Lipid control of membrane protein folding

Lead Research Organisation: Imperial College London
Department Name: Dept of Chemistry

Abstract

Proteins are the 'worker molecules' in life. Cells in our bodies sense and communicate with the outside world via proteins embedded in membranes that surround the cells. These membrane proteins constitute about a third of the proteins in our bodies, and over half of the current targets for new medicines. Unfortunately, there is a limited understanding of how membrane proteins work at a molecular level. This is because they are notoriously unstable outside the membrane and it is difficult to obtain sufficient amounts for scientific study. Recent advances are, however, beginning to alter this situation. We aim to devise new methods to study a phenomenon known as 'folding'. Genes carry the code for proteins, but puzzles remain in deciphering how genetic information is translated into functional proteins. Proteins begin as a string of amino acids, which then have to fold-up to a particular shape in the right part of the body. If this folding fails, disaster strikes and the proteins malfunction. The most glaring gaps in knowledge of this folding phenomenon come with membrane proteins. We propose to forge new territory by devising new methods to understand membrane protein folding. We intend to do this by focussing on the other main component of membranes: lipids. These lipid molecules are the basic building blocks of membranes, effectively the bricks that make up these biological walls and they influence how their neighbouring proteins behave. We already know that certain lipid properties can be used to alter the protein folding and we now aim to understand the precise details of this control.

Technical Summary

Lipids participate in many biological processes and actively influence membrane function. Elastic properties of lipid bilayers, including curvature and lateral pressure are increasingly being recognised as key controlling factors. We have shown that these lipid properties alter the folding of proteins within membranes. Our hypothesis is that lipids actively control folding. Here, we propose to elucidate the exact control mechanism and identify the lipid properties that manipulate the rate and yield of folding for helical membrane proteins. We will exploit the lipid control mechanism to study the folding of potassium and mechanosensitive channels. We aim to measure lipid elastic parameters under biological conditions; at present these parameters are only known in very artificial environments. We will measure them in the presence of buffers, membrane proteins and in asymmetric bilayers. We will also measure protein folding in lipid bilayers under the same conditions. By judicious alterations of the lipid compositions we can then determine correlations between specific lipid elastic properties and folding events. We combine state of the art approaches in Physics and Biochemistry. We have developed high throughput methods to measure lipid parameters and pioneered a range of techniques to monitor folding in lipids. We will also use novel methods to create lipid vesicles with asymmetric lipid compositions, to study the influence of asymmetry on folding. Understanding the origin of lipid control is crucial to predicting the properties of biological membranes that maintain the fold and function of membrane proteins, and thus mimicking these critical properties in vitro. We propose that bilayer asymmetry is a key factor. The different lipid compositions of the inner and outer leaflets of the bilayer will produce an asymmetric distribution of lateral forces that is likely to be vital to stabilising integral membrane proteins, which too have asymmetric structures.

Publications

10 25 50
 
Description 1. Development and application of new platform technologies for the measurements of the mechanical properties of lipid bilayers (e.g. membrane rigidity, curvature, tension, stress) in the absence/presence of membrane proteins. By combining all these techniques we have been able to study how the size, charge and headgroup types on lipids and the length and number of their tails affect their mechanical properties and then correlate this with the effect that this has upon protein folding. This enabled the demonstration of lipid bilayer mediated control and protein-protein communication through a lipid bilayer. This is in turn has been possible as we have new microfluidic platforms for incorporating and manipulating membrane proteins. This is a crucial breakthrough as our increased understanding of this interplay between membrane mechanics and protein folding means we can design lipid environments that replicate those found in living cells. This will aid the design of new drug molecules as these are in the main designed to target membrane proteins and this design process relies on the latter being embedded in lipid membranes that replicate the environment they would see in real cells.

2. Design and implementation of microfluidics strategies for manufacturing symmetric and asymmetric vesicles of defined composition and curvatures. This enabled us to make measurements of the effect of membrane asymmetry on the mechanical properties of lipid bilayers.

