ElectroProtein - Atomic-scale electrodynamics of protein-liquid interfaces
Lead Research Organisation:
University of Manchester
Department Name: Physics and Astronomy
Abstract
Electrostatic and electrodynamic properties are fundamental physical properties that strongly influence biomolecular structure and functions. In particular, they regulate protein functions of fundamental biological importance, including enzymatic catalysis, protein-protein/ligand interactions, and transport of ions in protein channels. Importantly, they are strongly influenced by the properties of interfacial water layers near the protein surface or confined inside ion channels, which are different than in bulk water. Despite the huge impact, little is known about these properties due to the lack of experimental tools able to probe them on the molecular scale. Standard techniques such as nuclear magnetic resonance, dielectric-sensitive fluorescence microscopy, and impedance spectroscopy, lack the required spatial/temporal resolution or are influenced by several parameters that impede direct measurement. Furthermore, theorists struggle to predict these properties and need experimental data to benchmark their theories. A technique able to measure them on the molecular scale in their native liquid environment is much needed. Scanning Dielectric Microscopy (SDM) is a scanning probe microscopy technique recently introduced to probe these properties on the nanoscale. The challenge is now to push its resolution down to the molecular level and apply it to biological relevant molecules. In this project, the fellow will build on previous breakthroughs of the supervisor combining SDM and advanced 2D crystal technology to probe the electrodynamics of protein-liquid interface in liquid environment. The results of this action will allow gaining new fundamental knowledge and formulating better theories of proteins' functioning during complex biological processes.
Publications
Advincula XR
(2024)
Ice interfaces: general discussion.
in Faraday discussions
Backus EHG
(2024)
Soft matter-water interface: general discussion.
in Faraday discussions
Benaglia S
(2024)
Atomic-scale structure of interfacial water on gel and liquid phase lipid membranes.
in Faraday discussions
Benaglia S
(2025)
Quantification of solvation forces with amplitude modulation AFM
in Journal of Colloid and Interface Science
Read H
(2024)
Structure and thermodynamics of supported lipid membranes on hydrophobic van der Waals surfaces.
in Soft matter
| Description | The project successfully advanced the understanding of interfacial and confined water at solid interfaces, as well as protein-liquid interfaces, through Scanning Dielectric Microscopy (SDM). The research objectives were met overall, leading to novel insights and scientific outputs. In WP1 (SDM of Interfacial and Confined Water), all objectives were successfully achieved. The project delivered significant findings on the electrodynamic and dielectric properties of interfacial water at 2D hydrophobic/hydrophilic surfaces and water confined within 2D nanopores and nanochannels. Atomically flat 2D solid membranes with ultra-thin channels, only a few angstroms thick, were successfully fabricated and employed to characterise the behaviour of confined water. These findings resulted in three major scientific outputs. One paper, currently under submission in Nature and available on Arxiv (10.48550/arXiv.2407.21538), details the electrodynamic properties of water confined within hydrophobic pores. Two additional manuscripts are in preparation for submission this year, focusing on the electrodynamic properties of water and ions confined within hydrophilic nanopores. In summary, we obtained new information on the electrodynamic properties (conductivity and dielectric constant) of water at the interface of hydrophobic/hydrophilic materials, and how the electrical properties of nanoscale pores change depending on the type of intercalated ions. The planned milestones (M1, M2) were successfully completed, leading to key research deliverables (D1.1, D1.2). The results are on the way to be published as high-impact publications, further supporting the significance of our findings. In WP2 (SDM of Protein-Liquid Interfaces), the project aimed to probe the electrodynamic properties of water at the interface of biological molecules, particularly proteins. The initial focus was on the characterisation of self-assembled monolayers (SAMs) (D2.1, M3), and we chose to use lipid membranes with tuneable headgroups. These samples were successfully produced by the fellow and characterised in terms of their electrical properties and hydration structure. An initial study on their behaviour on metallic substrates resulted in a scientific publication (10.1039/D4SM00365A). The structural characterisation of hydration structures on these SAMs led to a second publication (10.1039/D3FD00094J). Additionally, the quantification of interfacial hydration forces resulted in a third article (10.1016/j.jcis.2025.01.131), introducing a novel method for accurately measuring hydration forces at interfaces. These three works laid the foundation for precise quantification of the electrical properties of hydration structures at lipid membranes, with a final publication in preparation for this year. The second milestone and deliverable (M4, D2.2) were partially achieved. Membrane proteins were successfully produced on substrates for electrical characterisation, and the SDM setup was refined to characterise hydrated membrane proteins in humid air. Performing experiments directly in a liquid medium was challenging due to experimental complexity. Hence, we proceeded to implement a set-up that allows us to keep the probe in air but on hydrated protein samples. Experiments on fully hydrated proteins are ongoing, and the study is now being extended beyond membrane proteins to include globular proteins. Data collection is in progress, with a publication expected next year. Overall, we expect WP2 to lead to 5 publications, three of which published in high-impact journal. |
