Photoinduced electron transfer in DNA photolyase

该奖项由化学系实验物理化学项目资助，支持坦普尔大学罗伯特·斯坦利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，这是其作为遗传存储介质所必需的特性。尽管这种稳定性，DNA不断地受到各种环境因素的破坏。其中，紫外线(UV)是最具诱变性的光之一，导致相邻的嘧啶类化合物发生交联，生成环丁基嘧啶二聚体(CPD)。所有生物体都有很强的修复CPD的能力。DNA光解酶(PL)是一种结合和修复CPD的蛋白质，具有极高的特异性。独特的是，光解酶利用可见光作为能源。一旦被结合，CPD在催化光子吸收后大约20亿分之一秒内被修复。这种光子被蛋白质结合的黄素腺嘌呤二核苷酸(FAD)吸收，FAD是一种维生素B2的衍生物，它将一个电子传递到CPD。这一关键的超快电子转移步骤还没有被很好地理解。斯坦利教授和他的学生将使用最先进的生物物理、生化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素蛋白的研究，以发现电子转移的方向，从而揭示初始电子受体的身份。突变体将被用来阐明黄素周围的氨基酸在修复反应中的作用。这种方法，再加上使用斯塔克光谱学对激发态电子结构的分析，将提供关于光解酶如何以及为什么起作用的最清晰的图景。更广泛地说，斯塔克光谱在光致发光中的应用将直接适用于其他光驱动的黄素蛋白(例如蓝光光受体)，它们利用不同的光化学机制，但与光致发光一样，开始于黄素中的光驱动电荷重新分配。这些蛋白质中的一些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引导(光磁接收)进行迁徙的能力。已知还有其他蛋白质在基因水平上调节光合作用。两个合作者加入了这一努力。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。拉斐特学院的伊冯·金特教授和她的本科生研究小组将对修饰的蛋白质进行电化学测量，以衡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和宾夕法尼亚州伊斯顿的拉斐特学院的一批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。所有参与者将在一年两次的“小型会议”中展示他们的成果，轮流在费城和伊斯顿举行，杜克大学的团队将通过视频会议参与进来。PI指导了许多年轻的科学家，包括女性和代表不足的少数族裔成员。为了让非科学观众了解科学对社会的重要性，PI几年来一直密切参与坦普尔的草皮工作人员计划。这个项目混合了非科学领域的本科生和理科专业的本科生的演讲，以产生多学科的互动。

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.