UV CROSSLINKING AS TOOL FOR MODELING PROTEIN NUCLEIC ACID INTERACTIONS

蛋白质-核酸的相互作用是许多 生物过程，包括转录、复制和 蜂窝维护。然而，到目前为止，这种情况还很少。 已发表的这些类型的配合物的X射线和核磁共振结构。 这些化合物的光化学交联是一种很有吸引力的方法。 来分析这些类型的交互作用。光子是“零长度”的 交联剂，这意味着它们在反应性之间产生共价键 没有分子桥的物种。我们有直接的实验 有证据表明，交联肽-核苷酸物种可以完全 以质谱学为特征。我们已经成功地使用了 蛋白质-寡核苷酸复合体的质谱学分析 在polβ和寡核苷酸d(ATATATA)之间形成，以及 胰酶来源的多肽-寡核苷酸复合体的研究 《消化》。我们还成功地确认了最近的核磁共振结果 关于蛋白质结合部位。我们正在积极追求使用 纳秒脉冲激光紫外光交联，原则上可以， 探索微秒和毫秒时间内的特定交互作用 比例。正是在这一点上，计算机的设施 图形实验室是最有用的。作为此的解决方案结构 蛋白质-寡核苷酸体系已经解决，我们可以补充 核磁共振数据通过提供对分子动力学的洞察和 这类系统的拓扑结构。我们希望利用分子动力学研究 来探索具体的相互作用 纳秒时间刻度，并将该信息与 我们的交联结果，以及溶液结构，以 提供了一系列复杂事件的更完整图景 发生在这些类型的系统中。

Protein-nucleic acid interactions lie at the heart of many biological processes, including transcription, replication, and cellular maintenance. To date, however, there have been very few published X-ray and NMR structures of these types of complexes. Photochemical crosslinking of these complexes is an attractive method for analyzing these types of interactions. Photons are "zero-length" crosslinkers, which means they induce covalent bonds between reactive species without a molecular bridge. We have direct experimental evidence that crosslinked peptide-nucleotide species can be fully characterized by mass spectrometry. We have been successful in using mass spectrometry to analyze the protein- oligonucleotide complex formed between pol beta and oligonucleotide d(ATATATA), as well as that of peptide-oligonucleotide complexes arising from a tryptic digest. We have also had success in confirming recent NMR results regarding the protein-binding site. We are actively pursuing using nanosecond-pulsed laser UV crosslinking, which could, in principle, explore specific interactions at the microsecond and millisecond time scale. It is at this point where the facilities at the Computer Graphics Lab are of most use. As the solution structure of this protein-oligonucleotide system has been solved, we can complement the NMR data by providing insight to the molecular dynamics and the topology of such systems. We hope to use molecular dynamics studies of this recently-solved complex to explore specific interactions at the nanosecond time scale, and use the information in conjunction with our crosslinking results, as well as the solution structure, to provide a more complete picture of the complex series of events that take place with these types of systems.