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

基本信息

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
项目类别：
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

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

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项目摘要

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.

蛋白质功能周期期间发生的运动通常涉及曲折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，因为打破伸展强的骨干氢键本质上并不是微不足道的。那是了解膜蛋白构象变化，我们需要学习骨干运动由序列编码。我们建议审查跨膜螺旋在孤立的螺旋中和全长膜蛋白中均变形通过一系列实验方法，结合了详细的分子动力学模拟。具体目的是：目标1。序列如何改变改变H键的转移，H键强度和动力学在孤立的TM螺旋中？从TM Helix“主持人”开始，我们将介绍“客人” 氨基酸和测量（1）I，I+4到I，I+3 H-键通过NMR方法的变化；（2）通过EPR方法改变螺旋动力学；（3）主链H键的改变通过测量H/D同位素效应来实现优势。为了获得机械理解，我们将通过分子动力学（MD）模拟研究序列效应。这项工作将阐明如何在TM螺旋中对骨干动力学进行编码。 AIM 2。主链H键和动态的变化如何影响功能？测试 TM螺旋灵活性在通过螺旋构象信号转导中的重要性，我们将研究KVAP通道。我们将引入骨干“酯”突变和侧链突变以改变主链氢键和柔韧性。骨干动力学的改变氢键将通过实验验证以及对电压感应的影响调查。实验观察到的更改的详细可视化将通过 MD模拟。