Structural and Dynamics in Allosteric Gene Regulation

描述(申请人提供)：对基因表达的严格控制对于正常的细胞发育、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-蛋白质相互作用来调节基因表达的机制。为了了解变构基因的调控，我们在芽孢杆菌中选择了一个生化上容易控制的基因调控系统：通过寡聚体TRAP和抗TRAP蛋白控制色氨酸生物合成基因。具体地说，本建议重点描述了(1)变构激活剂色氨酸对环状TRAP 11-mer蛋白的正调控和(2)抗TRAP 3-/12-mer对TRAP的负调控的结构和动力学基础。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apo和Trp激活的TRAP的溶液结构(目标1)、动力学和热力学(目标2)以及一个结构性活性突变体，并通过测量TRAP激活的动力学(目标3)来验证这一假设。我们将使用核磁共振光谱和小角X射线散射(SAXS)来确定溶液的结构和动力学，并使用量热和光谱方法来表征色氨酸介导的陷阱活化的热力学和动力学。II.我们假设抗TRAP(AT)以不对称的方式与TRAP结合，蛋白质动力学对于三聚体/十二聚体AT对十一聚体(11-聚)陷阱的特异性识别至关重要。为了深入了解AT使陷阱失活的机理，我们将(目标4)表征AT的溶液结构和动力学，(目标5)研究它们之间的相互作用，并建立一个陷阱-AT复合体的三维模型。了解基因表达的调控对于阐明许多疾病的分子机制是至关重要的一步，这些疾病是由基因调控不当引起的，如癌症。蛋白质动力学和变构是紧密联系在一起的；因此，从机制上理解通过改变蛋白质行为来实现调控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。

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.