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

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。 二级结构元素在跨膜螺旋的情况下， 指导这些构象变化可能特别重要，因为 并且伸展强骨架氢键在能量上不是微不足道的。从而 了解膜蛋白构象的变化，我们需要了解如何骨干 动作由序列编码。我们建议仔细研究 在分离的螺旋和全长膜蛋白中的跨膜螺旋畸变 通过一系列实验方法，再加上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？从一个TM螺旋“主机”开始，我们将介绍“客人” 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） EPR方法发现的螺旋动力学改变;（3）骨架氢键的改变 通过测量H/D同位素效应的强度。为了获得机械的理解，我们将 通过分子动力学（MD）模拟研究序列效应。这项工作将阐明 骨架动力学是如何在TM螺旋中编码的 目标2.主链氢键和动力学的改变如何影响功能？测试 TM螺旋柔性在通过螺旋的构象信号转导中的重要性，我们 将研究KvAP频道。我们将介绍主链“酯”突变和侧链 突变以改变主链氢键和柔性。主链动力学的改变 和氢键将通过实验验证， 研究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。

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.