The role of closely-associated lipids in membrane protein structure and function

All cells within living organisms require membranes to function, as they act as a barrier between the cell and the outside environment and also define specific compartments within the cell. To allow small and large molecules to enter and leave the cell, and allow the cell to talk to its neighbours, the membranes also contain proteins that function as receptors, transporters and channels. Membrane proteins are responsible for a broad range of diseases and often act to import and export drugs within the cell. Their importance is highlighted with 60% of therapeutic targets being membrane proteins; however, our understanding of their structure and function lags significantly behind their soluble counterparts. One significant hurdle in studying membrane proteins is that they are rarely stable outside the membrane environment. Therefore, we need to use mimics of the membrane, for example detergents to stabilise the proteins. These membrane mimics are often a crude approximation of the native environment, and membrane proteins do not behave as they would in the native membrane. One reason is that the lipids that make up the membrane are also involved in the function and regulation of membrane proteins. Although the lipids in the membrane have been shown to be important for structure, function and regulation of membrane proteins, only in the last few years have technologies been developed that allow us to better study these interactions. This is highly significant within the therapeutic industries as obtaining high quality medicines is often underpinned by a robust and accurate model systems for assays, with structural information aiding in the modification of drug scaffolds.
This project has been developed in collaboration with the industrial partners GSK and UCB who will also support the research financially and by providing guidance and access to their facilities. We propose to use cutting edge technology to analyse the lipids associated with specific membrane proteins (mass spectrometry), conduct structural studies (electron microscopy) and use new membrane protein stabilising scaffolds. This will allow us to ask a number of important questions. The first is how do different methodologies for extracting membrane proteins from their native membrane affect their stability and function? We will next address how the different ways of stabilising membrane proteins may affect the structure and also the efficiency by which a reliable structure can be obtained by electron microscopy. The third question will address which lipids are found tightly associated with membrane proteins and do these lipids change with different expression and extraction methods? These three important questions will be answered using three model systems that represent a GPCR receptor (A2A) that allows the cell to respond to external stimuli, a transporter (AcrB) that exports, amongst other things, antibiotics and is involved in antibiotic resistance and a channel that transports potassium ions and is involved in cell homeostasis (BK channel). By using a broad set of exemplar membrane proteins we can start to understand if the observations we see are unique to a certain family of proteins or translates across a broader area into a range of membrane proteins.
To assist with the work the team consists of experts in membrane protein biochemistry, electron microscopy, mass spectrometry, pharmacology and tools for extracting membrane proteins. Moreover, we are collaborating with two major pharmaceutical companies, GlaxoSmithKline and UCB, which highlights the importance of this work not just within the academic field, but also in industry. The industrial support will allow us to translate our research to a very broad audience as it has implications on not just our fundamental understanding of membrane proteins but also in producing more efficient structural biology pipelines and more robust and accurate downstream assays, facilitating the drug design process.

Membrane proteins represent over 30% of the genome and make up ~60% of therapeutic targets playing key roles in cells, from receptors to maintaining chemical and proton gradients, energy production and cellular transport. However, our structural and biochemical understanding of membrane proteins has lagged behind that of their soluble counterparts. A significant hurdle in studying membrane proteins has been in stabilising them outside of their native membrane environment with the closely associated lipids playing a key role in stability, function and/or regulation. However, these are often removed after extraction from the membrane.
By combining cutting edge electron microscopy (EM) and mass spectrometry (MS) we will compare the effect of both extraction route and expression level on the composition of closely associated lipids and its effect on stability and structure. The first objective will investigate the stability of three model membrane protein systems in detergent, nanodisc and styrene maleic acid co-polymer. Objective two will use EM to investigate how membrane protein structure is affected by different scaffolds in terms of architecture and also in the efficiency of structure solution. The third objective will use MS to identify closely associated lipids with proteins, following different extraction methods, which can be correlated with the stability and structural data form the first two objectives. Moreover, objective three will investigate the difference in composition of closely associated lipids with different levels of protein expression. Combined, this program of work will provide new and important insights into the role of closely associated lipids on the stability and function of membrane proteins and will generate better models for structural studies and biochemical analysis. This work has been developed with, and is supported by a collaboration with UCB and GSK highlighting its importance both in an academic and industrial setting.

Membrane proteins represent over 30% of the genome and make up 60% of therapeutic targets. They play vital roles in cells in health and disease, from acting as receptors in signalling pathways, maintaining chemical and proton gradients, to energy production and cellular transport. However, our structural and biochemical understanding has lagged behind that of their soluble counterparts. A significant hurdle in studying membrane proteins has been in stabilising them outside of their native membrane environment. Therefore, in collaboration with GlaxoSmithKline and UCB, the proposal sets out to better understand the role of lipids for protein structural and functional studies.

In the long term, this proposal could deliver significant benefits to society, the economy and individuals through enabling the development of novel therapeutics to treat a wide variety of diseases caused by defects or changes in membrane proteins. For example, ~40% of therapeutics target GPCRs and two of the most common monogenetic diseases, cystic fibrosis and polycystic kidney disease are caused by mutations of plasma membrane proteins. Patients, healthcare professionals and society will benefit from improved treatment options, reducing mortality, morbidity and economic contribution. The general economy and specifically pharmaceutical companies will benefit through the development of new treatments, increasing revenue and securing competitive market positions.

In the medium term, the anticipated beneficiaries are the collaborating companies on this proposal, as the research will enable them to optimise single particle cryo-EM studies to obtain structures that better reflect the native membrane bound state. Moreover, through mass spectrometry we can identify the tightly bound lipids and understand the make-up of the local membrane environment. This will facilitate the development of novel small molecule therapeutics through enhanced understanding of structure-function relationships in near-native environments, reducing the number of small molecules which fail to bind to the native membrane protein. Moreover, it will provide a more robust environment for membrane protein assays which has downstream implications in high throughput small molecule screening.

In the short-term, the main beneficiaries will be the academic community (addressed in the academic beneficiaries section), industrial partners, researchers on the grant and the general public. The industrial partners will benefit as the first to access the results, and to work with the research team to inform the development of this work. Importantly, this grant will strengthen the collaborative link between UoL, UCB and GSK permitting a transfer of knowledge and expertise between the groups and lead to future collaborative projects. Knowledge of the native structure and associated lipids for the model systems (BK & A2A) will be of significant value. All the researchers on the grant will benefit through working in a cutting edge research environment with transfer of knowledge and expertise between the academic and industrial sites. The PDRA will gain from a broad training in this highly integrated project (MS and EM). This will produce highly skilled scientists at the forefront of structural biology and the expanding field of studying the role of native lipids in structure/function. This will enable them to make a valuable and practical contribution to the continued growth of this type of cross-disciplinary cutting edge research activity in the UK. Master's and undergraduate students will also benefit from this proposal through being able to undertake high quality innovative research projects. The general public and school children will benefit through increased understanding of the role of membrane proteins in causing human diseases, and how scientific advances may lead to new treatments.