Time Resolved Fluorescence Spectroscopy

时间分辨荧光光谱是生物化学的有力工具；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。我们使用单色氨酸突变体的FRET来测量色氨酸和病毒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形成*准分子*的能力，在激发态下，瞬态（NS）交联产生绿色荧光，而没有它们的地方只有紫色。这些准分子在确定链转移所需的四聚体的组装方面提供了一种有用的寡聚化措施。我们为这种困难的酶准备了可增强溶解度的突变，并使用超离心以盐依赖的方式定量DNA诱导的组装。我们继续为FRET和准分子制备标记的单半胱氨酸版本。我们的计划是建立一个距离的“支架”来定义复合物，以帮助药物设计。我们还完成并发表了在溶剂“口袋”内的~400飞秒平板状分子（苝和四烯，大小与色氨酸相似）的振动研究，为蛋白质的类似研究做准备。这种振动的测量解决了长期以来关于各向异性起源低于0.40（“0缺陷”）的争议。我们已经完成了分子动力学和量子力学模拟。布鲁克斯和吴证实了振动，而不是内部振子位移，导致了我们的瞬态项。我们继续对多肽进行飞秒上转换研究，以量化早期（可能是电子弹射）事件（导致溶剂化电子），这些事件解释了我们之前在多肽和蛋白质中看到的“准静态自猝灭”。我们开始使用IIAGlc蛋白突变体在330fs-200ps时间尺度上进行蛋白溶剂化研究。我们继续与LCE合作研究分离心肌线粒体- NADH的主要燃料状态。我们通过不同的荧光寿命来区分NADH的自由和结合种群，并且我们在氧化还原状态和区室浓度的变化中量化了这些储存库，最近导致了相关烟酰胺结合位点的亲和力模型。

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.