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.
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