Conformation, dynamics and reactivity of enhanced Hb

描述(由申请人提供)： Hb的聚乙二醇化和诱变操纵及其组合是积极的策略 正在探索它们在产生基于Hb的氧气载体(HBOC)方面的潜力。这些 正在实施改进，试图开发出将 血管活性和有毒反应物的产生，同时保持充足的氧气供应 到纸巾。这些修饰策略如何在分子水平上改变Hb的性质 没有得到很好的解决。相应地，我们建议探索单一和组合 通过聚乙二醇化和诱变操作修饰Hb的生物物理后果。 局部和全球水平上的构象变化、蛋白质动力学、配基结合 影响使用无细胞Hb作为血液替代品的动力学和反应将被曝光 并进行探索。 这项工作将在两个实验室进行，乔尔·弗里德曼博士(AECOM)和乔尔·弗里德曼博士(Dr.Joel Friedman)。 西莉亚·博纳文图拉(博福特，杜克大学)。正在研究的修改后的HBS将被检查 在不同的条件下，以澄清潜在的结构-功能关系。哈佛商学院将成为 在溶液中，在溶胶-凝胶中，在海藻糖玻璃中，在使我们能够 确定血红素环境和配基结合的能量障碍的差异。Dr。 弗里德曼的努力将主要包括测量共振拉曼光谱、荧光、 双基复合和其他快速动力学过程和瞬时吸收。Dr。 Bonaventura的实验室将在配体(O2)方面表征相同的改良HBS 和NO)的协同结合和对阴离子效应物的反应，S亚硝化形式的形成和稳定性，以及SH反应性的变化，氧化过程，使用改进的 光谱电化学法和快速混合法。作为一名经验丰富的 互动团队，Friedman博士和Bonaventura博士将剖析基本控制的特征 控制配体进出活性中心的机制，变构阴离子控制 并阐明了控制氧化毒性的因素和 HbOCs的NO依赖相互作用。

DESCRIPTION (provided by applicant): PEGylation and mutagenic manipulation of Hb and their combination are strategies actively being explored for their potential in generating Hb based oxygen carriers (HBOCs). These modifications are being implemented in an attempt to develop materials that minimize vasoactivity and the production of toxic reactants while maintaining adequate oxygen delivery to tissues. How these modification strategies alter the properties of Hb on a molecular level has not been well addressed. Accordingly, we propose to explore the single and combined biophysical consequences of modifying Hb by PEGylation and mutagenic manipulation. Alterations of conformation on a local and global level, protein dynamics, ligand-binding kinetics, and reactions that affect the use of cell-free Hb as a blood substitute will be exposed and explored. The work will be conducted in two laboratories, those of Dr. Joel Friedman (AECOM), and of Dr. Celia Bonaventura (Beaufort, Duke University). The modified Hbs under study will be examined under varied conditions to clarify underlying structure-function relationships. Hbs will be compared in solution, in sol-gels, and in trehalose glass under conditions that will enable us to identify differences in the heme environment and in energy barriers for ligand binding. Dr. Friedman's efforts will largely entail measurements of resonance Raman spectra, fluorescence, geminate recombination and other fast kinetic processes and transient absorption. Dr. Bonaventura's laboratory will characterize the same modified Hbs with regard to ligand (O2 and NO) binding cooperatively and responses to anionic effectors, formation and stability of S-nitrosated forms and alterations in SH reactivity, oxidative processes, using improved spectroelectrochemical methods and rapid-mixing methods. As an experienced and interactive team, Drs. Friedman and Bonaventura will dissect features of the basic control mechanisms that govern ligand entry and exit from the active site, the allosteric anionic control of heme and SH-group reactivity, and clarify the factors that govern the oxidative toxicity and NO-dependent interactions of HBOCs.