PROTEIN-PROTEIN INTERACTIONS AND REGULATION

蛋白质-蛋白质相互作用和调节

• 批准号: 2180122
• 负责人: CATHERINE A ROYER
• 金额: $ 15.14万
• 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/2180122
• 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讨基本原理