Structural and Dynamics in Allosteric Gene Regulation

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。拟议研究的广泛、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。为了了解变构基因调控，我们在芽孢杆菌中选择了生化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。具体来说，该提案重点关注以下方面的结构和动态基础：(1) 环形 TRAP 11-mer 蛋白通过其变构激活剂色氨酸进行正向调节，以及 (2) Anti-TRAP 3-/12-mer 对 TRAP 进行负向调节。 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 apo 和 Trp 激活的 TRAP 以及组成型活性突变体的溶液结构（目标 1）以及动力学和热力学（目标 2），并通过测量 TRAP 激活的动力学（目标 3）来检验这一假设。我们将采用核磁共振波谱和小角 X 射线散射 (SAXS) 来确定溶液结构和动力学，并采用量热和光谱方法来表征色氨酸介导的 TRAP 激活的热力学和动力学。二.我们假设 Anti-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 灭活 TRAP 的机制，我们将（目标 4）表征 AT 的溶液结构和动力学，（目标 5）研究它们的相互作用并开发 TRAP-AT 复合物的三维模型。了解基因表达的控制是阐明由不当基因调控引起的许多疾病（例如癌症）分子机制的关键一步。蛋白质动力学和变构密切相关。因此，从机制上理解如何通过改变蛋白质行为来实现调节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.