Structure-function relations in Rieske-type proteins

Rieske型蛋白质的结构-功能关系

• 批准号: 7025645
• 负责人: SERGEI A DIKANOV
• 金额: $ 21.99万
• 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 2007-09-03
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7025645
• 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.

铁硫蛋白本质上是普遍存在的，并且发生在大多数代谢途径中，并且对确定其物理化学特性的参数的理解是生物化学的核心问题。 最有趣的是2FE-2S簇，它们是通过两个半胱氨酸和两个组氨酸对铁的配体的共同特征。 其中包括BC1（及相关）配合物的Rieske Iron-硫蛋白（ISP），以及几种Rieske型细菌蛋白，包括Sulfolobus sp的古细菌（SDX）。该项目中的菌株7和Rieske型铁氧还蛋白（ARF）将在该项目中进行研究。 这些蛋白质的一个显着特征是，氧化还原电位的广泛变化，从-100至300 mV，尽管结扎相似，但它们具有2FE -2S簇的表现。 最近的晶体结构显示了该范围两个极端的蛋白质簇结合结构域中配体的相似方向。 这意味着每个特定簇的氧化还原电位和蛋白水解特性受蛋白质环境的其他独特特征的控制。 为了了解蛋白质的功能，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。 我们建议通过使用先进的磁共振技术来研究上面三个Rieske型簇簇的蛋白质环境，并强调2-D ESEEM探索邻里磁磁中心与核旋转之间的相互作用。 我们将分析数据以提供结构信息，并将其与ISP和萘-1,2-二氧酶结构的晶体学进行了比较。 光谱结果将提供限制，以精确确定簇环境，包括组氨酸和半胱氨酸配体的协调，氢键的存在和非协调的硝化物以及溶剂的可及性。 通过使用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蛋白水解特性的局部特征，它们在反应机制中的作用以及在不同菌株中产生功能性修饰的变化。 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中的类似问题提供见解。