PROTEIN-PROTEIN INTERACTIONS AND REGULATION

蛋白质-蛋白质相互作用和调节

• 批准号: 2180121
• 负责人: CATHERINE A ROYER
• 金额: $ 13.99万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-12-01 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2180121
• 关键词: DNA binding protein; bacterial genetics; chemical binding; chemical stability; conformation; dimer; fluorescence spectrometry; fluorescent dye /probe; gel mobility shift assay; gene induction /repression; genetic operator element; genetic regulation; lasers; light scattering; mutant; oligonucleotides; polymerization; protein denaturation; protein folding; protein purification; protein structure function; stoichiometry; thermodynamics; transcription factor; tryptophan

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.

本提案中的实验旨在探索基本原理 色氨酸与色氨酸结合的物理化学机制 大肠杆菌的阻遏蛋白会抑制其目标操纵子。的 特别感兴趣的是蛋白质-蛋白质相互作用在 调控转录。结构基础模型 连接、寡聚和 DNA 结合之间的变构连接将 来自一系列的热力学和动态特性 阻遏蛋白突变体表现出改变的功能特性。 该提案的具体目标是 l) 充分描述 已显示的突变体的连接、寡聚和 DNA 结合 表现出改变的辅阻遏物和 DNA 结合； 2）调查变化 这些突变蛋白的动态行为； 3）确定其亲属 稳定性并表征其折叠特性。互动 多肽链之间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， 发展与转型。 调节转录的能力 治疗上是非常可取的，但最终取决于 了解其调节的潜在物理机制。 一个 对原型系统（例如色氨酸阻遏物）的详细研究将 为实验和理论框架做出了重大贡献 研究特征不太明确但具有医学重要性的真核生物 转录调节因子。 非变性 PAGE、光散射、色谱法 和沉淀技术将用于确定物种数量和 化学计量。时间分辨和稳态的结合 荧光光谱将用于获得寡聚、配体 结合和 DNA 结合谱以及评估动态 折叠蛋白质的特性、稳定性和完整性。这些 荧光方法将涉及监测稳态和时间 解析内在蛋白质荧光的强度和各向异性 以及与蛋白质或共价结合的外在荧光团 脱氧核糖核酸。由于其实验多功能性和灵敏度，荧光 光谱学特别适合研究这种复杂的物质 大分子相互作用。