APPLICATION OF MASS SPECTROMETRY TO STRUCTURAL BIOLOGY

工作总结：最近的工具 基质辅助激光等质谱学的研究进展 解吸和电喷雾电离，使质量 光谱学家用显著的方法研究生物分子 比以前可能的更高的MR。这些结合在一起 大型生物聚合物的化学加工技术，如 蛋白质现在能够使用质谱学来发挥作用 在结构生物学领域发挥着重要作用。在这方面， 结构生物学不仅指的是确定 翻译后修饰的主要序列和位置，但 探索分子和络合物的三级结构也是如此。 我们目前正在进行几个项目。简短地描述 其中一些如下：？第一个项目是开发 探测蛋白质与蛋白质间非共价复合体的能力 DNA，使用酵母转录因子Gcn4。这些蛋白质 含有一个基本的DNA结合域和一个亮氨酸拉链 二聚化结构域，二聚体与dsDNA特异性结合 形成四分子非共价络合物。使用ESI，我们拥有 首次观察到这种特殊的四分子络合物 BV女士我们希望最终能包括DNA：蛋白质足迹 对这个项目进行实验。？第二个项目是确定 人P53抑癌蛋白上的残基 翻译后磷酸化对辐射诱导的反应 DNA损伤(与LMC的Merrick合作)。我们有 在人类体内发现10种不同的P53磷酸化异构体 乳腺上皮细胞与重组P53我们假设 P53的特定磷酸化模式决定了决定 作为对DNA损伤的反应，有丝分裂停滞或凋亡。vbl.使用 到目前为止，我们已经明确地确定了一种 磷酸化位点。？催化芯的结构测定 蛋白磷酸酶5(一种丝氨酸/苏氨酸磷酸酶 可能介导某些脂类对离子通道活性的影响)，即 在枯草杆菌毒素消化后从监管区域释放以及如何 这个序列与胰酶释放的类似核心不同。这个 枯草杆菌酶和胰酶裂解片段的序列已被 下定决心。该项目是与NIEHS/LST合作的 和普渡大学。组。？蛋白质的结构测定 中间体中参与席夫碱形成的残基 由Pol beta在碱基切除修复中形成(与S. 威尔逊，LSB)？TTP上磷酸化残基的鉴定， 一种RNA结合蛋白及其上残基的鉴定 通过光诱导与RNA交联化的蛋白质 交联剂(与P.Blackset，DIR)。

Summary of Work: Recent instrumental developments in mass spectrometry, such as matrix-assisted laser desorption and electrospray ionization, have enabled mass spectrometrists to investigate biological molecules with significantly higher Mr than previously possible. The combination of these techniques with chemical processing of large biopolymers such as proteins now enables the use of mass spectrometry to play a significant role in the realm of structural biology. In this respect, structural biology not only refers to the determination of the primary sequence and sites of post-translational modifications, but also to probing the tertiary structure of molecules and complexes. We are currently working on several projects. Short descriptions of some of these follows: ? The first project is to develop the capability of probing non-covalent complexes between proteins and DNA, using the yeast transcription factor GCN4. These proteins contain a basic DNA-binding domain and a leucine zipper dimerization domain, and the dimers specifically bind dsDNA to form a tetramolecular noncovalent complex. Using ESI, we have observed, for the first time, such specific tetramolecular complexes bv MS. We hope, eventually, to include DNA:protein footprinting experiments into this project. ? A second project is to identify the residues on the human p53 tumor suppressor protein post-translationally phosphorylated in response to radiation induced DNA damage (in collaboration with Merrick, LMC). We have found 10 different phosphorylated isoforms of p53 in human mammary epithelial cells and recombinant p53. We hypothesize that the specific phosphorylation pattern of p53 determines the decision for mitotic arrest or apoptosis in response to DNA damage. Using MS/MS we have so far unequivocally identified one phosphorylation site. ? Structure determination of the catalytic core of protein phosphatase 5 (a serine/threonine phosphatase which may mediate the effect of some lipids on ion channel activity) that is released from a regulatory region upon subtilisin digestion and how this sequence differs from the similar core released by trypsin. The sequences of the subtilisin and trypsin cleaved fragments have been determined. This project is in collaboration with the NIEHS/LST and Purdue Univ. groups. ? Structure determination of the protein residues involved in Schiff base formation in the intermediate formed in base excision repair by Pol beta (Collaboration with S. Wilson, LSB) ? Identification of phosphorylated residues on TTP, an RNA binding protein, and identification of residues on this protein that are crosslinked with RNA using a photoinducible crosslinking reagent (with P. Blackshear, DIR).