Time Resolved Fluorescence Spectroscopy

时间分辨荧光光谱是生物化学的一种强有力的工具，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，我们继续对DNA-蛋白质相互作用进行合作研究。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己整合到人类DNA中的酶。我们使用带有单一色氨酸突变体的FRET来测量Trp和病毒DNA末端之间的特定位点距离，包括3‘加工过程中的动力学测量。我们还测试了Py-Maleimide标记的整合酶突变体在激发状态下形成*激发子*的能力，即瞬时(NS)交联物，在没有它们的情况下产生绿色荧光。这些准分子提供了一种低聚的测量方法，可用于确认链转移所需的四聚体的组装。我们为这种困难的酶准备了溶解性增强的突变，并使用超速离心法以盐依赖的方式量化了DNA诱导的组装。我们继续为FRET和激发子准备标记的单半胱氨酸版本。我们的计划是建立一个定义复合体的距离的“脚手架”，以帮助药物设计。我们还完成并发表了对片状分子(大小类似于色氨酸)的~400飞秒振动的研究，为在蛋白质中进行类似的研究做准备。这种振动的测量解决了关于低于0.40的各向异性起源(“ro缺陷”)的长期争论。我们已经与Brooks和Wu博士一起完成了分子动力学和量子力学模拟，证实了导致我们的瞬变项的是振动，而不是内部振子位移。我们继续对多肽进行飞秒上转换研究，以量化早期(可能是电子抛射)事件(导致溶剂化电子)，这些事件解释了我们以前在多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，在330ps-200ps的时间尺度上开始了对蛋白质溶剂化的研究。我们继续与LCE合作研究分离的心肌线粒体-NADH的主要燃料状态。我们的努力通过NADH的不同荧光寿命来区分自由和结合的群体，并且我们在氧化还原状态和隔室浓度变化时对这些储存库进行了量化，最近导致了一个相关烟酰胺结合位点亲和力的模型。

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.