NMR SOLUTION STRUCTURE OF THE MMOB COMPONENT

MMOB 组件的 NMR 解决方案结构

• 批准号: 2521187
• 负责人: George T. Gassner
• 金额: $ 3.02万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-28 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2521187
• 关键词: active sites; calorimetry; catalyst; chemical kinetics; enzyme complex; enzyme mechanism; enzyme structure; ionic bond; methane monooxygenase; nuclear magnetic resonance spectroscopy; oxygenases; protein structure; site directed mutagenesis

The soluble methane menooxygenase system isolated from M. capsulatus (Bath) consists of a reductase (MMOR), coupling protein (MMOB), and a hydroxylase (MMOH). The 16.5 kDa MMOB component has a profound influence on the type of chemistry catalyzed at the active site of MMOH. In a still unknown mechanism, MMOB directs the evolution of a series of oxygen intermediates that lead to the insertion of an atom of molecular oxygen into a CH bond of methane. In the absence of MMOB, MMOH still accepts electrons from substrate specificity of MMOH. This broad substrate specificity makes MMOH valuable system in both aqueous phase organic synthesis, and in the detoxification of contaminated environmental sites. High resolution homonuclear and heteronuclear NMR experiments will be used to solve the solution structure of MMOB. The three dimensional NMR structure will provide greater insight into the way in which MMOB tunes the reactivity potentials of the MMOH iron center. Searches based on the complementarity of the topologies and electrostatic surface potentials of MMOH and MMOB will be used in conjunction with chemical cross-linking data to identify probable MMOB binding sites on the surface of MMOH. Proposed binding sites will be confirmed through titration calorimetric studies involving synthetic peptides representative of secondary structural motifs in interface region. Individual amino acid side chains involved in the complex formation will be identified in high resolution NMR studies of complexes of MMOB with synthetic peptides. Amino acid side chains found to be involved in the binding interaction will be sequentially mutated to alanine. The relative contribution of individual amino acid side chains the macroscopic stability of the complexes formed between MMOB and MMOH will be measured based on calorimetric measurements.

从M. capsulatus （Bath）由还原酶（MMOR）、偶联蛋白（MMOB）和还原酶（MMOR）组成。 羟化酶（MMOH）。16.5 kDa MMOB成分影响深远 在MMOH的活性位点催化的化学类型。在还 未知的机制，MMOB指导一系列氧的演变， 导致插入一个氧原子的中间体 甲烷的CH键。在没有MMOB的情况下，MMOH仍然接受 来自MMOH的底物特异性的电子。这个广阔的基底 特异性使得MMOH在水相和有机相中都是有价值的体系 合成，并在受污染的环境场所的解毒。 将使用高分辨率的均相和异相NMR实验 解决MMOB的解决方案结构。三维核磁共振 结构将提供更深入的了解MMOB调谐的方式 MMOH铁中心的反应潜力。基于 的拓扑结构和静电表面电位的互补性 MMOH和MMOB将与化学交联数据结合使用 以鉴定MMOH表面上可能的MMOB结合位点。提出 结合位点将通过滴定量热研究确认 涉及代表二级结构基序的合成肽 在界面区域中。参与蛋白质合成的单个氨基酸侧链 复杂的形成将确定在高分辨率NMR研究 MMOB与合成肽的复合物。发现氨基酸侧链 将依次突变为 丙氨酸单个氨基酸侧链的相对贡献 MMOB和MMOH之间形成的复合物的宏观稳定性 将基于量热测量进行测量。