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

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.

Lipid bilayers represent one of the most important classes of supramolecular assemblies for life forms on earth. Our novel MD-EPR analysis tool will, for the first time, bring together state-of-the-art MD modelling of lipid bilayer systems with EPR in order to investigate dynamics and molecular organisation in lipid membranes. The knowledge generated by this project will contribute to our understanding of molecular interaction mechanisms which are responsible for the dynamics and self-assembly of phospholipids at the lipid bilayers and in protein-lipid complexes. It will also provide critical insights into the role cholesterol plays in these processes. Since lipid bilayers play significant roles in many important bio-molecular processes (in membrane transports, signal transduction, apoptosis) such knowledge is vital for advances in biological and medical research in general. Thus over a longer period this will contribute to the enhancement of the quality of life and health.
Understanding intermolecular interactions in the lipid membranes is also vital for designing novel lipid based materials with desired properties for pharmaceutical applications. Thus in a short term pharmaceutical companies working on the development of lipid-based drug delivery systems such as "trigger release liposomes" would benefit from the knowledge generated by our research.
The outcomes of the project would also be of relevance to industrial companies working on the rational design of hybrid nanomaterials containing lipids for biomedical and technological applications (lipid bilayer coated nanomaterials such as noble metal and silica nanoparticles, semiconductor quantum dots and carbon nanotubes).
Another outcome of the project will be novel advanced software for the prediction of EPR spectra from MD simulations of lipid bilayers with spin probes. Our simulation program will be able to predict directly from MD simulations the effects of hydrophobicity and oxygen permittivity on the EPR data. These new functionalities will be integrated within the general MD-EPR simulation suite and will be available from a dedicated website. This will contribute to the dissemination of our results and will increase the international impact of our research.
Since the project will contribute to fundamental understanding of structure and self-assembly of biological membranes, which are essential elements of all living cells, it is important to EPSRC's key theme Healthcare Technologies. It is also directly related to research area "Biophysics and Soft matter Physics" as well as has relevance to the 'Physical Sciences Grand Challenges' strategic priority, in particular 'Understanding the physics of life'.
Finally, EPSRC has recently made a large strategic investment into the development of new functional materials with lipid bilayer membranes by the UK groups based in London, Nottingham, Leeds, Durham and Cambridge (Programme grant EP/J017566/1). Our MD and EPR studies will complement the application of other physical techniques to study lipid bilayers and the work of research groups in the UK.