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组成部分。它们在许多重要的膜生物机制中发挥关键作用，如为活细胞提供能量，组织和调节酶活性，促进信息转导，甚至为生物合成和信号分子提供底物。人们普遍认为，膜不形成均匀的流体脂相，相反，脂质根据不同的条件被组织成相分离的动力域。分子相互作用、热力学和系统组成效应的知识对于理解不同脂质在生物膜的重要生命过程中所起的作用至关重要。这一知识对于设计基于脂质体（由脂质双层组成的人工囊泡）的药物输送系统也很重要。一个例子是“触发释放脂质体”，其中温度敏感脂质体可以被设计成具有相分离结构域的方式，以便在触发时释放其内容。在目前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）首先提供了关于脂质膜和脂质双分子层系统的迁移性和有序性的信息。自旋探针是一种特殊设计的化学试剂，携带稳定的未配对电子，可以在复杂的部分有序分子系统中引入，以报告周围分子的秩序和动力学。它们可以探测双层的不同深度/部分，也可以附着在嵌入的肽和蛋白质上。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。自旋标签对磁场的方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是一种技术，可以作为非常快速的分子运动的快照，并且可以在比十亿分之一秒短的时间内解决引入的自旋探针的分子重新定向动力学。然而，对信息丰富而复杂的EPR线形的分析需要充分的计算机仿真。目前的方法依赖于简化的参数化运动模型，需要用多个可调参数拟合EPR谱。在许多情况下，这种方法不能提供光谱的明确解释，从而无法得出关于多组分脂质双分子层的运动和秩序的明确结论。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分子层在内的复杂分子和生物分子系统的分子建模有了根本性的改进。MD模拟现在更快，更准确，使研究人员能够使用实际结构预测复杂的分子现象。该项目将MD和EPR结合在一起，并将首次尝试直接从MD的结果模拟生物膜的EPR光谱。这种方法的优势是双重的。首先，它将提供改进，并将有助于解释生物膜的EPR。其次，我们的MD-EPR方法将作为脂质双层模拟的高级计算模型的测试平台。我们将利用东英尼亚大学的独特专业知识，对自旋标记生物分子进行EPR和原子MD模拟，并在达勒姆大学提供的大型系统上进行粗粒度模拟。我们将使用一种新的MD-EPR方法来解决理解分子相互作用、热力学和系统组成对脂质结构域形成和动力学的影响、跨膜蛋白周围脂质的组织和动力学以及胆固醇作为脂质双分子层稳定剂的作用等关键问题。

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.