Nanoscale organisation of water and ions at bio-interfaces: consequences on anti-infective peptide adsorption

Recent findings have shown that water and simple metal ions can spontaneously create correlated, ordered networks at the surface of minerals in solution. Preliminary results based on atomic force microscopy (AFM) suggest that similar effects occur at the surface of lipid bilayers. Due to the soft nature of biointerfaces, the existence of ordered interfacial water/ion structures would have deep implications for regulating biomembranes' mechanical properties, shape and local fluidity. It would also have implications in mediating the interactions of membrane-binding molecules such as biomarker, drugs and peptides, and for charge transfer in bioenergetics.
This proposal exploits recent advances in the field of AFM to explore the interplay between the local structure of the interfacial liquid (water and ions) and the behaviour of model membranes when undergoing a phase transition. The study will be conducted by AFM in solution and with molecular or single hydrated ion precision. The results will provide sub-nanometre maps of interfacial liquid structures, track their evolution locally as the membrane undergoes a gel to fluid transition, establish their influence on the membrane properties (elasticity, fluidity), and quantify their implications in mediating the adsorption of anti-invective peptides.
In the first part of the project, specific regions of the bilayer surface will be imaged and probed (mechanical properties mapping) by AFM while the temperature of the solution is progressively increased using a precise environment controller. The mutual effect that the melting membrane and the interfacial ionic structures have on each other will be monitored throughout the process, including upon subsequent cooling of the membrane. The proposed research examines the influence of parameters such as membrane lipid composition and the type of ions present in the solution on the formation and evolution of interfacial structures. Interfaces involving binary mixtures of dissimilar lipids will also be studied in order elucidate the role played by membrane physical and chemical singularities such as domain edges on interfacial processes.
Subsequently, the influence of interfacial effects on membrane-binding peptides will be studied in biologically relevant conditions. Experiments will be conducted in the presence of antimicrobial/anti-infective peptides (Temporins) known to insert into the membrane. The goal will be to determine whether the interfacial effects observed in the previous phases of the project can influence the nanoscale interactions between antimicrobial peptides and membranes, in particular the binding and subsequent insertion of single peptides. The process will be carefully monitored, starting from gel-phase membranes (no insertion) to fluid membranes, paying particular attention to the spatial location of inserting peptides and a potential temporal correlation with a prior interfacial singularity.
The findings of this project will be highly novel and are likely to provide the first direct observation of interface-mediated processes at biointerfaces.

The proposed research will impact different groups of people in the UK and internationally. The main potential beneficiaries are academic researchers, the pharmaceutical industry, the scientists directly involved into the project, and, on a longer term, society as a whole.

(i) Academic researchers (immediate impact)
Results from the proposal will rapidly impact across several fields of research such as molecular biophysics, membrane biology, physiology, medical sciences and bio-nanotechnology. The main impact stems from the ability to objectively quantify the influence of interfacial water and ions on biological interactions and molecular processes at interfaces. Results will contribute towards a better understanding of biological interfaces, and help build a more complete picture. They should open new research avenues for the research community and provide experimental basis for the development of mesoscopic scale (1nm-100nm) computer simulations.
Findings will also impact medical research, in particular the development of anti-infective, anti-bacterial and anti-microbial peptides. Most drugs currently available on the market target biomembrane (membrane proteins) but the effect of the interfacial liquid is usually ignored. Research initiated by the proposed work will provide important information towards the development of the next generation of antibiotics that takes into account or even exploits the interfacial liquid.
In bio-nanotechnology research, results could provide additional parameters in the development of lipid-based structures with a set of desired properties, either structural or interfacial. Results could be extended to liquid-based self-assembly of synthetic soft matter systems, for example in organic and polymer chemistry.
The academic community is usually best reached through scientific publications, conferences and workshops. High-profile open access publications as well as multiple presentations at international conference in the UK and abroad have been planned in order to best disseminate the project's results. All papers and datasets will be made freely available through the Durham University (DU) online repository. Additional cross-disciplinary dissemination and discussions will be achieved by the means of short workshops held by the DU Biophysical Science Institute, and that gathers scientists from biology, physics, chemistry and mathematics. These types of interdisciplinary workshops have been highly successful in the past.

(ii) Industry (5-10 years)
Pharmaceutical research and hence companies producing drugs are likely to benefit from the proposed research. The impact is however expected on a longer term and will depend on efficient dissemination and acceptance of the results by the academic community. The emphasis has therefore been placed first on academic impact.

(iii) People directly involved in the proposal (immediate)
The project will be conducted by a postdoctoral research assistant (PDRA) with help from the PI. Given the novelty and the large potential impact of the findings on the scientific community, this project should strongly benefit their careers. The PDRA will be working in a highly interdisciplinary environment and will be able to broaden his/her knowledge on each aspect of the project. (S)he will be presenting his/her results at conferences, and will be eligible for a large selection of postgraduate courses available at DU. His/her training will equip him/her with a highly valuable set of skills: expertise with atomic force microscopy measurements in liquid, molecular biophysics, biomembranes, and basic knowledge of peptide research.

(iv) Society as a whole (> 10 years)
In the long term, society will benefit from the proposed research and its consequences mainly through improved medical and pharmaceutical research. A better understanding of biointerfaces may also make its way to the education system, for example in biochemistry textbooks.