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的能力。多肽和金属协同作用，产生基本的生物分子结构、调节的分子稳定性以及氧化还原、酸碱和光化学，否则在有限的性质或有机化合物中是无法获得的。为了评估金属蛋白金属离子专一性的决定因素及其与蛋白质构象稳定性和氧化还原性质的关系，我们建议设计一种双位点识别/报告生物无机构象稳定性和氧化还原性质我们建议设计一个基于特征良好的铜还蛋白模板的两位点识别报告生物无机系统，该系统将允许对金属结合和蛋白质构象的光谱评估。结合在蛋白质表面的设计的Ru(II)和Re(I)光敏剂与结合在蛋白质活性部位的金属离子之间的能量/电子转移将被监测，以确定一系列过渡金属离子与血液库珀蛋白之间的亲和力常数。通过取代金属离子的系统扰动，以及通过合理定向突变和从组合突变体库中选择天青素和氨兰模板，将允许深入研究金属离子特异性、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一种混合无机/生物无机传感器文库铺平道路，该传感器可以与外源过渡金属发生选择性反应，从而产生对外源金属类型、氧化状态和浓度的独特光学信号诊断。

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.