VIBRATIONAL STUDIES OF ENERGY TRANSDUCING PROTEINS

能量转换蛋白的振动研究

• 批准号: 6192238
• 负责人: DAVID F. BOCIAN
• 金额: $ 18.44万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6192238
• 关键词: Raman spectrometry; Rhodospirillales; bacterial pigment; biophysics; chemical bond resonance; chemical kinetics; chlorophyll; cofactor; conformation; electric field; electron density; electron transport; electronic spectra; photochemistry; photosynthetic bacteria; photosynthetic reaction centers; protein structure function; vibration

The specific aims of the proposed research are to characterize the structural, vibronic, and electronic properties of the functionally important cofactors in reaction center (RC) proteins. The principal investigative tool is resonance Raman (RR) spectroscopy. The first major objective is to characterize the properties of the bacteriochlorophyll/bacteriopheophytin (BCh1/BPh) in genetically modified bacterial RCs that exhibit unusual electron-transfer properties. The genetic modifications include replacements near the primary electron donor (P), both accessory BChls, and the BPh on the photophysically, active L branch of the protein (which is the primary electron acceptor). The studies focus on three general classes of genetically modified RCs: (1) Mutants in which the hydrogen bonding interactions and/or the electric fields in the vicinity of BCh1L, BCh1M, and BPhL are altered by addition/deletion of amino acid residues near ring V of the BCh1/BPh macrocycle. A particular focus of these studies concerns the effects of placing (potentially) charged residues near the photoactive cofactors. (2) Double (and higher order) mutants which incorporate the replacements characteristic of class 1 into a background in which BPhL is replaced with a BCh1 molecule (beta-type RCs). (3) Mutants in which the histidine axial ligands to the BChls of P are replaced by non-ligating glycine residues (cavity mutants). In all cases the RR studies will be conducted on RCs whose detailed electron-transfer kinetics have been elucidated via time-resolved optical experiments. The second major objective is to conduct RR studies aimed at refining the structure of oxygen- evolving complex in photosystem (PS) II RCs. The particular target of these studies is the manganese cluster, which directly mediates the water-splitting/oxygen-evolution reaction. The focus will be the low-frequency region of the spectrum where manganese-ligand vibrations are expected to occur. Toward this end, RR data will be acquired for PSII in which isotopic labels (2H, 18O, 15N, 37C1-/37C1- ) have been incorporated and/or essential ions such as Ca+2 and C1- have been exchanged (for example, Sr+2 for Ca+2 or Br- for C1-). The long-term objective of the studies on bacterial and PSII RCs is to determine how the physical properties of the cofactors (structure, conformation, electron-density distribution) govern and/or reflect their functional characteristics (electron transfer/charge separation across the biological membrane; water splitting/oxygen evolution).

本研究的具体目的是表征反应中心（RC）蛋白中功能重要的辅因子的结构、振动和电子特性。主要的研究工具是共振拉曼光谱（RR）。第一个主要目标是表征转基因细菌RCs中细菌叶绿素/细菌叶绿素（BCh1/BPh）的特性，这些细菌RCs表现出不同寻常的电子转移特性。遗传修饰包括主要电子供体(P)附近的替换，两个辅助bchl，以及光物理上的BPh，活性的蛋白质L分支（这是主要的电子受体）。研究主要集中在三大类转基因RCs上：(1)突变体中BCh1/BPh大环V环附近氨基酸残基的添加/删除改变了BCh1L、BCh1M和BPhL附近的氢键相互作用和/或电场。这些研究的一个特别焦点是在光活性辅助因子附近放置（可能）带电残基的影响。(2)双重（和高阶）突变体，将1类的替换特征合并到BPhL被BCh1分子取代的背景中（β型RCs）。(3) P的组氨酸轴向配体被非连接的甘氨酸残基取代的突变体（空腔突变）。在所有情况下，RR研究将在RCs上进行，其详细的电子转移动力学已通过时间分辨光学实验阐明。第二个主要目标是开展旨在完善光系统（PS） II RCs中进化氧复合物结构的RR研究。这些研究的特定目标是锰簇，它直接介导水裂解/析氧反应。重点将是频谱的低频区域，在那里锰配体振动预计会发生。为此，将获取PSII的RR数据，其中包含同位素标记（2H, 18O, 15N, 37C1-/37C1-），并且/或交换了Ca+2和C1-等必需离子（例如，Sr+2交换Ca+2或Br-交换C1-）。细菌和PSII RCs研究的长期目标是确定辅因子的物理性质（结构、构象、电子密度分布）如何控制和/或反映其功能特征（电子转移/跨生物膜的电荷分离；水分裂/析氧）。