Tertiary Structure and Binding of Azurin

一个多方面的生物物理方法概述了从天青蛋白，将导致直接影响蛋白质的构象稳定性的物理因素，提高认识，以获得结构，动力学和生物信息。这些信息对于理解为什么蛋白质在实现其活性构象后展开或错误折叠至关重要，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。天青蛋白的三级结构将采用化学、物理、光化学和静电方案进行扰动，以改变构象亚态的能量和分布。在溶胶-凝胶材料中的天青蛋白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。由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。天青蛋白具有对三级结构的变化敏感的单一掩埋色氨酸（Trp），而Cu（II）离子在可见光区经历强电荷转移吸收，其涉及半胱氨酸残基。Trp荧光和电荷转移吸收在我们的实验设计中起着关键作用，因为它们探测了它们的局部环境和蛋白质的三级结构。该项目的一个目标是实现一种创新的，基于激光的光谱技术的融合，以获得动态和结构 资料：（1）将Gd（III）离子取代到载脂蛋白天青蛋白中将允许我们检查Gd（III）的发射光谱，其显示对应于金属离子与蛋白质中的配位位点之间的特定相互作用的振动带。(2)记录低分辨率的拉曼光谱，我们将研究振动光谱是如何影响扰动的三级结构的蛋白质。(3)测量Trp荧光寿命，我们将寻求动态信息。(4)通过研究短脉冲激光引发的一氧化氮/天青蛋白双生复合反应Az + NO → Azo.NO，我们期望了解伴随天青蛋白之间“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蛋白/NO双生复合实验。

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.