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、蛋白质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。我们继续使用马来酰亚胺标记的整合酶突变体来形成*激发子*，在激发状态下产生瞬时(Ns)交联物，在没有它们的情况下只存在紫色的绿色荧光。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。我们的方案是建立一个定义复合体的距离的“脚手架”，以帮助设计适当的作用于三元结构而不是活性部位的抑制药物，从而避免VD(J)宿主的破坏。 我们重新审查了DNA的A-链弯曲研究(MS重新提交)，并继续使用核苷酸类似物来揭示DNA结构的局部破坏(例如，碱基翻转)。 我们继续并扩大了我们对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作用，发现只有1.5ps的“散装水”响应。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，发现它受到QSSQ的影响。其他人则将光谱漂移解释为 整个系统是由蛋白质中的水解吸引起的，而实际上QSSQ在光谱一侧的选择性损失导致了这个范围的错误漂移。我们已经提交了一份手稿，澄清了局部猝灭是主要的机制，对解吸模型提出了质疑。 我们发表了与LCE合作研究心肌线粒体的主要燃料-NADH的状态。我们的努力通过不同的荧光寿命来区分NADH的游离态和结合态，并在氧化还原状态和隔室浓度变化期间对这些库进行了量化。我们正在我们的新的双光子荧光寿命显微镜设备(与Micro Core合作)中扩展这些研究，并获得完整分离心肌细胞线粒体中NADH结合水平的图像。最近，我们一直在用nadh负载的囊泡重新审视这些测量，以量化荧光团的聚集和潜在的同质转移效应，这可能会扭曲自由/结合比。 在极端的亚细胞条件下。 我们组装了一台具有成像功能的双光子荧光(互相关)光谱仪 并在小分子和转基因细胞的荧光蛋白上进行了测试。特别是， 核转运蛋白被发现具有显著不同的迁移率(能力 横向扩散)在细胞质与胞核之间。我们开始检测LTRDNA上的整合酶组装 使用相同的系统。FCCS是量化稀疏分子迁移率和化学计量的有用工具 蛋白质在溶液中或在细胞中。

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, protein 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 continued using 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 design appropriate inhibitory drugs that act on ternary structure rather than active site, thus avoiding VD(J) host disruption. We revisited A-tract bending studies of DNA (ms resubmitted) and continued using nucleotide analogs to reveal local disruptions in DNA structure (e.g., base flipping). We have continued and expanded our 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, and finding only a 1.5ps "bulk water " response. More important, we have also reexamined the work done by others on proteins such as Monellin, finding it is subject to QSSQ. Others interpreted the spectral shift as a property of the whole system, caused by water desorbing from protein, when in fact the selective loss on one side of a spectrum by QSSQ leads to a false shift in this range. We have submitted a manuscript to clarify that local quenching is the dominant mechanism, calling the desorbtion model into question. We published collaborative studies with LCE into the status of a primary fuel of heart muscle mitochondria- NADH. Our efforts distinguish free and bound populations of NADH by their different fluorescence lifetimes, and we had quantified these reservoirs during changes in redox state and compartmental concentration. We are extending these studies in our new 2-photon fluorescence lifetime microscopy facility (collaboration with Microscopy Core) and obtaining images of NADH binding levels in the mitochondria of intact isolated cardiac myocytes. Recently, we have been carefully revisiting these measurements with NADH loaded vesicles to quantify aggregation of fluorophores and potential homotransfer- effects that might distort the free/bound ratio under extreme subcellular conditions. We assembled a 2-photon Fluorescence (Cross) Correlation Spectrometer with imaging capabilities and tested it on small molecules and on fluorescent proteins in transfected cells. In particular, nuclear transporter proteins were found to have significantly different mobilities (ability to diffuse laterally) in cytoplasm vs. nucleus. We began examining integrase assembly on LTR DNA with the same system. FCCS is a useful tool for quantifying mobility and stoichiometry of sparse proteins either in solution or in a cell.