LASER SPECTROSCOPY OF TRIPLET STATES IN PROTEINS

光学光谱技术已经对研究的重大影响， 蛋白质结构和机制。 然而，只有一小部分 工作采用基于三重态的光谱学。 拟议 研究将结合联合收割机的技能和设施的生物制药学家（ 首席研究员）和一名激光光谱学家（共同负责人 研究人员）开发几种基于激光的方法， 一个广泛的计划的基础上应用三重态 分子生物物理学中的重要问题。 丰富的 关于蛋白质结构和相互作用的重要新信息， 希望通过这种方法找到解决方案。 将采用的方法 开发的系统包括：（a）快速获取系统（基于 单个激发脉冲）的磷光衰减动力学， 实时监测蛋白质的结构转变， 展开和重折叠：（B）使用扩散增强的福斯特型能量 从内禀三重态供体转移到研究蛋白质结构 和相互作用，通过绘制磷光的距离， （c）时间积分和时间 解析圆偏振磷光（CCP）的详细应用 蛋白质构象的研究以及构象变化， 发生在与三重态寿命相当的时间尺度上;（d）激光- 用于确定三重态的基于三重态-三重态吸收 低磷光产率系统中的衰减模式。 延长 包括三重态的时间分辨圆二色性， 三重态跃迁将提供与导出的信息互补的信息。 从CPP。 这些方法将使用选择的模型酶来开发 由于它们的稳定性、磷光性质和结构 信息，并将适用于几个重大问题， 分子生物物理学 这些包括酶中的构象异构， 重折叠中间体之间的结构变异性，亚基缔合 寡聚酶和酶-酶复合物形成模式。 另一个有趣的应用将是结构的研究， 修饰负责酶之间的差异记录 从年轻和年老动物的组织中提纯。

Optical spectroscopic techniques have had a major impact on the study of protein structure and mechanisms. However, only a small fraction of the work has employed triplet-state based spectroscopies. The proposed research will combine the skills and facilities of a biophysicist (the principal investigator) and a laser spectroscopist (co-principal investigator) to develop several laser-based methodologies which will form the basis of an extensive program to apply the triplet-state spectroscopies to important problems in molecular biophysics. A wealth of significant new information on protein structure and interactions in solutions is expected from this approach. The methodologies to be developed include: (a) A system from the rapid acquisition (based on a single excitation pulse) of phosphorescence decay kinetics allowing for real-time monitoring of structural transitions in proteins as involved in unfolding and refolding: (b) Use of diffusion-enhanced Forster-type energy transfer from intrinsic triplet-state donors to study protein structure and interactions though mapping of the distances of phosphorescent residues from the surface of the protein; (c) Time integrated and time resolved circularly polarized phosphorescence (CCP) to applied in detailed studies of protein conformations as well as conformational changes that occur on a time scale comparable to the triplet state lifetime; (d) Laser- based triplet-triplet absorption to be used to determine triplet state decay patterns in systems with low phosphorescence yield. An extension of this approach to include time-resolved circular dichroism of triplet- triplet transitions will provide information complementary to that derived from CPP. These approaches will be developed using model enzymes chosen by virtue of their stability, phosphorescence properties and structural information and will be applied to several significant problems in molecular biophysics. These include conformational isomerism in enzymes, structural variability among refolding intermediates, subunit association in oligomeric enzymes and enzyme-enzyme complex formation patterns. Another interesting application will be the study of the structural modifications responsible for the documented differences between enzymes purified from tissues of young and old animals.