Structural and Dynamics in Allosteric Gene Regulation

描述（由申请人提供）：严格控制基因表达对正常细胞发育、对环境信号的反应、生长和增殖至关重要。该研究的长远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一个生化可调控的基因调控系统：通过低聚TRAP和Anti-TRAP蛋白控制色氨酸生物合成基因。具体来说，本研究的重点是表征(1)环形TRAP 11-mer蛋白的变构激活剂色氨酸对其的正调控，以及(2)Anti-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激活TRAP。我们将通过比较载脂蛋白和trp激活的TRAP的溶液结构（目标1）、动力学和热力学（目标2）以及组成活性突变体，并通过测量TRAP激活动力学（目标3）来验证这一假设。我们将使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，并使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。2。我们假设Anti-TRAP （AT）以不对称的方式结合TRAP，蛋白质动力学对于三聚体/十二聚体AT特异性识别非美聚体（11-mer） TRAP至关重要。为了深入了解AT对TRAP失活的机制，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由基因调控不当引起的疾病（如癌症）的分子机制的关键一步。蛋白质动力学和变构是密切相关的；因此，对调节如何通过改变蛋白质行为实现的机制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。

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.