3. Development of microfluidic strategies for manufacturing droplet interface bilayers (individual units and networks) that can be used as a platform for studying the correlation between membrane composition and membrane activity/function. Since its development this technology is now being used to manufacture biological circuitry and artificial cell assemblies and has stimulated collaborations with industry in the fields of drug-membrane translocation and membrane-protein folding.
Exploitation Route See above. The platform technologies developed during this project are already being exploited in the industrial sector in a wide range of applications from drug-translocation/non-specific binding studies, drug design through membrane protein assays and surfactant phase behaviour studies. The development of novel microfluidic strategies for the high-throughput construction of droplet interface bilayers (DIBs) and associated reconstitution strategies for the incorporation of membrane proteins has attracted a great deal of interest from industry. We are now exploiting their potential for studying the effects of membrane composition on the translocation rates of small molecules with a view to understanding how these barriers control transport in animals and plants. In the case of animals this is particularly significant as these in-vitro models are now showing great promise with respect to their ability to predict non-specific binding of drug molecules in animals systems. At present such measurements can only effectively be made using animal models in conjunctions with positron emission tomography. The DIB strategies we have developed have the potential to replace these animals based experiments in the long term. The microfluidic strategies for manufacturing droplets interface bilayers (DIB)/vesicles and associated reconstitution strategies for membrane proteins has also stimulated collaborations in the fields of membrane protein folding (Pfizer plc) and agrichemical translocation in plant systems (Syngenta).



The versatility of the high-throughput SAXS system has stimulated two follow-on collaborations with Proctor and Gamble (P&G) looking at phase behaviour studies and the use of SAXS to study biological systems such hair fibres. These collaborations are directly supported by P&G through two P&G funded PDRA positions and two PhD studentships.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology,Other

 
Description The development of novel technologies for manufacturing artificial membranes with controlled levels of asymmetry and composition has led provided a new methodology for studying protein-membrane interactions in high-throughput and under controlled conditions. In addition it has enabled studies of drug-membrane translocation studies which has attracted a great deal of interest from industry (pharma, personal care and agriscience sectors). The automated SAXS systems has transformed our ability to study the properties of lipid systems in high-throughput and has stimulated a number of collaborations with the personal care sector.
First Year Of Impact 2012
Sector Agriculture, Food and Drink,Chemicals,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Frontier Manufacturing: Scaling up synthetic biology
Amount £5,158,504 (GBP)
Funding ID EP/K038648/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 10/2018
 
Description Sculpting Dynamic Amphiphilic Structures
Amount £4,821,721 (GBP)
Funding ID EP/J017566/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 06/2012 
End 05/2017
 
Title Microfluidic strategies for high-throughput formation of droplet interface bilayers 
Description Microfludic DIB construction 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Development of platform technologies for studying drug-membrane interactions in collaboration with industry. Construction of compartmentalised artificial cells. Studies of small molecule translocation mechanisms in partnership with industry 
 
Description Phase behaviour studies 
Organisation Procter & Gamble
Country United States 
Sector Private 
PI Contribution Development of high-throughput SAXS technology
Collaborator Contribution Application of SAXS technology to in-house challenges
Impact Currently trialling technology
Start Year 2013
 
Description Small-molecule membrane translocatio processes 
Organisation Syngenta International AG
Department Syngenta Ltd (Bracknell)
Country United Kingdom 
Sector Private 
PI Contribution Development of DIB technology that can be applied to studies of translocation
Collaborator Contribution Application of DIB technology in industrial context
Impact Currently trialling translation potential of the technology, multi-disciplinary collaboration.
Start Year 2013
 
Title Fluctuation mode technology for analysis of lipid systems 
Description Fluctuation mode analysis (include capture and image analysis software) for characterising mechanical properties and lamellarity of vesicles as a function of membrane composition (in the abscence and presence of proteins) 
Type Of Technology Software 
Year Produced 2012 
Impact No actual Impacts realised to date 
 
Title High-throughput SAXS Analysis Systems for undertaking lipid phase behaviour studies 
Description An automated laboratory based x-ray beamline with a multi-capillary sample chamber capable of undertaking small angle X-ray scattering measurements on up to 104 samples at a time as a function of temperature between 5 and 85°C has been developed. The modular format of the system enables the user to simultaneously equilibrate samples at 8 different temperatures with temperature control to +/-0.05 degrees C. Beamline control including sample exchange and data acquisition has been fully automated using a custom-designed Labview framework leading to 100s/1000s independent SAXS measurements with a monthly device duty cycle. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2012 
Impact Collaboration with Proctor and Gamble looking at phase behaviour of structured products. 
 
Title Membrane pressure sensing lipid based probes 
Description Development and application of fluorescent lipid based probes that are able to measure the pressure within lipid bilayers as a function of depth (lipids based upon both dipyPC and red/green BODIPY phosphatidylcholine motifs studied). 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2011 
Impact No actual Impacts realised to date 
 
Description Invited Oral Presentation, Advanced School in Soft Matter, University of Leeds, 24-27th, March 2013 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Oral presentation, Advanced School in Soft Matter, University of Leeds, 24-27th, March 2013 Three invited oral presentations

awareness of tech to new end users
Year(s) Of Engagement Activity 2013