RUI: Disulfide Bond Reduction: A Powerful Chemical Probe for the Study of Structure-Function Relationships in the Hemocyanins

9972667多酚血蓝蛋白是一种高相对分子质量的含铜蛋白质，在许多节肢动物和软体动物中充当氧气载体。尽管氧结合在两种蛋白质的双核铜中心上，但蛋白质的四级结构有很大的不同。晶体结构分析表明，二硫键是两种血蓝蛋白的共同特征。最近的研究表明，血蓝蛋白的二硫键对于其氧结合部位的完整性是必不可少的。这项工作的第一个目标是确定二硫键在维持两类血蓝蛋白中氧结合位点完整性方面的一般重要性。用分光光度法测定氧结合功能和活性中心铜的损失。由于二硫键断裂导致的结构变化将通过凝胶电泳、高效液相色谱、发光光谱和圆二色谱进行分析。这项工作的第二个目的是利用二硫键还原和恢复时氧结合功能的丧失和恢复作为化学探针来确定氧亲和力的亚基间接触和变构调节如何影响节肢动物和软体动物血蓝蛋白氧结合部位的构象和稳定性。大多数动物生活在富氧环境中，依赖有氧代谢。绝大多数都有专门用于将氧气输送到组织内部的特定蛋白质。虽然血红蛋白是研究最广泛的氧转运蛋白，但其他氧转运蛋白也被与血红蛋白一样多的不同物种使用。血蓝蛋白是一种高相对分子质量的含铜蛋白质，在许多节肢动物和软体动物中充当氧气载体。血蓝蛋白引起了研究人员的兴趣，不仅是因为它们的生理作用，还因为它们的结构和功能随着环境条件的变化而发生明显的变化。在这个项目中，一种简单而强大的化学工具，即在蛋白质二硫键还原和恢复时可逆地失去和恢复功能的能力，被用来探索控制血蓝蛋白生理上重要的结构-功能关系的内部和环境因素。控制血蓝蛋白的组装、稳定性和功能的因素具有普遍意义，因为复杂的聚集体进行了许多具有生物学意义的反应，其组装、稳定性和功能类似地受pH、金属结合、特定离子和代谢物的控制。

9972667TophamHemocyanins are high-molecular-weight, copper-containing proteins that serve as oxygen carriers in many arthropods and molluscs. Although oxygen is bound to dinuclear copper centers in both, the quaternary structure of the proteins differ dramatically. Crystal structure analyses show that disulfide bonds are a common feature in both hemocyanin types. Recently, it has been demonstrated that the disulfide bonds of Limulus hemocyanin are essential to the integrity of its oxygen-binding sites. The first objectiveof this work is to determine the general importance of disulfide bonds in maintaining the integrity of the oxygen-binding sites in both classes of hemocyanins. The loss of oxygen-binding function and active-site copper is determined spectrophotometrically. Structural changes that result from the cleavage of the disulfide bonds will be analyzed by gel electrophoresis, high performance liquid chromatography, luminescence spectroscopy, and circular dichroism. A second objective of this work is to use the loss and recovery of oxygen-binding function upon reduction and restoration of the disulfide bonds as a chemical probe to determine how intersubunit contacts and allosteric regulators of oxygen affinity affect the conformation and stability of the oxygen binding sites of arthropod and mollusc hemocyanins.Most animals live in an oxygen-rich environment and rely upon aerobic metabolism. The vast majority have specific proteins specialized for the transport of oxygen to interior tissues. Although hemoglobins are theoxygen transport proteins most extensively studied, other oxygen transport proteins are used by as many different species as the hemoglobins. Hemocyanins are high-molecular-weight, copper-containing proteins that serve as oxygen-carriers in many arthropods and molluscs. The hemocyanins have attracted the interest of researchers, not only because of their physiological role, but also because of the appreciable alterations that occur in their structure and function in response to changes in environmental conditions. In this project, a simple, yet powerful, chemical tool, the ability to reversibly lose and regain function upon thereduction and restoration of protein disulfide bonds, is used to probe the internal and environmental factors that govern physiologically important structure-function relationships in the hemocyanins. The factorsthat govern the assembly, stability, and function of the hemocyanins are of general significance in view of the many reactions of biological importance conducted by complex aggregates whose assembly, stability, and function are similarly controlled by pH, metal binding, specific ions, and metabolites.