Time Resolved Fluorescence Spectroscopy

时间分辨荧光光谱

• 批准号: 6541692
• 负责人: JAY R KNUTSON
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6541692
• 关键词: DNA; DNA binding protein; bioenergetics; biomedical equipment development; chemical kinetics; conformation; fluorescence resonance energy transfer; fluorescence spectrometry; human immunodeficiency virus; integrase; intermolecular interaction; laser spectrometry; molecular assembly /self assembly; nicotinamide adenine dinucleotide; nucleic acid repetitive sequence; protein binding; protein folding; stop flow technique; time resolved data; virus integration; virus protein

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.

时间分辨荧光光谱是生物化学的强大工具;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整合酶的寡聚化和DNA结合，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FRET与单色氨酸突变体来测量Trp和病毒LTR末端（长末端重复序列）之间的位点特异性距离。我们发现关键位点的两种距离的混合物，并且我们正在努力将它们分配给复合物的不同功能状态（游离，结合和3 '加工）。我们还开始使用标记的单半胱氨酸蛋白质版本。我们的计划是建立一个距离的“支架”，以帮助我们设计合适的药物。我们开始研究translin的结构和组装，translin是一种在重组/修复中重要的DNA结合蛋白。我们了解到它是一种稳定的八聚体，并且使用单Cys突变体，我们发现了只能在尾-尾组装中形成的准分子的形成。ssDNA复合物的研究已经开始。我们还研究了β-聚合酶活性位点的局部灵活性和蛋白质诱导的DNA弯曲A相DNA或正常DNA与因子HU。 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UV上转换荧光计，我们表明内部转换快于100 fs，表明fs测量蛋白质中的Trp将产生关于局部堆积和介电环境的新数据。我们还测量了溶剂“口袋”内板状分子（类似于Trp）的400飞秒局部振动，为蛋白质内的类似研究做准备。 3)我们开始合作研究心肌线粒体的主要燃料- NADH的状态。我们的初步努力区分自由和绑定群体的NADH不同的荧光寿命，我们正在朝着量化这些水库与能量状态。