Redox incuced Metal Transfer Reaction of Metalloproteins

氧化还原引起金属蛋白的金属转移反应

• 批准号: 6359162
• 负责人: FEIMENG ZHOU
• 金额: $ 13.59万
• 依托单位: CALIFORNIA STATE UNIVERSITY LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6359162
• 关键词: atomic force microscopy; chemical group; computer data analysis; conformation; electrochemistry; electrodes; electron transport; metalloproteins; metallothionein; molecular film; oxidation reduction reaction; protein transport; scanning electron microscopy; scanning tunneling microscopy; surface coating; surface plasmon resonance; surface property

In the proposed study, various analytical and surface analysis techniques will be employed to characterize and study redox-active proteins immobilized onto metal surfaces. Particular emphasis will be placed on the electrochemical studies of metallothioneins (MTs) and the metal transfer processes accompanying with their electrochemical redox reactions. Metal/electrode surfaces will be modified in such a way that facile electron transfer (ET) reactions are facilitated without significantly altering the stable or physiologically relevant protein structures. A few useful compounds have been identified for electrode modification. Several important parameters that are related to the biological functions of MTs will be determined. These parameters include the redox potential of MT, the number of electroactive groups per MT molecule, and the heterogeneous ET rate constant. Another focus of this proposal will be on the quantification of metals released by surface-confined MT molecules upon electrochemical redox reactions. The general strategy for the proposed work is to verify an existing methodology or to establish a new procedure through the study of a model protein (e.g., cytochrome c for the redox potential measurement and for the study of heterogeneous ET and ferredoxin III for the redox- induced metal transfer). The specific plan will include the application of the following techniques: (1) Using atomic force microscopy and scanning tunneling microscopy to probe the surface coverage and orientation of immobilized MT molecules; (2) Quantifying the amount of protein attachment with quartz crystal microbalance; (3) Studying the redox properties of MT using voltammetrically based techniques (e.g., mediated thin-layer voltammetry and scanning electrochemical microscopy); (4) Examining possible structural changes associated with the MT redox reactions with electrochemical surface plasmon spectroscopy; and (5) Investigating the metal-transfer processes induced (modulated) by the redox reactions of immobilized MT molecules using electrochemistry combined on-line with atomic spectroscopic techniques. The systematic study based on our mufti-technique approach should provide new insight to the understanding of the intricate relationship between the MT redox reaction and the subsequent metal transfer in a cellular milieu. The accurate measurement of the MT redox potential will help elucidate the role of certain biological redox couples (e.g., glutathione) in the MT metal transfer process, whereas the quantification of redox-active groups and metals can allow us to determine the number of redox-active groups accessible for redox reactions and the labile metals for metal transfer in a MT molecule. Finally, we hope to probe the surface configuration and structure of the immobilized MT molecules and use the results to explain the observed redox behavior of MT (e.g., the heterogeneous ET rate and the number of redox-active groups involved).

在本研究中，将采用各种分析和表面分析技术来表征和研究固定在金属表面上的氧化还原活性蛋白。重点将放在金属硫蛋白（MTs）的电化学研究和金属转移过程伴随其电化学氧化还原反应。金属/电极表面将以这样一种方式进行修饰，即在不显著改变稳定或生理相关的蛋白质结构的情况下，促进易电子转移（ET）反应。一些有用的化合物已被确定用于电极修饰。将确定与mt的生物学功能有关的几个重要参数。这些参数包括MT的氧化还原电位，每个MT分子的电活性基团的数量和非均相ET速率常数。本提案的另一个重点将是在电化学氧化还原反应中由表面受限的MT分子释放的金属的量化。拟议工作的总体策略是通过研究模型蛋白（例如，用于氧化还原电位测量的细胞色素c和用于氧化还原诱导的金属转移的异质ET和铁氧化还蛋白III的研究）来验证现有方法或建立新的程序。具体计划将包括以下技术的应用：(1)利用原子力显微镜和扫描隧道显微镜探测固定化MT分子的表面覆盖和取向；(2)用石英晶体微天平定量蛋白质附着量；(3)利用基于伏安法的技术（如介导薄层伏安法和扫描电化学显微镜）研究MT的氧化还原性质；(4)利用电化学表面等离子体光谱技术检测与MT氧化还原反应相关的可能结构变化；(5)利用电化学在线结合原子光谱技术研究固定化MT分子氧化还原反应诱导（调节）的金属转移过程。基于我们的多技术方法的系统研究将为理解细胞环境中MT氧化还原反应与随后的金属转移之间的复杂关系提供新的见解。MT氧化还原电位的精确测量将有助于阐明某些生物氧化还原偶（如谷胱甘肽）在MT金属转移过程中的作用，而氧化还原活性基团和金属的定量可以使我们确定可用于氧化还原反应的氧化还原活性基团的数量以及MT分子中金属转移的不稳定金属。最后，我们希望探索固定化MT分子的表面构型和结构，并利用这些结果来解释观察到的MT的氧化还原行为（例如，多相ET速率和参与氧化还原活性基团的数量）。