Time Resolved Fluorescence Spectroscopy

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。今年， 1)我们继续合作研究DNA-蛋白质相互作用。 我们发表了一项关于translin的结构和组装的研究，translin是一种在重组/修复中重要的DNA结合蛋白。我们使用时间分辨的各向异性，发现它是一个稳定的八聚体，其特征在于每个半胱氨酸的反应性。然后，使用单半胱氨酸突变体，我们发现形成的芘准分子（纳秒瞬时交联），只能形成一个“尾到尾”的四级结构。translin-ssDNA复合物中的关键距离的研究也已经通过FRET（福斯特共振能量转移）进行。 我们研究了β-聚合酶的N螺旋（控制活性位点保真度的片段）的局部灵活性和其8 kD裂解酶结构域的片段旋转。我们发现局部运动的时间尺度适合于这些结构元素，我们发现每个局部运动“冻结”在不同的时间在催化循环的β-pol. We相关的事实，即聚合酶也作为一个线性的“电机”进行时，出版物有待轻微的修订。 我们的主要目标仍然是HIV整合酶的寡聚化和DNA结合，HIV整合酶是艾滋病病毒将自身整合到人类DNA中的酶。我们采用FRET与单色氨酸突变体来测量色氨酸和病毒DNA末端之间的位点特异性距离。关于这一点的手稿正在等待批准。最近，我们为这种困难的酶制备了溶解度增强突变，并且我们使用超离心来定量我们以前在光学上看到的DNA诱导的聚集。我们继续准备标记的单半胱氨酸版本的FRET和准分子。我们的计划是建立一个“支架”的距离，定义复杂的，以帮助药物设计。 2)我们完成了对溶剂“口袋”内板状分子（二萘嵌苯和并四苯，大小与色氨酸相似）的~400飞秒振动的研究，为蛋白质的类似研究做准备。这种天平动的测量解决了长期存在的关于各向异性起源低于0.40（“ro缺陷”）的争论。我们已经开始与布鲁克斯和吴博士进行分子动力学模拟，以开发一个更合适的非指数天平动模型。我们已经开始对肽进行飞秒上转换研究，以了解早期电子喷射事件（导致溶剂化电子）是否有助于解释我们以前在肽和蛋白质中看到的“准静态自猝灭”。 3)我们继续与LCE合作研究分离的心肌线粒体的主要燃料- NADH的状态。我们的努力区分自由和绑定群体的NADH不同的荧光寿命，我们已经量化了这些水库在能量状态和房室浓度的变化。

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, 1) We continued collaborative studies into DNA-protein interactions. We published a study of the structure and assembly of translin, a DNA-binding protein important in recombination/repair. We used time-resolved anisotropy to find it is a stable octamer and characterized the reactivity of each of its cysteines. Then, using single-Cys mutants, we found formation of pyrene excimers (nanosecond transient crosslinks) that could only form in a 'tail to tail' quaternary structure. Studies of key distances in translin-ssDNA complexes have also been made via FRET (Forster Resonance Energy Transfer). We studied both the local flexibility of the N helix of beta-polymerase (a segment that controls active site fidelity) and the segmental rotation of its 8kD lyase domain. We found local motions had timescales appropriate for those structural elements, and we found each local motion "freezes" at different times in the catalytic cycle of beta-pol. We related this to the fact that the polymerase also acts as a linear "motor" when processive; publication is pending minor revision. Our main target remains 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. A manuscript about this is pending clearance. Recently, we prepared solubility-enhancing mutations for this difficult enzyme, and we used ultracentrifugation to quantify DNA -induced aggregation we had previously seen optically. 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. 2) We completed 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 settles longstanding controversy about anisotropy origins below 0.40 (the "ro defect"). We have begun molecular dynamics simulations with Drs. Brooks and Wu to develop a more appropriate model for the nonexponential libration. We have begun femtosecond upconversion studies of peptides to learn if early electron ejection events (leading to solvated electrons) help explain the "quasistatic self-quenching" we had previously seen in peptides and proteins. 3) We continued collaborative studies with LCE into the status of a primary fuel of isolated heart muscle mitochondria- NADH. Our efforts distinguished free and bound populations of NADH by their different fluorescence lifetimes, and we have quantified these reservoirs during changes in energy state and compartmental concentration.