HEME PROTEIN STRUCTURE AND FUNCTION

描述：(逐字摘自申请者的摘要)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在DNA裂解的定位识别中的作用 目前对位点识别的理解是基于一种模型，即配体 药物中的铁、氨基嘧啶、DNA中的鸟嘌呤与H键。然而， Tallysmycin(TLM)，一种含有相同金属配体的BLM同系物 另外一个L-塔尔糖远离金属结合部位，有不同的结合部位 特异性，而去除伪四肽A中的双噻唑部分 (PTP)取消了站点专一性。这表明金属结合部位 仅凭这一点并不能管理网站认知度。因此，我们进行了一系列实验 目的在于了解DNA在FeTLM和PTP中的位置识别。 推测TLm与BLm在结构上的差异 表现在对药物与DNA结合的直接影响和间接作用 对金属结合位电子分布和含氧量的影响 铁的配体，从而改变活性。这项研究将包括核磁共振 DNA存在和不存在时HoO-Co(III)TLm的研究 O_2-Co(II)和Fe(III)TLM的ESEEM研究 用特定的氢化寡核苷酸来确定与 金属与脱氧核糖的靶向C4，H以及(1)作用机理 TLM和PTP活性络合物的形成和结构 质谱仪和(2)在反应中形成的产物的速率和化学计量比 在单周转和多次周转实验中，用DNA将活动与 动力学能力和反应机理。第二个重点是开展环境保护责任， ESEEM和HYSCORE(2D ESEEM)对Fe-Co杂化脱氧钴亚基的研究 R、T和新提出的T‘(单寡化)中的血红蛋白(HBs) 和勤奋的四聚体，其中同一二聚体中的两个亚基被连接) 第四纪状态。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，以检验存在第四纪的假设 与Monod-Wyman-Changeux中的T和R态结构不同的态 两态模型，并且伴随着第四纪结构的变化 仅在阿尔法亚基的金属中心发生结构变化。

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.