Time Resolved Fluorescence Spectroscopy

时间分辨荧光光谱

• 批准号: 6817752
• 负责人: JAY R KNUTSON
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6817752
• 关键词: DNA; DNA binding protein; biomedical equipment development; fluorescence resonance energy transfer; fluorescence spectrometry; human immunodeficiency virus; integrase; intermolecular interaction; laser spectrometry; virus integration; virus protein

Time-Resolved Fluorescence Spectroscopy is a powerful tool for biochemistry; it can provide unique insights into the structure, assembly and flexibility of complex macromolecules. This year, we continued collaborative studies into DNA-protein interactions. We remain interested in the oligomerization and DNA binding of HIV-integrase, the enzyme used by the AIDS virus to incorporate itself into human DNA. We employed FRET with single-tryptophan mutants to measure site-specific distances between Trp and the end of the viral DNA, including kinetic measurement during 3' processing.. We also tested the ability of pyrene maleimide-labeled integrase mutants to form *excimers*, transient (NS) crosslinks in the excited state that create a green fluorescence where only violet was present without them. These excimers provide a measure of oligomerization useful in affirming the assembly of tetramers required for strand transfer. We prepared solubility-enhancing mutations for this difficult enzyme, and we used ultracentrifugation to quantify DNA -induced assembly in a salt-dependent fashion. We have continued preparation of labeled single-cysteine versions for FRET and excimers. Our scheme is to build a "scaffold" of distances that define the complex, to help drug design. We also completed and published studies of the ~400-femtosecond librations of platelike molecules (perylene and tetracene, with sizes similar to tryptophan) inside solvent "pockets" to prepare for similar studies in proteins. Measurement of this libration settled a longstanding controversy about anisotropy origins below 0.40 (the "ro defect"). We have completed molecular dynamics and quantum mechanical simulations with Drs. Brooks and Wu that confirm the libration, and not internal oscillator displacement, causes our transient term. We have continued femtosecond upconversion studies of peptides to quantify early (possibly electron ejection) events (leading to solvated electrons) that explain the "quasistatic self-quenching" we had previously seen in peptides and proteins. We began studies of protein *solvation* on the 330fs-200ps time scale, using mutants of IIAGlc protein. We continued collaborative studies with LCE into the status of a primary fuel of isolated heart muscle mitochondria- NADH. Our efforts distinguish free and bound populations of NADH by their different fluorescence lifetimes, and we have quantified these reservoirs during changes in redox state and compartmental concentration, recently leading to a model for the affinity of relevant nicotinamide binding sites.

时间分辨荧光光谱是生物化学的强大工具;它可以为复杂大分子的结构，组装和灵活性提供独特的见解。今年，我们继续合作研究DNA-蛋白质相互作用。我们仍然对HIV整合酶的寡聚化和DNA结合感兴趣，HIV整合酶是艾滋病病毒用来将自身整合到人类DNA中的酶。我们使用具有单色氨酸突变体的FRET来测量Trp与病毒DNA末端之间的位点特异性距离，包括3'加工期间的动力学测量。我们还测试了芘马来酰亚胺标记的整合酶突变体在激发态下形成 * 准分子 *、瞬时（NS）交联的能力，该交联产生绿色荧光，其中只有紫色存在而没有它们。这些准分子提供了可用于确认链转移所需的四聚体组装的低聚化的量度。我们为这种困难的酶制备了溶解度增强突变，并且我们使用超离心以盐依赖性方式定量DNA诱导的组装。我们继续准备标记的单半胱氨酸版本的FRET和准分子。我们的计划是建立一个“支架”的距离，定义复杂的，以帮助药物设计。我们还完成并发表了关于溶剂“口袋”内板状分子（二萘嵌苯和并四苯，大小与色氨酸相似）的~400飞秒振动的研究，为蛋白质中的类似研究做准备。这种天平动的测量解决了一个关于各向异性起源低于0.40（“ro缺陷”）的长期争论。我们与布鲁克斯和吴博士一起完成了分子动力学和量子力学模拟，证实了引起瞬态项的是天平动，而不是内部振荡器位移。我们继续对肽进行飞秒上转换研究，以量化早期（可能是电子喷射）事件（导致溶剂化电子），这解释了我们以前在肽和蛋白质中看到的“准静态自猝灭”。我们使用IIAGlc蛋白的突变体，开始在330 fs-200 ps时间尺度上研究蛋白质 * 溶剂化 *。我们继续与LCE合作研究分离的心肌线粒体的主要燃料- NADH的状态。我们的努力区分自由和绑定群体的NADH通过其不同的荧光寿命，我们已经量化这些水库在氧化还原状态和房室浓度的变化，最近导致相关的烟酰胺结合位点的亲和力的模型。