Tertiary Structure and Binding of Azurin

概述了一种从天青素中获得结构、动力学和热力学信息的多方面生物物理方法，这将有助于更好地理解直接影响蛋白质构象稳定性的物理因素。这些信息对于理解为什么蛋白质在达到活性构象后展开或错误折叠是必不可少的，因为这些问题与阿尔茨海默氏症和其他神经退行性疾病有关。为了改变构象亚态的能量和分布，采用化学、物理、光化学和静电方法对天青的三级结构进行微扰。天青在溶胶-凝胶材料中的固定化将在以下背景下进行探索 机械地限制蛋白质的构象状态。所有这些扰动引起的结构效应将通过获得热力学和光谱信息来表征。天青具有单一的、埋藏的色氨酸(Trp)，它对三级结构的变化很敏感，而Cu(11)离子在可见光区域经历了涉及半胱氨酸残基的强烈电荷转移吸收。色氨酸荧光和电荷转移吸收在我们的实验设计中都发挥了关键作用，因为它们探测了各自的局部环境和蛋白质的三级结构。该项目的一个目标是实施一种创新的、基于激光的光谱技术的混合，以获得动力学和结构 信息：(1)将Gd(111)离子替换为脱氧天青素将使我们能够检测Gd(111)的发射光谱，其显示的振动带对应于金属离子与蛋白质中配位位置之间的特定相互作用。(2)记录低分辨率拉曼光谱，我们将研究振动光谱是如何在扰动蛋白质的三级结构时受到影响的。(3)测定色氨酸荧光寿命，寻求动态信息。(4)研究由短脉冲激光引发的一氧化氮/天青双酯复合反应，我们期望了解伴随着天青键形成的分子内重组的动力学。 和一氧化氮。利用激光闪光光解技术进行了天青/NO双酯复合实验。

A multifaceted biophysical approach is outlined to obtain structural, dynamical and thermodynamical information from azurin that will lead to an improved understanding of the physical factors that directly affect the conformational stability of proteins. This information is essential to understand why proteins unfold or misfold after achieving their active conformation, as these problems are associated to the Alzheimer's and other neurodegenerative disorders. The tertiary structure of azurin will be perturbed employing chemical, physical, photochemical and electrostatical protocols in order to shift the energy and distribution of the conformational substates. The immobilization of azurin in sol-gel materials will be explored in the context of mechanically constraining the conformational states of the protein. The structural effects caused by all these perturbations will be characterized by obtaining thermodynamic and spectroscopic information. Azurin has a single, buried tryptophan (Trp) that is sensitive to changes in the tertiary structure, whereas the Cu(ll) ion undergoes a strong charge-transfer absorption in the visible region that involves a cystein residue. Both the Trp fluorescence and the charge-transfer absorption play a key role in our experimental design because they probe their local environments and the tertiary structure of the protein. A goal of this project is to implement a blend of innovative, laser-based spectroscopic techniques to obtain dynamical and structural information: (1) Substituting the Gd(lll) ion into apo azurin will permit us to examine the emission spectra of Gd(lll), which displays vibrational bands that correspond to specific interactions between the metal ion and the coordination sites in the protein. (2) Recording low resolution Raman spectra we will examine how the vibrational spectra are affected upon perturbing the tertiary structure of the protein. (3) Measuring Trp fluorescence lifetimes we will seek dynamic information. (4) Studying the nitric oxide/azurin geminate recombination reaction, Az + NO-->Azo.NO, initiated using short laser pulses, we expect to learn about the dynamics of the intramolecular reorganization that accompany the formation of the "bond' between azurin and nitric oxide. The azurin/NO geminate recombination experiment will be carried out using the laser flash photolysis technique.