Conformational states of membrane proteins: Technology development for bioscience

Lead Research Organisation: University of Aberdeen
Department Name: School of Medical Sciences


Although life requires water, life must be able to control the flow of water. As an example, if a person (70% water) jumps into a swimming pool they do not dissolve like a sugar cube. This is because we have a barrier between the swimming pool water and the water inside our cells. This barrier is the membrane or lipid bilayer, it is made of oily compounds. The bilayer is essential it keeps important things in and poisonous things out. All organisms have these bilayers. On their own bilayers would simply block all transport, thus we could not take up nutrients nor could we get rid of waste. Proteins embedded in this membrane are thus needed to act as gate keepers to control movements of ions, nutrients, waste and proteins across the lipid bilayer. These proteins are also the telephone connections between one cell and another. The nerve impulse in humans transmits trigger release of a transmitter (small chemical) from the neuron. The transmitter interacts with another cell by promoting some change in the cell, each cell uses membrane proteins as part of this process. Membrane proteins that control ions movement across the bilayer are called ion channels. They must, like a tap, be able to fully closed to stop leaks but they must also open when required. Many diseases are caused by membrane proteins not working properly. If we are to treat these diseases we need to understand how membrane proteins work. Protein crystallography has transformed our understanding of proteins. It allows us to see every atom in the structure and understand a great deal about the function of the protein. This scientific approach has led to the development of many new drugs. However, this technique can only see one state of the protein at a time. We propose to develop a new approach that will allow us to see in detail exactly how membrane proteins move between the open and closed states. We have chosen to study the pain receptors in humans and the osmotic stress survival proteins in bacteria. These are important systems with obvious medical benefits, treatment of pain and design of new antibiotics.

Technical Summary

Membrane proteins undergo significant conformational changes during their gating. Quantitating these changes is a significant challenge but is vital to our understanding of the molecular biology. We will use EPR in combination with site directed spin labeling to measure these changes. Unpaired electron spins interact with each other over long distances. This can be used to derive the distance between the spins via measurement of the dipolar coupling. For distances up to 15 Å this can be achieved with conventional continuous wave (cw) EPR methods, for distances in the critical 15 to 80 Å regime, pulsed techniques as Pulsed Electron-Electron Double Resonance (PELDOR or DEER) are the method of choice. In the PELDOR time trace, a fixed distance manifests itself as a periodic modulation whose frequency can be converted back into the distance (implemented in the program DEERAnalysis2008). Since the PELDOR experiment is not a single molecule experiment, random intermolecular spin-spin coupling occurs superimposing an exponential decay which has to be removed. However, for this ill-posed back transformation to work reliably and with high precision it is crucial to obtain PELDOR data with observable modulation. We identified several in steps in sample preparation and processing that improve signal to noise and we have obtained preliminary data showing that we can make accurate measurements. We will label the ion channels MscS, MscL, ASIC1a and FaNaC to derive distance measurements in the different conformational states that accompany the closed to open structural transitions and also make meausrements of other crucial states, such desensitisation and inactivation. Making such measurements requires the analysis of selected channel mutants. The PELDOR sudies will be complemented with state-of-the-art measurements of the activity of the mutant channels in the laboratories of the applicants, ensuring that no significant peturbations of structure arise from mutagensis.

Planned Impact

Who will benefit from this research? This research will benefit the biological and medical research communities in both industry and academia. Membrane proteins are a key target for the pharmaceutical industry and UK has a strong record in pharmaceutical and biotechnology research. The academic community recognizes that membrane biology is underdeveloped and holds significant opportunities for future research. How will they benefit from this research? We propose to develop a new integrated approach combining PELDOR (an emerging EPR technology), X-ray crystal analysis, electrophysiology and molecular biology to characterize the conformational states of membrane proteins as they change during function. The genetic basis of over 70% of major diseases has been shown to result from a defect in one or more membrane proteins. Bacterial membrane systems have provided novel targets for therapeutics and it is in this sector of bacterial and fungal pathogenesis that new targets are being discovered as a consequence of more rapid advances in the characterization of membrane proteins. Further, many current therapeutics target membrane proteins as part of their action. For example and directly relevant to this proposal many treatments for high blood pressure (a common ailment) block the sodium channel ENaC. These 'old' drugs were developed before there was any molecular understanding of the channel. There is clear need for new more effective therapies for many diseases and new drugs requiring new scientific understanding. It is likely that the majority of such new drugs will target membrane proteins and their processes. In addition, membrane proteins are also important sensors, diagnostics and biotransformation platforms. Membrane protein biology and biochemistry is therefore an important and impactful area. This proposal will lead to a significant increase in the scientific understanding of ion channel function, transmembrane signaling and membrane transport. By doing this work in the UK we will ensure the impact of the scientific advancement is realized by UK Biotech and Pharmaceutical Industry. What will be done to ensure that they benefit from this research? We will engage with the immediate beneficiaries, (The science community and Health Care Industry) through a mixture of public presentations at companies, professional conferences and to the general public. We will also host workshops at St Andrews in structural biology, membrane functions and in EPR. The structural biology workshop has been running in St Andrews since 1999 and every two years up to 40 PhD students from across the UK come to this week long residential course. The course covers experimental and detailed theory. The two last advanced European Summer Schools on EPR were co-organized by Schiemann (funded by the EU) and trained graduate scientists from across the EU in modern EPR techniques. Such courses ensure the widespread dissemination of new techniques. We will promote public engagement by generating press releases for the University press offices. Our University web sites are an excellent method for the general public to see what research is being carried out with BBSRC funding. We will make ourselves available to international, national and local media organizations to talk about the work. We will manage the regular meetings of the groups involved such that in addition to the science focus, they will also explore ways of communicating the impact of the science to the widest possible audience. The impact of the work will be spearheaded by Liu, Schiemann, Booth and Naismith. The project requires the collaboration of Professor Eric Linguelia (CNRS, Valbone, France), Dr Graham Smith (St Andrews) and Professor Mark Sansom (Oxford). We will collaborate with the UK Membrane Protein Laboratory to ensure the widest possible dissemination of the results to the community. We will also distribute experimental materials (clones and reagents) to other labs.


