PROTEIN-PROTEIN INTERACTIONS AND REGULATION

这一方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 大肠杆菌的抑制子导致其目标操纵子的抑制。的 特别令人感兴趣的是蛋白质-蛋白质相互作用在 调节转录。的结构基础的模型 连接、寡聚和DNA结合意志之间的变构联系 是由一系列的热力学和动力学表征而来的 表现出功能特性改变的阻遏物的突变体。 这项建议的具体目的是为了充分描述L) 已显示的突变体的连接、寡聚和DNA结合 显示辅阻遏子和DNA结合的变化；2)研究 这些突变蛋白的动态行为；3)确定它们的相对 稳定性，并表征其折叠性能。互动 多肽链之间的相互作用已被证明可以调节基因表达 在真核生物和原核生物中，影响亲和力和 参与生长的转录调节蛋白的特异性， 发展转型。调节转录的能力 在治疗上是非常可取的，但最终将取决于 了解其调节的潜在物理机制。一个 对一个原型系统的详细研究，如Trp阻遏器Will 对实验和理论框架做出重大贡献 研究特征不是很好但医学上很重要的真核生物 转录调控因子。天然PAGE、光散射、层析 和沉积技术将用于识别物种数量和 化学计量学。时间分辨和稳态的组合 将荧光光谱用于获得齐聚物、配体 结合和DNA结合图谱以及评估动态 折叠蛋白的性质、稳定性和完整性。这些 荧光方法将涉及监测稳态和时间- 内源蛋白质荧光的分辨强度和各向异性 以及外源荧光团共价结合到蛋白质或结合到 DNA由于它的实验通用性和敏感性，荧光 光谱学特别适合于研究这种络合物。 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.