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。 1)今年，我们继续对DNA-蛋白质相互作用进行合作研究。我们的主要目标是HIV整合酶的寡聚和DNA结合，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LTR(长末端重复序列)末端之间的特定位点距离。我们发现关键位点有两个距离的混合物，我们正在努力将它们分配给复合体的不同功能状态(自由、结合和3‘加工)。我们还开始使用这种蛋白质的标记单半胱氨酸版本。我们的计划是建立一个复合体所能容纳的距离的“脚手架”，以帮助我们设计合适的药物。我们开始研究翻译蛋白的结构和组装，这是一种在重组/修复中起重要作用的DNA结合蛋白。我们了解到它是一个稳定的八聚体，利用单半胱氨酸突变体，我们发现只有在尾巴组装中才能形成准分子。对单链DNA复合体的研究已经开始。我们还研究了具有HU因子的A相链DNA和正常DNA在β-聚合酶活性部位和蛋白质诱导的DNA弯曲作用部位的局部灵活性。2)我们完成并发表了色氨酸的飞秒动力学研究，色氨酸是蛋白质中最重要的天然荧光团。使用我们独特的紫外上转换荧光仪，我们显示了内转换快于100fS，这表明FS测量蛋白质中的色氨酸将产生关于局部堆积和介电环境的新数据。我们还测量了片状分子(类似色氨酸)在溶剂“口袋”内400飞秒的局部振动，为蛋白质内部的类似研究做准备。3)我们开始合作研究心肌线粒体的主要燃料-NADH的状态。我们的初步努力通过NADH的不同荧光寿命区分了自由和结合的人群，我们正在朝着量化这些储存库与能量状态的方向迈进。

Time-Resolved Fluorescence Spectroscopy is a powerful tool for biochemistry; it can provide unique insights into the structure, assembly and flexibility of complex macromolecules. 1) This year, we continued our collaborative studies into DNA-protein interactions. Our main target was 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 LTR (long terminal repeat) We found a mixture of two distances for key sites, and we are working to assign them to different functional states of the complex(free, bound and 3'processed). We have also begun using labeled single-cysteine versions of the protein. Our scheme is to build a "scaffold" of distances that the complex fits in, to help us design appropriate drugs. We began studies into the structure and assembly of translin, a DNA-binding protein important in recombination/repair. We learned it is a stable octamer and , using single-Cys mutants, we found the formation of excimers that could only form in a tail-tail assembly. Studies of ssDNA complexes have begun. We also studied local flexibility in the active site of beta-polymerase and protein-induced DNA bending of phased-A tract DNA or normal DNA with factor HU. 2) We completed and published studies of the *femtosecond* dynamics of tryptophan, the most important natural fluorophore in proteins. Using our unique UV upconversion fluorometer, we showed that internal conversion was faster than 100fs, showing that fs measurements of Trp in protein will yield new data about local packing and dielectric environment. We have also made measurements of the 400-femtosecond local librations of platelike molecules (similar to Trp) inside solvent "pockets" to prepare for analogous studies inside proteins. 3) We began collaborative studies into the status of a primary fuel of heart muscle mitochondria- NADH. Our preliminary efforts distinguished free and bound populations of NADH by their different fluorescence lifetimes, and we are moving toward quantifying these reservoirs versus energy state.