Proton transport and proton-coupled transport

Project Information

PROTON

Grant agreement ID: 860592

DOI

10.3030/860592

Project closed

EC signature date 9 August 2019

Start date 1 September 2019

End date 31 July 2024

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 3 920 811,13

EU contribution

€ 3 920 811,13

Coordinated by

UNIVERSITAT LINZ
Austria

Periodic Reporting for period 2 - PROTON (Proton transport and proton-coupled transport)

Reporting period: 2021-09-01 to 2024-07-31

Summary of the context and overall objectives of the project

Secure, competitive, and sustainable energy production is a major challenge facing human societies. Biomimetic solutions such as the development of new biofuel cells are hampered by our thus far incomplete understanding of proton transfer reactions. The same holds for health threats to humanity: Curing diseases like cancer, obesity, chronic gastritis, gastric and duodenal ulcers, requires molecular details of yet unresolved proton transfer reactions.
We aim to clarify the molecular reaction mechanism in the confines of interfacial water layers and proteinaceous cavities with emphasis on the arrangement and mobility of proton relay moieties. Achieving this requires an interdisciplinary, multi-level approach comprising cutting-edge technologies like second harmonic imaging, single-molecule and time-resolved fluorescence microscopy and spectroscopy, advanced calculations of proton transfer, bioengineering of membrane channel and transporter-containing systems, synthetic design of biomimetic proton channels, solving protein structures and rational drug design.

Proton transfer is crucial in numerous biological and chemical processes, e.g. in cellular proton pumps or in hydrogen fuel cells. Even though their empirical study began with the origin of chemistry, many details of the proton transfer mechanism are still unresolved and understanding the way in which confined water mediates proton dynamics remains a fundamental challenge in chemistry and biochemistry. Transmembrane proton gradients are essential to life on earth as they are intricately linked to both photosynthesis and synthesis of adenosine triphosphate (ATP, the energy currency of life). Yet, once protons have crossed the membrane, they do not freely exchange with protons on the receiving site. An energy barrier with the height of ~30 kT opposes their release into the bulk. The mainly entropic nature of the barrier ensures high lateral proton mobility. However, besides being attributed to structured water, the molecular origin of that barrier remained thus far elusive. Yet, newly developed label-free and charge-sensitive dynamic imaging techniques of lipid membrane hydration, hydration of active protein sites as well as their dipolar relaxation dynamics now offer the possibility to explore the interplay between structural features of the hydration shell and proton migration on the millisecond time scale. Likewise, technically demanding ab-initio molecular dynamics (MD) simulations of protons adjacent to lipid bilayers also promise insight into the molecular proton migration mechanism. By levering on these new methods for (i) visualising proton surface transport as well as (ii) assessing its energetics and combining them with approaches for deciphering the structure of G-protein-coupled receptors (GPCRs) and other proton-dependent membrane protein, the PROTON project will perform ground-breaking work in this field.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The PROTON program successfully recruited, enrolled, and trained 15 PhD students, providing them with a comprehensive scientific and professional development experience.

The PROTON network's research dissemination was robust, with PhD students presenting their findings at 16 international conferences and workshops. A total of 35 publications have been produced so far, with 12 more in preparation. The ESRs published high-ranking journals, including renowned outlets such as the Journal of the American Chemical Society (JACS), Proceedings of the National Academy of Sciences (PNAS), and Angewandte Chemie.

ESR2, Stefania Brescia, resolved a debate about the mechanism of proton transfer in the HV1 channel, clarifying the essential role of intraluminal amino acid residues. Her work also sheds light on the origin of Hv1’s proton selectivity.
ESR3, Iga Jakóbowska, developed an FCCS-based assay to measure inhibitor binding in the PfFNT proton transporter, advancing studies of antimalarial targets.
ESR4, Seonwoo Lee, used high-throughput microscopy to study proton permeability in lipid membranes, offering insights into energy loss through unintended proton leakage.
ESR5, Anna Maznichenko, investigated the role of ordered water on interfacial proton transport and narrowed down the localisation of the proton release barrier.
ESR6, Nathan Hugo Epalle, engineered FNT mutants to assess proton and water transport, discovering selective water permeability, suggesting the potential for aquaporin mimics.
ESR7, Bhav Kapur, created assays for the proton-sensing receptor GPR68, revealing pH-sensitive structural changes that could aid in drug design.
ESR10, Bingxin Chu, studied size-dependent effects in second harmonic scattering for nanoparticles, thus characterising the hydration shell of nanoparticles.
ESR11, Satyaranjan Bharambar Biswal, modeled proton conduction in collagen, highlighting unique hydration layers that guide proton diffusion.
ESR12, Iuliana-Marilena Andrei, and ESR17, Paras Raju Wanjari, developed pillar[5]arene-based synthetic proton channels, achieving selective ion transport.
ESR13, Alejandro Martínez León, used Cryo-EM models to study PfFNT’s structure, identifying potential inhibitors for antimalarial drug development.
ESR14, Abhinav Abhinav, and ESR18, Isabel Králová, studied the pH-gated conformational changes of HpUreI, an essential bacterial protein.
ESR15/19, Honey Jane, explored how lipid composition affects proton transfer on membranes, showing acidic lipids enhance this process near proton channels.
ESR16/20, Giorgia Roticiani, investigated proton transport in mitochondrial proteins, revealing mechanisms critical to cellular energy regulation.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

PROTON provided training to 15 PhD students, equipping them with cutting-edge, multidisciplinary scientific expertise in proton migration and reaction systems. Their training included five major training schools, which covered a range of scientific techniques and methodologies, such as single-molecule research and nanoscience techniques such as fluorescence microscopy and atomic force microscopy (AFM), scientific visualization and data analysis tools, advanced fluorescence techniques to study hydration and mobility, including second harmonic imaging, computational methods in drug discovery, including the use of artificial intelligence and molecular simulations. The soft skills courses included gender bias in science, project management, scientific writing, and the principles of open science. The entrepreneurship and innovation course was particularly impactful, offering PhD students hands-on experience in creating and pitching business plans. These soft skills courses were designed to prepare the students for future careers in both academia and industry.

The scientific results of the PROTON network span both fundamental research and practical applications. Among the key achievements are the development of artificial selective channels, innovative online tools for drug design, and new insights into facilitated and spontaneous membrane permeability to protons. Using a novel technique, label-free high-throughput wide-field second harmonic (SH) microscopy, researchers were able to detect transient proton wires in lipid bilayers for the first time. PROTON also established efficient assays for crucial transporters, such as the human Hv1 and the malaria-related PfFNT, making significant contributions to public health research.

Proton transport in biomembranes