PHOTOCHEMICAL ASSAYS OF CUPREDOXIN THERMODYMANICS--NOVEL METAL ION SENSORS

生命依赖于金属离子增强蛋白质化学性质的能力。多肽和金属协同作用，产生必要的生物分子结构，调节分子稳定性和氧化还原，酸/碱，光化学，否则无法实现与有限的性质或有机化合物。为了评估金属蛋白金属离子特异性的决定因素及其与蛋白质构象稳定性和氧化还原性质之间的关系，我们建议设计一个双位点识别/报告生物无机构象稳定性和氧化还原性质的系统。我们建议设计一个基于表征良好的铜氧还蛋白模板的双位点识别报告生物无机系统，这将允许光谱评估金属结合和蛋白质构象。将监测附着在蛋白质表面的Ru(II)-和Re(I)-光敏剂和结合在蛋白质活性位点的金属离子之间的能量/电子转移，以确定一系列过渡金属离子和血铜蛋白之间的亲和常数。通过合理定向诱变和选择组合突变文库，通过取代和azurin和amicyanin模板对金属离子进行系统扰动。将允许深入检查金属离子特异性，氧化态偏好和天然折叠稳定性之间的关系。从这些研究中获得的见解将为开发混合无机/生物无机传感器库铺平道路，这些传感器可以选择性地与外源过渡金属反应，并在此过程中产生诊断外源金属类型，氧化状态和浓度的独特光信号。

Life depends on the ability of metal ions to augment the chemistry of proteins. Polypeptide and metals interact synergistically to engender essential biomolecular structures, modulated molecular stabilities sand redox-, acid/base-, and photochemistry otherwise unattainable with the limited properties or organic compounds. To evaluate the determinants of metalloprotein metal-ion specificity and their relationships to protein conformational stability and redox properties we propose to design a two-site recognition/reporter bioinorganic conformational stability and redox properties we propose to design a two-site recognition reporter bioinorganic system based on well-characterized cupredoxin templates, which will allow spectroscopic evaluation of metal binding and protein conformation. Energy/electron transfer between designed Ru(II)- and Re(I)- photosensitizers attached to the protein surface and metal-ions bound in the protein active site will be monitored to determine affinity constants between a series of transition metal ions and blood cooper proteins Systemic perturbation of the metal ions, by substitution, and of azurin and amicyanin templates, by both rationally directed mutagenesis and selection from combinatorial mutant libraries, will allow in-depth examination of the relationships among metal-ion specificity, oxidation-state preference, and native fold stability. The insight gained from these studies will pave the way for the development of a library of hybrid inorganic/bioinorganic sensors that can selective react with exogenous transition metals and in doing so produce unique optical signals diagnostic of exogenous metal type, oxidation state, and concentration.