10 25 50
Description Controversy had surrounded the relevance of the crystal structures for the mechanosensitive channels MscS. Two other predicted structures that are based on non-crystallographic techniques had been posited and a new approach was needed to complement the structural data and, if possible, provide some discrimination between alternative structural models. We sought to use PELDOR to analyse the distances in both detergent-solubilised MscS, which was the source material for crystallography, and subsequently to investigate MscS reconstituted into lipid bilayers. PELDOR distance measurements were recorded from spin-labels attached to cysteine mutants on key secondary structure elements that were chosen to discriminate between the conflicting models for MscS structure. The activity of the mutant channels in in vivo physiological assays and their electrical behaviour, measured using patch-clamp physiology, was verified to ensure the mutations did not affect gating behaviour. The measurements were tested against in silico models generated from the MscS modified with the spin label and complemented by solving crystal structures for the modified channels. Surprisingly, the PELDOR measurements matched more closely to those from an open crystal structure, indicating that the detergent-solubilised MscS assumes the open state (Pliotas et al., 2012). In contrast, the original crystal structure (obtained by the Rees group) is in a closed state. Analyses of reconstituted proteins were undertaken in two bilayer systems; nanodiscs and bicelles (Ward et al., 2014). The distance measurements between multiple spin-labeled cys mutants in each bilayer system were less unequivocal and suggest mixed forms in the reconstituted samples.
Exploitation Route Our crystallography of the spin label-modified protein led to a higher resolution structure that displayed additional densities that can be modelled as lipids occupying 'voids' created by the arrangement of the helices in the heptameric MscS. We have subsequently built, and tested, a model for the structural transition during gating that leads to stable channel opening, matched by smaller 'voids' in the open state, due to lipid removal into the bulk membrane phase (Pliotas et al., 2015).
Sectors Chemicals

Description Membrane proteins represent the majority of drug targets but the resolution of their structure to facilitate targeted drug discovery represents a major challenge. In addition to the static information provided by crystallography one also requires an understanding of the conformational changes these proteins undergo during their functional cycles. The main impacts of this grant have been three-fold. Firstly, the benefit to academia in expanding and improving techniques to study conformational changes in membrane proteins, in different platforms, namely detergent-solubilised or in bilayer mimics. This grant allowed us to quantify distances between selected residues in the model systems and led to resolution of conflicting structural models for the MscS channel in detergent, and subsequently in bicelles and nanodiscs. This technological and intellectual advance in understanding contributed to our recently published model of the role of lipids in mechanosensation (Pliotas et al, 2015). These findings are of broader interest in the academic community because quantification of the molecular rearrangements that occur in membrane proteins is lacking and research funded by this grant has added to the repertoire of data and techniques that can be used. Secondly, a number of technical advances were essential to the programme and these are reflected in additional publications by a subgroup of the original consortia. Finally, this grant enabled active interdisciplinary collaboration between biologists, chemists and biophysicists at Aberdeen and St Andrews. As part of this collaboration researchers were trained in state-of-the art techniques and thus provided potential societal and economic impact by enhancing the skills of these researchers for their future careers.
First Year Of Impact 2012
Sector Chemicals
Impact Types Societal,Economic

Title Plasmids 
Description Created mutant plasmids used for structure-function studies 
Type Of Material Technology assay or reagent 
Provided To Others? Yes  
Impact Impacted on my own and other groups research