VIBRATIONAL STUDIES OF ENERGY TRANSDUCING PROTEINS

能量转换蛋白的振动研究

• 批准号: 6525600
• 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/6525600
• 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).

拟议的研究的具体目标是表征反应中心（RC）蛋白质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（RR）光谱。 第一个主要目标是表征转基因细菌RC中的细菌叶绿素/细菌脱镁叶绿素（BCh 1/BPh）的特性，这些转基因细菌RC表现出不寻常的电子转移特性。 遗传修饰包括在初级电子供体（P）附近的替换，两个辅助BChl，以及在蛋白质的具有免疫活性的L分支（其是初级电子受体）上的BPh。研究集中在三个一般类别的转基因RC：（1）突变体，其中的氢键相互作用和/或附近的电场BCh 1 L，BCh 1 M，和BPhL的BCh 1/BPh大环的V环附近的氨基酸残基的添加/删除改变。 这些研究的一个特别的重点是将（潜在的）带电残基附近的光活性辅因子的影响。(2)双（和更高阶）突变体，其将1类特征的替换纳入BPhL被BCh 1分子替换的背景中（β型RC）。(3)其中P的BChl的组氨酸轴向配体被非连接甘氨酸残基取代的突变体（空腔突变体）。在所有情况下，RR研究将在RC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。 第二个主要目标是进行RR研究，旨在改进光系统II RC中放氧复合物的结构。 这些研究的特定目标是锰簇，其直接介导水裂解/析氧反应。 重点将是低频区域的频谱，锰配体的振动预计会发生。 为此，将采集PSII的RR数据，其中同位素标记（2 H、18 O、15 N、37 C1-/37 C1-）已掺入和/或必需离子如Ca+2和C1-已交换（例如，Sr+2交换Ca+2或Br-交换C1-）。 对细菌和PSII RC的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）如何支配和/或反映其功能特性（电子转移/跨生物膜的电荷分离;水分解/氧释放）。