| Exploitation Route | The outcomes of this project have the potential to advance multiple disciplines. The findings have provided new experimental data that are key for our understanding of the structural and electrodynamic properties of water at interfaces. They will enable theoretical scientists to benchmark their mean-field and atomistic theories. A key impact of the project lies in the broader application of Scanning Dielectric Microscopy (SDM). By demonstrating its potential for studying biological interfaces, we have opened new avenues for exploring hydration and electrostatics in biomolecules. Other research groups can now build upon these methodologies, expanding SDM applications across biological and chemical systems. Beyond academia, these insights contribute to the development of next-generation materials and devices. The knowledge gained has direct implications for nanofluidic systems and membranes used in water purification and energy storage. Companies working in 2D materials-based nanotechnology and membrane science can use these findings to enhance material design and performance. In biotechnology, the newly developed methods for quantifying hydration structures at biological interfaces could improve biomolecular sensing technologies and protein stability studies. Finally, the methodological framework developed in this project offers relevant educational benefits. By integrating these techniques into university curricula and training programs, students and early-career researchers can gain hands-on experience with cutting-edge research approaches for molecular interface studies. |
| Sectors | Chemicals Electronics Energy |
| Description | Training for MPhys students, PhD students and postdoctoral researchers |
| Geographic Reach | National |
| Policy Influence Type | Influenced training of practitioners or researchers |
| Impact | The trainees underwent comprehensive training sessions, acquiring new skills and refining their abilities. Through these learning experiences, they have attained a high level of proficiency across different research techniques, essential to their ongoing Master and/or PhD topics and scientific projects. This will be crucial for both the production of scientific articles and the successful completion of their MPhys project and PhD thesis. |
| Description | Training for PhD students and postdoctoral researchers |
| Geographic Reach | National |
| Policy Influence Type | Influenced training of practitioners or researchers |
| Impact | The trainees underwent comprehensive training sessions, acquiring new skills and refining their abilities. Through these learning experiences, they have attained a high level of proficiency across differen research techniques, essential to their ongoing PhD topics and scientific projects. This will be crucial for both the production of scientific articles and the successful completion of their PhD thesis. |
| Title | Method for atomic scale visualisation of water molecules on soft biological membrane interfaces |
| Description | A novel method utilising Atomic Force Microscopy (AFM) was developed to investigate the hydration of biological membranes. In particular, we used three-dimensional AFM (3D AFM) which allows for a volumetric reconstruction of the interface between the aqueous solution and the lipid bilayer. This method underwent optimisation to enable visualisation of the molecular structure of water at the interface of zwitterionic biomembranes across different thermodynamic phases. The main innovation lies in its capability to visualise the arrangement of water molecules at the interface of liquid-phase lipid membranes. The protocol, published as scientific publication, contains three significant novelties. Firstly, it demonstrates the ability of AFM to probe the morphology of biomolecules and interfacial water with sub-molecular resolution. In particular, measurements can be tuned to allow for a direct observation of the nanometric morphological features of the lipids (heads and tails) in addition to the layered structures formed by the interfacial water molecules. Moreover, it shows that the molecular arrangement of water at the lipid surface depends on the lipid thermodynamic phase, identifying that water is organised in multiple hydration layers on both the solid-ordered and liquid-disordered lipid phases. Finally, it demonstrates for the first time the presence of oscillatory hydration layers at the interface of liquid phase lipid membranes, which have remained elusive in previous attempts. This achievement was made possible through the optimisation of AFM cantilevers and initial set-up parameters, including fine-tuning the applied force and acquisition speed. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2023 |
| Provided To Others? | Yes |
| Impact | The method can now be expanded to investigate hydration water arrangment across various lipid membranes and biological molecules. Several PhD students within our research group have begun utilising the newly implemented method for precisely this purpose, marking a significant advancement in our research projects. |
| Description | Water at lipid interfaces. |
| Organisation | Free University of Berlin |
| Country | Germany |
| Sector | Academic/University |
| PI Contribution | Our group produced new experimental data regarding the dielectric properties of interfacial near different lipids layers. |
| Collaborator Contribution | A collaboration has been established with the group of Prof. Roland Netz, Freie Universität Berlin (Germany), world leading research in atomistic simulations of water at biological interfaces and in particular its dielectric properties. His group has provided atomic simulations for the same systems, which were key to the interpretation of the results. |
| Impact | New data interpretation of our experimental findings. |
| Start Year | 2024 |
| Title | Matrix Force Reconstruction for AM AFM |
| Description | This is a novel numerical matrix-based Force Reconstruction Method specifically designed for Amplitude Modulation-Atomic Force Microscopy. The proposed matrix-based FRM, differently from standard FRMs, can reconstruct the tip-sample force for arbitrary value of the initial free amplitude, with no loss of information deriving from the specific choice of AFM experimental parameters or the force functional form. This method unlocks the full spectrum of physical phenomena encoded in the tip-sample interaction. An open access article describing the use of the software has been published. |
| Type Of Technology | New/Improved Technique/Technology |
| Year Produced | 2025 |
| Open Source License? | Yes |
| Impact | It is too early to judge the impact in the community since the algorithm has been just released. |
| URL | https://www.sciencedirect.com/science/article/pii/S0021979725001456#da005 |
| Description | Visit and talk at Italian Institute of Tachnology (CNST, Milano) |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | A visit was conducted at IIT Milano, where an oral presentation titled 'Probing the solid-liquid interface with atomic force microscopy' was delivered. The visit promoted discussion and opened new possible collaborations. |
| Year(s) Of Engagement Activity | 2023 |