HEME PROTEIN STRUCTURE AND FUNCTION

血红素蛋白结构和功能

• 批准号: 6283801
• 负责人: JACK PEISACH
• 金额: $ 43.19万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-05-01 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6283801
• 关键词: DNA damage; Raman spectrometry; amino group; analog; binding sites; bleomycin; cobalt; electron spin resonance spectroscopy; guanine; hemoprotein; hydrogen bond; hydrogen peroxide; iron; mass spectrometry; nuclear magnetic resonance spectroscopy; oligonucleotides; protein structure function; pyrimidine analog; stoichiometry

DESCRIPTION: (Verbatim from the Applicant's Abstract) One focus of this study is to further understand the role of specific domains of the activated form of the anticancer antibiotic bleomycin (BLM) in site recognition of DNA cleavage. Present understanding of site recognition is based on a model whereby a ligand of Fe in the drug, amino pyrimidine, H-bonds with guanine in DNA. However, tallysomycin (TLM), a BLM congener containing the same metal ligands and additionally an L-talose away from the metal binding site, has different site specificity, whereas removing the bithiazole moiety as in pseudotetrapeptide A (PTP) abolishes site specificity. This suggests that the metal binding site alone does not govern site recognition. A series of experiments are therefore proposed that aim at understanding site recognition in FeTLM and PTP with DNA. It is hypothesized that the difference in structure of TLM as compared to BLM is manifest in a direct effect on the binding of drug to DNA and an indirect effect on the electron distribution in the metal binding site and the oxygenous ligands of Fe, thereby altering activity. This study will include an NMR investigation of HOO-Co(III)TLM both in the presence and absence of DNA to elucidate the 3-D structure, an ESEEM study of O2-Co(II) and Fe(III)TLM with specifically deuterated oligonucleotides to determine the distance from the metal to the targeted C4,H of deoxyribose as well as (1) the mechanism of formation and the structure of the activated complexes of TLM and PTP using mass spec and (2) the rate and stoichiometry of products formed in the reaction in single and multiple turnover experiments with DNA to relate activity to kinetic competence and reaction mechanism. A second focus is to carry out EPR, ESEEM, and HYSCORE (2D ESEEM) studies of the deoxy Co subunits of Fe-Co hybrid hemoglobins (Hbs) that are in the R, T, and the newly proposed T' (monoligated and diligated tetramers in which two subunits in the same dimer are ligated) quaternary state. These studies will examine electron-nuclear coupling to the metal and its nitrogen ligands, as well as relative orientation of the axial His imidazole, in order to test the hypotheses that there exists a quaternary state structurally distinct from the T and R state in the Monod-Wyman-Changeux two-state model, and that quaternary structural change is accompanied by structural change in the metal center of the alpha-subunits only.

描述：（申请人摘要中的逐字记录）本研究的一个重点 是为了进一步了解激活形式的特定结构域的作用， 抗癌抗生素博莱霉素（BLM）在DNA切割位点识别中的作用。 目前对位点识别的理解是基于一种模型， 药物中的铁，氨基嘧啶，DNA中与鸟嘌呤的氢键。然而，在这方面， 他来霉素（TLM），一种含有相同金属配体的BLM同源物， 另外，L-塔罗糖远离金属结合位点，具有不同的位点， 特异性，而在假四肽A中去除双噻唑部分 (PTP)消除了位点特异性。这表明金属结合位点 并不能单独管理网站识别。因此，进行了一系列实验， 提出的目的是了解FeTLM和PTP与DNA的位点识别。 据推测，与BLM相比，TLM结构上的差异 表现为对药物与DNA结合的直接影响和对药物与DNA结合的间接影响。 对金属结合位上电子分布的影响和对含氧化合物 Fe的配体，从而改变活性。这项研究将包括核磁共振 HOO-Co（III）TLM在DNA存在和不存在下的研究， 阐明3-D结构，ESEEM研究O2-Co（II）和Fe（III）TLM， 特异性氘代寡核苷酸，以确定与 金属对脱氧核糖的靶向C4，H的作用以及（1） TLM和PTP的活化复合物的形成和结构， 质谱和（2）反应中形成的产物的速率和化学计量 在DNA的单次和多次周转实验中， 动力学能力和反应机理。第二个重点是进行生产者延伸责任审查， ESEEM和HYSCORE（2D ESEEM）研究了Fe-Co杂化材料的脱氧Co亚基 血红蛋白（Hb）在R、T和新提出的T'（单连接）中， 和其中同一二聚体中的两个亚基连接的双链四聚体） 四元态这些研究将检查电子核耦合到 金属及其氮配体，以及轴向的相对取向 他的咪唑，为了验证存在季铵化合物的假设， 在结构上不同于Monod-Wyman-Changeux中的T和R态的状态 两态模型，并且四级结构变化伴随着 仅在α亚基的金属中心发生结构变化。