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

点击翻译按钮获取中文摘要

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.