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、溶剂化和能量学进行合作研究。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。我们测试了马来酰亚胺标记的整合酶突变体在激发状态下形成*激发子*，瞬时(Ns)交联物的能力，在没有它们的情况下，只有紫色存在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，并继续为FRET和准分子酶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。 我们还完成了DNA的A-链弯曲研究(MS将提交)，开始研究正在修复的阶梯DNA螺旋上苯并[2]加合物的取向，并发表了用于DNA/RNA扩增和检测的独特杂交探针的研究。 我们继续研究在溶剂“口袋”内的片状分子的~400-飞秒振动(大小类似于色氨酸)，为蛋白质的类似研究做准备。我们与Brooks和Wu博士一起进行了分子动力学和量子力学模拟，证实了振动是亚ps瞬变项的一个可能的来源。 我们已经完成了多肽的飞秒上转换研究，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QSSQ“准静态自猝灭”。我们发现了极快(10-100ps)的非辐射事件，这些事件在蛋白质研究中也很重要，因为它们意味着有容易的电荷转移。我们使用IIAGlc蛋白的突变体，在330ps-200ps的时间尺度上继续研究蛋白质的溶剂化。更重要的是，我们还重新检查了其他人在Monellin等蛋白质上所做的工作，测试了它在这个范围内的任何QSSQ。 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。我们的工作通过NADH的不同荧光寿命来区分自由和结合的群体，我们已经在氧化还原状态和隔室浓度的变化过程中量化了这些库，最近导致了一个相关烟酰胺结合位点亲和力的模型和一份提交的手稿。我们在核心建立了一个双光子荧光寿命显微镜设备，并获得了完整的分离心肌细胞线粒体中NADH结合水平的图像，从而扩展了这些研究。

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, solvation, and energetics. 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 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 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 A-tract bending studies of DNA (ms to be submitted), began studies of the orientation of benzopyrene adducts on stepped DNA helices undergoing repair, and published studies of unique hybridization probes for DNA /RNA amplification and detection. We continued 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. We performed molecular dynamics and quantum mechanical simulations with Drs. Brooks and Wu that confirm the libration is a plausible source of the sub-ps transient term. We have completed femtosecond upconversion studies of peptides to quantify early (possibly electron ejection) events (leading to solvated electrons) that explain the QSSQ "quasistatic self-quenching" we had previously seen in peptides and proteins. We found extremely rapid (10-100ps) nonradiative events that are also important in protein studies, as they imply conformers with facile charge transfer. We continued studies of protein *solvation* on the 330fs-200ps time scale, using mutants of IIAGlc protein. More important, we have also reexamined the work done by others on proteins such as Monellin, testing it for any QSSQ in this range. 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 and a manuscript submitted. We extended these studies by building a 2-photon fluorescence lifetime microscopy facility in the core and obtaining images of NADH binding levels in the mitochondria of intact isolated cardiac myocytes.