Thermodynamic and kinetic studies of macromolec structure and enzymic mechanisms

大分子结构和酶机制的热力学和动力学研究

• 批准号: 8741362
• 负责人: Allen P Minton
• 金额: $ 20.29万
• 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/8741362
• 关键词: Accounting; Affinity; Amyloid; Amyloid Fibrils; Amyloid Proteins; Binding; Biochemical Reaction; Catalysis; Circular Dichroism; Collaborations; Crowding; Dependence; Fluorescence; Goals; Investigation; Kinetics; Ligands; Measurement; Methionine; Modeling; Molecular; Molecular Conformation; Nucleic Acids; Optics; Peptides; Secondary Protein Structure; Structure; Techniques; Thermodynamics; amyloid peptide; beta pleated sheet; biological systems; interest; intermolecular interaction; trimethyloxamine

(i) 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. This phenomenon is shown by peptides which have been shown to form amyloid with both parallel and anti-parallel beta sheet structure. (ii) In collaboration with the group of T. K. Dayie and B. Chen (Univ. of MD), we have investigated the effect of a small inert cosolute, trimethylamine oxide (TMAO), on the conformational changes of the SAM II riboswitch in the presence of increasing concentrations of the metabolite S-adenosyl-methionine (SAM) and Mg++. The free riboswitch was thought to be in an open or semi-random conformation in the absence of SAM and Mg++ and to become more folded and compact upon binding SAM and/or Mg++. Extensive measurements of the dependence of circular dichroism spectra of the riboswitch upon the concentrations of these small ligands may be quantitatively accounted for by a model, according to which SAM binds exclusively to a compact conformation. In the absence of SAM, the riboswitch seems to be a mixture of open and compact states. Low concentrations of Mg++ or TMAO bind to the open form(s), inhibiting the binding of SAM. At higher concentrations, cooperative binding of Mg++ or molecular crowding by TMAO, transform the riboswitch into the compact form, facilitating the binding of SAM. Further binding of Mg++ increases the affinity of the compact form of the riboswitch for SAM. Hydrodynamic measurements show that the riboswitch is compacted by concentrated TMAO.

(i) 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。这种现象通过肽表现出来，这些肽已被证明可形成具有平行和反平行β片层结构的淀粉样蛋白。 (ii) 与 T. K. Dayie 和 B. Chen（马里兰大学）小组合作，我们研究了一种小的惰性共溶物三甲胺氧化物 (TMAO) 对 SAM II 核糖开关构象变化的影响代谢物 S-腺苷甲硫氨酸 (SAM) 和 Mg++ 浓度不断增加。游离核糖开关被认为在不存在SAM和Mg++的情况下处于开放或半随机构象，并且在结合SAM和/或Mg++时变得更加折叠和紧凑。核糖开关的圆二色性光谱对这些小配体浓度的依赖性的广泛测量可以通过模型定量地解释，根据该模型，SAM仅与紧凑构象结合。在没有 SAM 的情况下，核糖开关似乎是开放状态和紧凑状态的混合体。低浓度的 Mg++ 或 TMAO 与开放形式结合，抑制 SAM 的结合。在较高浓度下，Mg++ 的协同结合或 TMAO 的分子拥挤，将核糖开关转化为紧凑的形式，从而促进 SAM 的结合。 Mg++ 的进一步结合增加了核糖开关的紧凑形式对 SAM 的亲和力。流体动力学测量表明核糖开关被浓缩的TMAO压实。