Molecular Dynamics and EPR spectroscopy on lipid bilayers: new approaches to study biological membranes

脂质双分子层是生物膜的主要构件。它们在许多重要的膜生物机制中发挥着关键作用，如为活细胞提供能量，组织和调节酶活性，促进信息的传递，甚至为生物合成和信号分子提供底物。人们普遍认为，膜不会形成均一的流体脂相，相反，脂类根据不同的条件被组织成不同的相分离的动态域。分子相互作用、热力学和体系组成效应的知识对于理解不同的脂类在生物膜中的重要生命过程中所起的作用至关重要。这一知识对于基于脂质体(由脂双层组成的人工囊泡)的药物输送系统的设计也很重要。一个例子是“触发释放脂质体”，其中温度敏感的脂质体可以被设计成具有相分离的结构域以在触发时释放其内容物。在目前应用于膜研究的生物物理技术中，氮氧化物自旋探针的电子顺磁共振(EPR)首次提供了关于脂膜和脂双层系统中的流动性和有序性的信息。自旋探针是一种特殊设计的化学试剂，可以携带稳定的未配对电子，可以引入复杂的偏序分子系统中，以报告周围分子的有序性和动力学。它们可以探测双层的不同深度/部分，也可以附着在嵌入肽和蛋白质上。因为电子有磁矩，所以它可以与外部磁场相互作用。EPR以谱线形状的形式测量这种相互作用。自旋标记对磁场的取向对这条线的形状有很大的影响，因此可以研究分子的迁移率、动力学和分布。EPR是一种作为非常快速的分子运动的快照的技术，可以在不到十亿分之一秒的时间内解析引入的自旋探测器的分子重定向动力学。然而，对信息丰富和复杂的电子顺磁共振线型的分析需要完全的计算机模拟。目前的方法依赖于简化的运动参数模型，并且需要用多个可调参数来拟合EPR谱。在许多情况下，这种方法并不能提供对光谱的明确解释，从而无法得出关于多组分脂双层的运动和有序性的明确结论。在过去的十年中，使用分子动力学(MD)模拟技术对包括脂质双层在内的复杂分子和生物分子系统的分子模型进行了根本性的改进。分子动力学模拟现在更快、更准确，使研究人员能够利用实际结构预测复杂的分子现象。这个项目将把分子动力学和EPR结合起来，并将首次尝试直接从分子动力学的结果模拟生物膜的EPR谱。这种方法的好处有两个。首先，它将对生物膜的EPR进行改进，并有助于对其进行解释。其次，我们的MD-EPR方法将作为高级计算模型的试验台，用于脂质双层模拟。我们将使用UEA在自旋标记生物分子的EPR和原子MD模拟中的独特组合，以及Durham.提供的大规模系统的粗粒度模拟。我们将使用一种新的MD-EPR方法来解决关键问题，即了解分子相互作用、热力学和体系组成对脂类结构域的形成和动力学的影响、跨膜蛋白周围的脂类的组织和动力学，以及胆固醇作为脂类双层稳定剂的作用。

Lipid bilayers are the main building blocks of biological membranes. They play a key part in many important biological mechanisms in membranes such as providing living cells with energy, organising and regulating enzyme activities, facilitating the transduction of information and even the supply of substrates for biosynthesis and for signalling molecules. It is widely accepted that membranes do not form homogeneous fluid lipid phases but, in contrast, lipids are organised into phase separated dynamical domains depending on various conditions. Knowledge of molecular interactions, thermodynamics and system composition effects are crucial for understanding the role which different lipids play in vital life processes in biological membranes. This knowledge is also important for the design of drug delivery systems based on liposomes (artificial vesicles composed of lipid bilayers). An example would be "trigger release liposomes" where temperature sensitive liposomes could be engineered in a way to have phase separated domains to release their content upon trigger.Of the biophysical techniques now being brought to bear on studies of membranes Electron Paramagnetic Resonance (EPR) of nitroxide spin probes was the first to provide information about mobility and ordering in lipid membranes and lipid bilayer systems. Spin probes, specially designed chemical agents that carry a stable unpaired electron, can be introduced within complex partially ordered molecular systems in order to report on the order and dynamics of surrounding molecules. They can probe different depths / parts of the bilayer and also be attached to embedded peptides and proteins. Because an electron has a magnetic moment it can interact with an external magnetic field. EPR measures this interaction in the form of spectral line shape. The orientation of the spin label to the magnetic field has a dramatic effect on this line shape and therefore molecular mobility, dynamics and distribution can be studied. EPR is a technique that acts as a snapshot of very fast molecular motions and can resolve molecular re-orientational dynamics of the introduced spin probe over times shorter than a billionth of a second. However, the analysis of the rich and complex in information EPR lineshape requires full computer simulation. Current approaches rely on simplified parametrised models of motion and require fitting of EPR spectra with multiple adjustable parameters. Such approaches in many cases do not provide an unambiguous interpretation of the spectra preventing definite conclusions about motion and order in multi-component lipid bilayers to be reached. The last decade has seen radical improvement in the molecular modelling of complex molecular and bio-molecular systems including lipid bilayers using Molecular Dynamics (MD) simulation techniques. MD simulations are now much faster and more accurate allowing researchers to predict complex molecular phenomena using actual structures.This project will bring together MD and EPR and will attempt for the first time simulation of EPR spectra of biological membranes directly from the results of MD. The advantage of such an approach is twofold. Firstly, it will provide the improvement and will facilitate the interpretation of EPR of biological membranes. Secondly, our MD-EPR methodology will serve as a test bed for advanced computational models for lipid bilayers simulations.We will use the unique combination of expertises from UEA in both EPR and atomistic MD simulations of spin labelled bio-molecules and coarse-grained simulations on large scale systems provided by Durham.We will use a novel MD-EPR methodology to address the key problems of understanding molecular interactions, thermodynamics and system composition effects on the formation and dynamics of lipid domains, the organisation and dynamics of lipids around trans-membrane proteins, and the role of cholesterol as a lipid bilayer stabiliser.