LASER SPECTROSCOPY OF TRIPLET STATES IN PROTEINS

光学光谱学技术对地球物理研究产生重大影响 蛋白质的结构和机制。然而，只有一小部分人 这项工作采用了基于三重态的光谱仪。建议数 研究将结合生物物理学家的技能和设施( 首席调查员)和一名激光光谱仪(共同负责人 调查员)开发几种基于激光的方法，这些方法将形成 应用三重态的广泛程序的基础 分子生物物理学中重要问题的光谱学。一大笔钱 关于蛋白质结构和相互作用的重要新信息 这种方法有望提供解决方案。方法论将是 开发的内容包括：(A)从快速获取系统(基于 单个激发脉冲)的磷光衰减动力学 实时监测蛋白质中的结构转变 展开和再折叠：(B)使用扩散增强的福斯特型能量 从固有的三态供体转移到蛋白质结构研究 通过绘制磷光的距离图和相互作用 蛋白质表面的残基；(C)时间积分和时间 分辨圆偏振磷光(CCP)的详细应用 对蛋白质构象以及构象变化的研究 发生的时间尺度与三重态寿命相当；(D)激光- 基于三重态-三重态吸收用于确定三重态 低磷光产额体系中的衰变模式。延期 这种方法包括时间分辨的三重圆二向色性- 三重态跃迁将提供与派生的信息互补的信息 来自CPP的。这些方法将使用所选的模型酶来开发 由于它们的稳定性、磷光性质和结构 信息，并将应用于以下几个重要问题 分子生物物理学。这些包括酶的构象异构性， 折叠中间体、亚基结合的结构变异性 在寡聚酶和酶-酶复合体形成模式中。 另一个有趣的应用将是研究结构 引起酶之间记录差异的修饰 从幼年和老年动物的组织中提纯。

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.