APPLICATION OF MASS SPECTROMETRY TO STRUCTURAL BIOLOGY

工作总结：最近的工具 质谱的发展，如基质辅助激光 解吸和电喷雾电离，使质量 光谱学家研究生物分子， 比以前可能的更高。结合这些 大型生物聚合物的化学加工技术， 蛋白质现在可以使用质谱法来发挥作用， 在结构生物学领域的重要作用。在这方面， 结构生物学不仅是指确定 一级序列和翻译后修饰位点，但 也适用于探测分子和复合物的三级结构。 我们目前正在进行几个项目。简要说明 其中一些如下：第一个项目是开发 探测蛋白质之间的非共价复合物的能力， DNA，使用酵母转录因子GCN 4。这些蛋白质 含有碱性DNA结合域和亮氨酸拉链 二聚体与dsDNA特异性结合， 形成四分子非共价复合物。使用ESI，我们有 首次观察到这种特殊的四分子复合物 我们希望，最终，包括DNA：蛋白质足迹 实验到这个项目。?第二个项目是确定 人p53肿瘤抑制蛋白上的残基 辐射诱导的后磷酸化 DNA损伤（与梅里克，LMC合作）。我们有 在人类中发现了10种不同的p53磷酸化亚型 乳腺上皮细胞和重组p53。我们假设 p53的特异性磷酸化模式决定了 有丝分裂停滞或细胞凋亡，以应对DNA损伤。使用 MS/MS，我们已经明确确定了一个 磷酸化位点。?催化核的结构测定 蛋白磷酸酶5（丝氨酸/苏氨酸磷酸酶， 可能介导某些脂质对离子通道活性的影响）， 在枯草杆菌蛋白酶消化后从调节区释放， 该序列不同于胰蛋白酶释放的类似核心。的 枯草杆菌蛋白酶和胰蛋白酶切割片段的序列已经被 测定该项目与NIEHS/LST合作 Purdue Univ.集团?蛋白质的结构测定 中间体中参与席夫碱形成的残基 在Pol β的碱基切除修复中形成（与S. Wilson，LSB）？TTP上磷酸化残基的鉴定， RNA结合蛋白，并鉴定其上的残基 使用光诱导蛋白质与RNA交联， 交联剂（用P.黑剪，P.D.）。

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).