Structure-function relations in Rieske-type proteins

Rieske型蛋白质的结构-功能关系

• 批准号: 6435616
• 负责人: SERGEI A DIKANOV
• 金额: $ 22.52万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6435616
• 关键词: X ray crystallography; bacterial proteins; chemical structure; electron spin resonance spectroscopy; ferredoxin; iron sulfur protein; membrane proteins; oxidation; photosynthetic bacteria; physical model; protein structure function; site directed mutagenesis

Iron-sulfur proteins are ubiquitous in nature and occur in most metabolic pathways, and an understanding of the parameters determining their physico-chemical properties is a central problem of biochemistry. Among the most interesting are the 2Fe-2S clusters that have as a common feature the liganding of the irons by two cysteines and two histidines. These include the Rieske iron-sulfur protein (ISP) of the bc1 (and related) complexes, and several Rieske-type bacterial proteins, including the archaeal sulredoxin (SDX) of Sulfolobus sp. Strain 7 and Rieske-type ferredoxin (ARF) from S. solfaricus to be investigated in this project. A remarkable feature of these proteins is the wide variation in redox potentials, from -100 to 300 mV, exhibited by the 2Fe-2S clusters, despite their similar ligation. Recent crystallographic structures show a similar orientation of ligands in the cluster binding domain in proteins at both extremes of this range. This implies that the redox potentials and protolytic properties of each particular cluster are controlled by other unique features of the protein environment. To understand how the proteins function, the factors influencing these parameters must be determined at the atomic level for each protein. We propose to investigate the protein environment of the three Rieske-type clusters above by using advanced magnetic resonance techniques, with an emphasis on 2-D ESEEM to explore the interaction between the paramagnetic centers and nuclear spins in the neighborhood. We will analyze the data so as to provide structural information, and compared this with the crystallographic structured of ISP, and of naphthalene- 1,2-dioxygenase. The spectroscopic results will provide constraints to allow precise determination of the cluster environment including coordination of histidine and cysteine ligands, presence of hydrogen bonds and non-coordinated nitrogens, and accessibility of solvent. By performing similar experiments with mutant strains generated by molecular engineering, we anticipate that we will be able to identify the local features of the protein environment that control the redox and protolytic properties of the clusters, their role in reaction mechanisms, and the changes that produce functional modification in different strains. The results will answer some fundamental questions about structural factors controlling the redox potentials of Rieske-type proteins, and provide insights to similar questions in other redox proteins.

铁硫蛋白在自然界中普遍存在，并存在于大多数代谢途径中，了解决定其物理化学性质的参数是生物化学的核心问题。 其中最有趣的是 2Fe-2S 簇，其共同特征是通过两个半胱氨酸和两个组氨酸配体铁。 其中包括 bc1（及相关）复合物的 Rieske 铁硫蛋白 (ISP)，以及几种 Rieske 型细菌蛋白，包括硫化叶菌属的古细菌硫氧还蛋白 (SDX)。本项目将研究来自 S. solfaricus 的菌株 7 和 Rieske 型铁氧还蛋白 (ARF)。 这些蛋白质的一个显着特征是 2Fe-2S 簇表现出氧化还原电位的广泛变化，从 -100 到 300 mV，尽管它们的连接相似。 最近的晶体结构显示，在该范围的两个极端的蛋白质中，簇结合结构域中的配体具有相似的方向。 这意味着每个特定簇的氧化还原电位和质子分解特性受蛋白质环境的其他独特特征控制。 为了了解蛋白质如何发挥作用，必须在每种蛋白质的原子水平上确定影响这些参数的因素。 我们建议利用先进的磁共振技术研究上述三个 Rieske 型簇的蛋白质环境，重点是 2-D ESEEM 来探索顺磁中心与邻近核自旋之间的相互作用。 我们将分析数据以提供结构信息，并将其与 ISP 和萘-1,2-双加氧酶的晶体结构进行比较。 光谱结果将提供限制条件，以允许精确确定簇环境，包括组氨酸和半胱氨酸配体的配位、氢键和非配位氮的存在以及溶剂的可及性。 通过对分子工程产生的突变菌株进行类似的实验，我们预计我们将能够识别控制簇的氧化还原和质子分解特性的蛋白质环境的局部特征、它们在反应机制中的作用以及产生的变化不同菌株的功能修饰。 结果将回答有关控制 Rieske 型蛋白质氧化还原电位的结构因素的一些基本问题，并为其他氧化还原蛋白质中的类似问题提供见解。