Structural and Dynamics in Allosteric Gene Regulation

变构基因调控的结构和动力学

• 批准号: 7470022
• 负责人: MARK P. FOSTER
• 金额: $ 28.8万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7470022
• 关键词: Anabolism; Bacillus (bacterium); Behavior; Binding; Calorimetry; Cells; Chemicals; Controlled Study; Coupled; Coupling; Data; Development; Disease; Dissociation; Fostering; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Growth; Heteronuclear NMR; Hydrogen; In Vitro; Kinetics; Lead; Ligand Binding; Ligands; Link; Malignant Neoplasms; Measures; Mediating; Methods; Molecular; Motion; Mutagenesis; NMR Spectroscopy; Play; Property; Protein Dynamics; Proteins; RNA Binding; Rate; Regulation; Regulator Genes; Relaxation; Research; Research Personnel; Residual state; Roentgen Rays; Role; Shapes; Signal Transduction; Solutions; Structure; System; TRAP Complex; Testing; Therapeutic; Thermodynamics; Titrations; Tryptophan; Vertebral column; Work; base; insight; mutant; prevent; programs; protein folding; protein protein interaction; research study; response; restraint; three dimensional structure; three-dimensional modeling

DESCRIPTION (provided by applicant): Tight control over gene expression is critical for normal cellular development, response to environmental signals, growth and proliferation. The broad, long-term objective of the proposed research is to understand the mechanisms by which cells use ligand-protein and protein-protein interactions to allosterically regulate gene expression. To understand allosteric gene regulation we have chosen a biochemically tractable gene regulatory system in Bacilli: control of tryptophan biosynthesis genes by the oligomeric TRAP and Anti-TRAP proteins. Specifically, this proposal focuses on characterizing the structural and dynamic basis for (1) positive regulation of the ring-shaped TRAP 11-mer protein by its allosteric activator, tryptophan, and (2) negative regulation of TRAP by the Anti-TRAP 3-/12-mer. I. We hypothesize that tryptophan binding activates TRAP by altering the protein's dynamic behavior. We will test this hypothesis by comparing the solution structures (aim 1) and dynamics and thermodynamics (aim 2) of apo and Trp-activated TRAP, as well as a constitutively active mutant, and by measuring the kinetics of TRAP activation (aim 3). We will employ NMR spectroscopy and small angle X-ray scattering (SAXS) to determine solution structure and dynamics, and calorimetric and spectroscopic methods to characterize thermodynamics and kinetics of tryptophan-mediated TRAP activation. II. We hypothesize that Anti-TRAP (AT) binds TRAP in an asymmetric fashion and that protein dynamics is critical for specific recognition of undecameric (11-mer) TRAP by trimeric/dodecameric AT. To provide insight into the mechanism of TRAP inactivation by AT, we will (aim 4) characterize the solution structure and dynamics of AT, (aim 5) study their interaction and develop a three-dimensional model of the TRAP-AT complex. Understanding the control of gene expression is a crucial step toward elucidating the molecular mechanisms of many diseases caused by improper gene regulation, such as cancer. Protein dynamics and allostery are intimately linked; therefore, mechanistic understanding of how regulation is achieved by changing protein behavior will help us to understand and ultimately control gene expression as part of a therapeutic strategy.

描述（由申请人提供）：对基因表达的严格控制对于正常细胞发育、对环境信号的反应、生长和增殖至关重要。这项研究的广泛、长期目标是了解细胞利用配体-蛋白质和蛋白质-蛋白质相互作用来变构调节基因表达的机制。为了理解变构基因调控，我们选择了一种在芽孢杆菌中生物化学上易于处理的基因调控系统：通过寡聚TRAP和Anti-TRAP蛋白控制色氨酸生物合成基因。具体而言，该提案侧重于表征（1）通过其变构激活剂色氨酸对环形TRAP 11-mer蛋白的正调控和（2）通过抗TRAP 3-/12-mer对TRAP的负调控的结构和动力学基础。I.我们假设色氨酸结合通过改变蛋白质的动态行为来激活TRAP。我们将通过比较载脂蛋白和色氨酸激活的TRAP以及组成型活性突变体的溶液结构（目的1）和动力学和热力学（目的2），以及通过测量TRAP激活的动力学（目的3）来测试这一假设。我们将采用核磁共振光谱和小角X射线散射（SAXS），以确定解决方案的结构和动力学，量热和光谱方法来表征热力学和动力学的Dahan介导的TRAP激活。二.我们假设抗TRAP（AT）以不对称方式结合TRAP，并且蛋白质动力学对于三聚体/十二聚体AT特异性识别十一聚体（11聚体）TRAP至关重要。为了深入了解AT对TRAP的失活机制，我们将（目的4）表征AT的溶液结构和动力学，（目的5）研究它们的相互作用并建立TRAP-AT复合物的三维模型。了解基因表达的控制是阐明许多疾病（如癌症）的分子机制的关键一步。蛋白质动力学和变构密切相关;因此，对如何通过改变蛋白质行为实现调节的机械理解将有助于我们理解并最终控制基因表达作为治疗策略的一部分。