PROTEIN-PROTEIN INTERACTIONS AND REGULATION

本提案中的实验旨在探索 色氨酸与色氨酸结合的物理化学机制 E.大肠杆菌导致其靶操纵子的阻遏。的 特别令人感兴趣的是蛋白质-蛋白质相互作用在 调控转录的结构基础的模型 连接、寡聚化和DNA结合之间的变构连接将 从热力学和动力学特性的一系列 阻遏物的突变体表现出改变的功能特性。 本提案的具体目标是：（1）充分描述 突变体的连接、寡聚化和DNA结合， 表现出改变的辅阻遏物和DNA结合; 2）研究 这些突变蛋白的动力学行为; 3）确定它们的相对 稳定性并表征其折叠性质。相互作用 多肽链之间的相互作用可以调节基因表达 在真核生物以及原核生物中，影响亲和力和 参与生长的转录调节蛋白的特异性， 发展和转型。 调节转录的能力 在治疗上是非常理想的，但最终将取决于 了解其调节的潜在物理机制。 一 对原型系统如Trp阻遏物的详细研究将 对实验和理论框架做出了重大贡献， 研究不太明确的，但医学上重要的真核生物 转录调节因子。 非变性PAGE、光散射、色谱法 和沉积技术将用于确定物种数量和 化学计量结合时间分辨和稳态 荧光光谱将用于获得低聚，配体 结合和DNA结合概况，以及评估动态 折叠蛋白质的性质、稳定性和完整性。这些 荧光方法将涉及监测稳态和时间- 内在蛋白质荧光的分辨强度和各向异性 和共价结合到蛋白质或 DNA.由于其实验的多功能性和灵敏度， 光谱学特别适合于研究这种复杂的 大分子相互作用

The experiments in this proposal are designed to probe the fundamental physical-chemical mechanisms by which tryptophan binding to the trp repressor of E. coli results in the repression of its target operons. Of particular interest is the role of protein-protein interactions in regulation transcription. A model for the structural basis of the allosteric linkages between ligation, oligomerization and DNA binding will be derived from the thermodynamic and dynamic characterization of a series of mutants of the repressor which exhibit altered functional properties. The specific aims of this proposal are to l) fully characterize the ligation, oligomerization and DNA binding of mutants which have been shown to exhibit altered corepressor and DNA binding; 2) investigate changes in the dynamic behavior of these mutant proteins; 3) determine their relative stabilities and characterize their folding properties. Interactions between polypeptide chains have been shown to modulate genetic expression in eukaryotes as well as prokaryotes, affecting both the affinity and the specificity of transcriptional regulatory proteins involved in growth, development and transformation. The ability to modulate transcription therapeutically is highly desirable, but will ultimately depend upon understanding the underlying physical mechanisms of its regulation. A detailed investigation of a prototypic system such as trp repressor will contribute significantly to the experimental and theoretical framework for studying less well-characterized, yet medically important eukaryotic transcriptional regulators. Native PAGE, light scattering, chromatography and sedimentation techniques will be used to identify species number and stoichiometries. A combination of time-resolved and steady-state fluorescence spectroscopy will be used to obtain oligomerization, ligand binding and DNA binding profiles as well as to assess the dynamic properties, stability and integrity of the folded proteins. These fluorescence approaches will involve monitoring the steady-state and time- resolved intensity and anisotropy of the intrinsic protein fluorescence and of extrinsic fluorophores covalently bound to either the protein or to DNA. Due to its experimental versatility and sensitivity, fluorescence spectroscopy is particularly well-suited to the study of such complex macromolecular interactions.