UV CROSSLINKING AS TOOL FOR MODELING PROTEIN NUCLEIC ACID INTERACTIONS

紫外交联作为蛋白质核酸相互作用建模工具

• 批准号: 6280152
• 负责人: DALLAS A CONNOR
• 金额: $ 0.01万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6280152
• 关键词: AIDS; biological products; biomaterials; biomedical resource; biotechnology; immunology; infection; inflammation; lymphatic system; technology /technique; virus

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.

蛋白质-核酸相互作用是许多生物学领域的核心。 生物过程，包括转录、复制和 细胞维护 然而，迄今为止，这种情况还很少 这些类型的配合物的公开的X射线和NMR结构。 这些配合物的光化学交联是一种有吸引力的方法 来分析这些类型的互动。 光子是“零长度”的 交联剂，这意味着它们诱导反应性之间的共价键 没有分子桥的物种。 我们有直接的实验 交联的肽-核苷酸种类可以完全 通过质谱法表征。 我们已经成功地利用 质谱分析蛋白质-寡核苷酸复合物 在pol β和寡核苷酸d（ATATATA）之间形成，以及 由胰蛋白酶引起的肽-寡核苷酸复合物 消化 我们还成功地证实了最近的核磁共振结果 关于蛋白质结合位点。 我们正在积极利用 纳秒脉冲激光UV交联，原则上， 探索微秒和毫秒级的特定交互 规模 正是在这一点上，计算机的设施 图形实验室是最有用的。 作为此问题的解决方案结构， 蛋白质-寡核苷酸系统已经解决，我们可以补充 NMR数据，通过提供洞察分子动力学和 这类系统的拓扑结构。 我们希望利用分子动力学研究 这个最近解决的复杂的探索特定的相互作用， 纳秒时间尺度，并结合使用信息， 我们的交联结果以及溶液结构， 提供了一个更完整的图片的复杂的一系列事件， 这些类型的系统。