Bridge 6: H-bond Dynamics and Alpha-Helix Conformational Flexibility

桥梁 6：氢键动力学和 α-螺旋构象灵活性

• 批准号: 9149310
• 负责人: JAMES U BOWIE
• 金额: $ 14.21万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-10 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9149310
• 关键词: Affect; Amides; Amino Acid Sequence; Amino Acids; Binding; Ca(2+)-Transporting ATPase; Coupled; Defect; Development; Electron Spin Resonance Spectroscopy; Elements; Environment; Esters; Event; Hydrogen Bonding; Imagery; Isotopes; Learning; Length; Measures; Membrane; Membrane Proteins; Methods; Modeling; Movement; Mutation; NMR Spectroscopy; Pattern; Peptide Synthesis; Physical Chemistry; Physiology; Potassium Channel; Protein Engineering; Proteins; Pump; Resolution; Role; Side; Signal Transduction; Spectrum Analysis; Stretching; Structure; Surface; System; Testing; Vertebral column; Work; alpha helix; design; experience; flexibility; insight; meetings; molecular dynamics; protein expression; protein function; voltage

The movements that occur during protein functional cycles often involve twists and bends of secondary structure elements. In the case of transmembrane helices, the mechanisms for directing these conformational changes are likely to be particularly important because breaking and stretching the strong backbone hydrogen bonds is not energetically trivial. Thus, to understand membrane protein conformational changes, we need to learn how backbone movements are encoded by the sequence. We propose to scrutinize the mechanisms of transmembrane helix distortion both in an isolated helix and in a full length membrane protein through a battery of experimental methods, coupled to detailed molecular dynamics simulations. The specific aims are: Aim 1. How do sequence changes alter H-bond shifting, H-bond strengths and dynamics within an isolated model TM helix? Starting with a TM helix “host”, we will introduce “guest” amino acids and measure (1) alterations in i, i+4 to i, i+3 H-bond shifts by NMR methods; (2) alterations in helical dynamics by EPR methods; and (3) alterations in backbone H-bond strengths by measuring H/D isotope effects. To obtain a mechanistic understanding, we will study the sequence effects via molecular dynamics (MD) simulations. This work will illuminate how backbone dynamics can be encoded in a TM helix. Aim 2. How do alterations in backbone H-bonding and dynamics affect function? To test the importance of TM helix flexibility in conformational signal transduction through a helix, we will study the KvAP channel. We will introduce backbone “ester” mutations and side chain mutations to alter backbone hydrogen bonding and flexibility. Alterations in backbone dynamics and hydrogen bonding will be validated experimentally and the effects on voltage sensing investigated. Detailed visualization of the experimentally observed changes will be explored via MD simulations.

蛋白质功能周期中发生的运动通常涉及蛋白质的扭曲和弯曲 二级结构元素。就跨膜螺旋而言，其机制 指导这些构象变化可能特别重要，因为打破 拉伸强主链氢键在能量上并不是微不足道的。因此，为了 了解膜蛋白构象变化，我们需要了解主链如何变化 动作由序列编码。我们建议仔细研究机制 分离螺旋和全长膜蛋白中的跨膜螺旋扭曲 通过一系列实验方法，结合详细的分子动力学模拟。 具体目标是： 目标 1. 序列变化如何改变氢键位移、氢键强度和动力学 在一个孤立的模型 TM 螺旋中？从TM螺旋“宿主”开始，我们将引入“客体” 氨基酸并通过NMR方法测量(1) i，i+4至i，i+3 H键位移的改变； (2) 通过 EPR 方法改变螺旋动力学； (3)主链氢键的改变 通过测量 H/D 同位素效应来确定强度。为了获得机械上的理解，我们将 通过分子动力学 (MD) 模拟研究序列效应。这部作品将照亮 如何在 TM 螺旋中编码主链动力学。 目标 2. 主链氢键和动力学的改变如何影响功能？测试 TM 螺旋灵活性在通过螺旋进行构象信号转导中的重要性，我们 将研究KvAP信道。我们将引入主链“酯”突变和侧链 突变改变主链氢键和灵活性。骨干动力学的改变 和氢键将通过实验验证以及对电压传感的影响 调查了。将通过以下方式探索实验观察到的变化的详细可视化 MD 模拟。