Thermodynamic and kinetic studies of protein structure and enzymic mechanisms

蛋白质结构和酶机制的热力学和动力学研究

• 批准号: 8349677
• 负责人: Allen P Minton
• 金额: $ 30.77万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349677
• 关键词: Amyloid Fibrils; Amyloid Proteins; Binding; Biochemical Reaction; Catalysis; Circular Dichroism; Crowding; Detection; Elements; Fluorescence; Functional RNA; Gene Expression; Goals; Investigation; Kinetics; Label; Ligand Binding; Magnesium; Methionine; Methods; Molecular; Nucleic Acids; Optics; Polymers; Proteins; Proteobacteria; Reaction; Secondary Protein Structure; Solutions; Structure; Techniques; Thermodynamics; Work; amyloid peptide; biological systems; interest; intermolecular interaction; protein structure; trimethyloxamine

(i) Methods are being developed for the detection of very low concentrations of flourescently labeled proteins in the presence of very high concentrations of unlabeled proteins and polymers. These techniques will be used to investigate non-specific protein/protein and protein/polymer interactions in solution. (ii) Optical studies on the structures of amyloid peptides and proteins are continuing.We are particularly interested in origin of the very intense CD spectrum which has been associated with the cross-beta structure of amyloid fibrils. (iii) Riboswitches are functional RNA elements typically located in the 5'-region of m-RNAs, involved in modulation of gene expression. A type II riboswitch from proteobacteria binds magnesium and S-adenosyl methionine (SAM). Previous work has shown that binding of these ligands produces a large decrease in molecular volume (Chen et al, in press). We predicted that these reactions should be very sensitive to the presence of molecular crowding agents. Binding of Mg++ and SAM to the riboswitch induces marked changes in it circular dichroism spectrum. High concentrations of the crowder trimethylamine oxide (TMAO) produces similar changes. Using this change for analysis, we can show that increasing concentrations of TMAO facilitates binding of SAM to the riboswitch.

(i) 正在开发在存在非常高浓度的未标记蛋白质和聚合物的情况下检测非常低浓度的荧光标记蛋白质的方法。这些技术将用于研究溶液中非特异性蛋白质/蛋白质和蛋白质/聚合物相互作用。 (ii) 对淀粉样肽和蛋白质结构的光学研究仍在继续。我们对非常强的 CD 光谱的起源特别感兴趣，该光谱与淀粉样蛋白原纤维的交叉 β 结构有关。 (iii) 核糖开关是功能性 RNA 元件，通常位于 mRNA 的 5' 区，参与基因表达的调节。来自变形菌的 II 型核糖开关结合镁和 S-腺苷甲硫氨酸 (SAM)。先前的研究表明，这些配体的结合会导致分子体积大幅减小（Chen 等人，待出版）。我们预测这些反应应该对分子拥挤剂的存在非常敏感。 Mg++ 和 SAM 与核糖开关的结合引起圆二色光谱的显着变化。高浓度的拥挤三甲胺氧化物 (TMAO) 也会产生类似的变化。利用这一变化进行分析，我们可以证明增加 TMAO 浓度有利于 SAM 与核糖开关的结合。