of adjacent pyrimidines to generate Cyclobutylpyrimidine Dimers (CPDs). All organisms have a robust ability to repair CPDs. DNA photolyase (PL) is a protein that binds and repairs CPDs with exquisite specificity. Uniquely, photolyase uses visible light as an energy source. Once bound, the CPD is repaired in about two billionths of a second after the absorption of the catalytic photon. This photon is absorbed by the protein-bound Flavin Adenine Dinucleotide (FAD), a vitamin B2 derivative which transfers an electron to the CPD. This critical ultrafast electron transfer step is not well understood. Professor Stanley and his students will use state-of-the-art biophysical, biochemical, and molecular biology methods to determine the details of this electron transfer mechanism at the molecular level. In particular, fluorescence Stark spectroscopy will be utilized for the first time in the study of flavoproteins to discover the direction of the electron transfer, thereby revealing the identity of the initial electron acceptor. Mutants will be made to elucidate the role of amino acids around the flavin in the repair reaction. This approach, coupled with an analysis of the excited state electronic structure obtained using Stark spectroscopy will provide the clearest picture of how and why photolyase functions. More broadly, this application of Stark spectroscopy to PL will be directly applicable to other light-driven flavoproteins (e.g. blue light photoreceptors) which utilize different photochemical mechanisms but which, like PL, begin with light-driven charge redistribution in the flavin. Some of these proteins may be responsible for regulating our circadian clock or providing birds with the ability to migrate using the Earth's magnetic field for guidance (photomagnetoreception). Still other proteins are known to regulate photosynthesis at the gene level. Two collaborators join this effort. Professor David Beratan of Duke University will provide computational models to guide the interpretation of the experiments. Professor Yvonne Gindt and her group of undergraduate researchers at Lafayette College will perform electrochemical measurements on modified proteins to gauge the effect of chemical and mutational changes on the redox properties of the FAD. Postdoctoral and graduate students will gain a breadth of experience that is a hallmark of biophysical chemistry, learning techniques that include Stark and subpicosecond ultrafast laser spectroscopy, enzymology, molecular biology, and modern computational methods. Important experiments will be performed by a cadre of talented undergraduate students both at Temple and at Lafayette College in Easton, PA. These budding scientists will gain research experience by purifying the photolyase protein and performing chemical and molecular biological modifications to probe the true function of the FAD. All participants will present their results twice a year in a "mini-conference" setting, to take place alternately in Philadelphia and Easton and involving the Duke group through video-conferencing. The PI has mentored many young scientists, including women and members of underrepresented minorities. To reach a non-scientific audience about the importance of science to society, the PI has been closely involved in the TURF-Crews program at Temple for several years. This program mixes presentations by undergraduates from non-scientific fields with those by science majors to engender multidisciplinary interactions.

该奖项由化学部实验物理化学项目资助，支持天普大学Robert Stanley教授研究电子转移介导的DNA修复。DNA是一种非常稳定的分子，这是它作为遗传储存介质所必需的特性。尽管DNA具有这种稳定性，但它仍不断受到各种环境因素的破坏。其中，紫外线（UV）是最具诱变性的，导致相邻嘧啶交联生成环丁基嘧啶二聚体（CPDs）。所有生物都有强大的cpd修复能力。DNA光解酶（PL）是一种结合和修复cpd的蛋白质，具有精细的特异性。独特的是，光解酶使用可见光作为能量来源。一旦结合，CPD在吸收催化光子后大约在十亿分之一秒内修复。这个光子被蛋白质结合的黄素腺嘌呤二核苷酸（FAD）吸收，这是一种维生素B2衍生物，它将一个电子转移到CPD。这个关键的超快电子转移步骤还没有被很好地理解。Stanley教授和他的学生将使用最先进的生物物理学、生物化学和分子生物学方法，在分子水平上确定这种电子转移机制的细节。特别是，荧光斯塔克光谱将首次用于黄酮类蛋白的研究，发现电子转移的方向，从而揭示初始电子受体的身份。将制造突变体来阐明黄素周围氨基酸在修复反应中的作用。这种方法，加上使用斯塔克光谱获得的激发态电子结构分析，将提供光解酶如何以及为什么起作用的最清晰的图像。更广泛地说，Stark光谱学在PL中的应用将直接适用于其他利用不同光化学机制的光驱动黄素蛋白（例如蓝光光感受器），但与PL一样，它们都是从黄素中的光驱动电荷再分配开始的。其中一些蛋白质可能负责调节我们的生物钟，或者为鸟类提供利用地球磁场进行迁移的能力（光磁接收）。还有一些已知的蛋白质在基因水平上调节光合作用。两位合作者加入了这项工作。杜克大学的David Beratan教授将提供计算模型来指导对实验的解释。拉菲特学院的Yvonne Gindt教授和她的本科生研究小组将对修饰蛋白进行电化学测量，以测量化学和突变变化对FAD氧化还原特性的影响。博士后和研究生将获得广泛的经验，这是生物物理化学的标志，学习技术，包括斯塔克和亚皮秒超快激光光谱学，酶学，分子生物学和现代计算方法。重要的实验将由坦普尔大学和宾夕法尼亚州伊斯顿的拉斐特学院的一群有才华的本科生进行。这些崭露头角的科学家将通过纯化光解酶蛋白并进行化学和分子生物学修饰来探索FAD的真正功能，从而获得研究经验。所有参与者每年将在一个“小型会议”中展示他们的研究结果，该会议交替在费城和伊斯顿举行，杜克大学的研究小组将通过视频会议参与其中。PI指导了许多年轻科学家，包括女性和未被充分代表的少数民族成员。为了让非科学观众了解科学对社会的重要性，PI多年来一直密切参与天普大学的TURF-Crews项目。这个项目将来自非科学领域的本科生的演讲与来自科学专业的演讲混合在一起，以产生多学科的互动。