Tertiary Structure and Binding of Azurin

本文概述了从azurin获得结构、动力学和热力学信息的多方面生物物理方法，这将有助于提高对直接影响蛋白质构象稳定性的物理因素的理解。这些信息对于理解蛋白质在达到活性构象后展开或错误折叠的原因至关重要，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。采用化学、物理、光化学和静电等方法对azurin的三级结构进行扰动，以改变构象亚态的能量和分布。azurin在溶胶-凝胶材料中的固定化将在以下背景下进行探讨

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.