Structural Biochemistry of Metalloprotein Biosensors

金属蛋白生物传感器的结构生物化学

• 批准号: 7183528
• 负责人: John A. Tainer
• 金额: $ 31.15万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7183528
• 关键词: Aconitate Hydratase; Address; Affinity; Architecture; Bacterial Proteins; Binding; Binding Proteins; Binding Sites; Biochemical; Biochemistry; Biosensor; Cells; Charge; Chemistry; Class; Complex; Coupled; Coupling; Crystallization; Crystallography; DNA Binding; DNA Binding Domain; DNA Sequence Rearrangement; Databases; Deposition; Depth; Detection; Electrons; Environment; Enzymes; Event; Feedback; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic; Goals; Homeostasis; Immune response; Immune system; In Vitro; Ions; Iron; Iron-Regulatory Proteins; Ligands; Link; Mammalian Cell; Mediating; Messenger RNA; Metabolic; Metabolism; Metal Binding Site; Metal Ion Binding; Metalloproteins; Metals; Molecular Conformation; Mutagenesis; Mutation; N-terminal; Nucleic Acid Binding; Nucleic Acids; Organism; Oxidation-Reduction; Oxidative Stress; Oxidative Stress Pathway; Oxygen; Pathogenesis; Peroxides; Play; Protein Conformation; Protein Databases; Proteins; RNA Binding; Range; Regulation; Role; Scaffolding Protein; Signal Transduction; Site; Spectrum Analysis; Stress; Structural Biochemistry; Structural Chemistry; Structure; Sulfur; Superoxides; Surface; Testing; Transcription Coactivator; Translations; Virulence Factors; X-Ray Crystallography; Zinc; base; combinatorial; density; design; human WNT2 protein; iron metabolism; mutant; novel; oxidation; pathogen; protein function; protein protein interaction; protein structure; response; sensor; small molecule; theories; uptake

DESCRIPTION (provided by applicant): This new proposal focuses on cross-disciplinary characterizations of protein metal ion based biosensors. These biosensors include key virulence factors that act in pathogenesis. All these proteins influence cellular metabolism through the metal-mediated control of gene expression by nucleic acid interactions. This function of these protein biosensors involves metal-induced protein conformational changes expected to be large and suitable for elucidation by the proposed crystallography characterizations. These structural efforts will be synergistically coupled with biochemistry, genetics, and spectroscopy results. Structural and mutational studies of the bacterial transcriptional regulators ferric uptake regulator (Fur) and the zinc uptake regulator (Zur) will reveal how metal ion binding is structurally linked to rearrangements of a DNA binding surface. Importantly, metal binding provides sufficient binding energy to drive protein conformational changes yet maintain sufficient affinity so that these changes are triggered at physiologically relevant concentrations for the control of bacterial pathogenesis. The superoxide stress regulator (SoxR) acts as a redox sensor to drive pathogen responses to oxidative stress induced by the environment and the mammalian immune response. Conformational changes that convert SoxR from a repressor to an activator of transcription are induced via the single electron oxidation of a [2Fe-2S] cluster. SoxR crystal structures in apo and in metal-bound, defined oxidation states will be combined with site directed mutants and density functional theory calculations of changes in partial charge distribution to delineate the mechanism by which this information is transduced through the protein architecture. The mammalian iron regulatory protein (IRP1) uses the assembly of a [4Fe-4S] cluster as an iron sensor to regulate the translation of genes involved in iron metabolism. Crystal structures of apo IRP in complex with its RNA binding target will reveal the means whereby the protein can stabilize two discrete conformations. Together, these aims will address mechanisms by which metal ion switches are coupled to protein conformational changes that allow pathogens and mammalian to sense small molecule ligands, redox states, and metal concentration within the cell.

描述（由申请人提供）：该新提案的重点是基于蛋白质金属离子生物传感器的跨学科特征。 这些生物传感器包括在发病机理中起作用的关键毒力因子。所有这些蛋白质通过核酸相互作用对金属介导的基因表达的控制影响细胞代谢。 这些蛋白质生物传感器的这种功能涉及金属诱导的蛋白质构象变化，预计会大，适合通过所提出的晶体表征阐明。 这些结构性努力将与生物化学，遗传学和光谱结果协同结合。细菌转录调节剂的结构和突变研究铁摄取调节剂（Fur）和锌摄取调节剂（ZUR）将揭示金属离子结合如何在结构上与DNA结合表面的重排结合。重要的是，金属结合提供了足够的结合能以驱动蛋白质构象变化，但保持足够的亲和力，以便在生理相关的浓度下触发这些变化以控制细菌发病机理。超氧化应激调节剂（SOXR）充当氧化还原传感器，以驱动病原体对环境引起的氧化应激的反应和哺乳动物免疫反应。通过[2FE-2S]簇的单电子氧化诱导SOXR从阻遏物转换为转录激活因素的构象变化。 APO和金属结合的SOXR晶体结构将与位置的突变体和密度功能理论理论结合，对部分电荷分布中变化的变化计算，以描述通过蛋白质结构传递该信息的机制。哺乳动物铁调节蛋白（IRP1）使用[4FE-4S]簇的组装作为铁传感器来调节与铁代谢有关的基因的翻译。 Apo IRP的晶体结构与其RNA结合靶标的复合物中，将揭示蛋白质可以稳定两个离散构象的手段。总之，这些目标将解决金属离子开关与蛋白质构象变化耦合的机制，从而使病原体和哺乳动物可以感知细胞内的小分子配体，氧化还原态和金属浓度。