Structures of photoactive proteins studied by (in-situ) neutron scattering

Duration of the project

01.06.2021. - 15.11.2021.

Countries and institutions involved in the project

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University of Tartu
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Humboldt University of Berlin
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Technical University of Berlin

Project manager

Prof. Dr. Jörg Pieper

Aim of the project

A proper understanding of protein function requires detailed knowledge about protein structure, which will be demonstrated for two attractive examples. First, photosystem I (PSI) is a remarkably stable membrane protein converting solar energy into electrical and finally storable chemical energy. PSI is of great biotechnological interest as it can be integrated into biohybrid photoelectrochemical cells. For electrode design, structural characterization of the complex formation between PSI and electron donors/acceptors like cytochrome c6, ferredoxin, and FNR is needed. Second, the orange carotenoid protein (OCP) is photoswitchable and undergoes a structural transition to its active state upon illumination. Despite many efforts, the active state structure is still elusive. In the absence of high-resolution structures for the latter examples, small-angle neutron scattering (SANS) is the technique of choice to investigate structures in aqueous solution, complex formation, or light-induced structural changes. Since PSI is a membrane protein, SANS studies require solubilization using deuterated detergents, which are “invisible” with proper contrast matching and do thus not affect the structure determination of the PSI complexes. In contrast, SANS studies of OCP require the design of a setup for in-situ illumination during the SANS experiments. Both applications will deliver unique structural information.

Main activities of the project

  1. The Institute of Physics, Tartu, Estonia, is responsible for preparing and performing the neutron scattering experiments (small-angle scattering) and for the construction of a setup for in-situ optical excitation of the samples. Finally, data analysis and the development of structural models will be carried out here.
  2. The Humboldt Universität zu Berlin, Germany, will prepare and supply complexes of PSI with different electron donors and acceptors for biotechnological applications. In this regard, it is absolutely essential for the success of the SANS experiments that PSI will be isolated using fully deuterated (“invisible”) detergents for SANS with contrast variation, because matching of protonated detergent is inherently incomplete and may result in artifacts in structural models derived from SANS data.
  3. The group at Technische Universität Berlin, Germany, will prepare samples of the orange carotenoid protein with different carotenoid content and complexes with fluorescence recovery protein using partial deuteration for SANS with contrast variation.
  4. The experiments will be carried out at MLZ Garching, Germany, using small-angle neutron scattering, in part with in-situ illumination, within the user service of the neutron scattering facility. One scientist of the Tartu group has to travel to Garching to carry out this experiment.
  5. The European Spallation Source ERIC (ESS) Lund, Sweden (ESS) is a collaborative effort of 17 European countries including Germany and Estonia to build a new generation neutron source with unparalleled neutron flux permitting novel types of neutron scattering experiments. The group of J.Pieper develops new in-situ methods for structure research with neutrons for ESS and especially also for instruments developed by German partners like MLZ Garching so that both, the Estonian and German contribution to ESS will benefit from the experiences gathered during this project.
  6. In view of the pandemic, we do not plan any meetings or workshops in-person to avoid travel but will hold regular video conferences to discuss the progress during the project period and a final online seminar to summarize the results.
  7. Because we will not encounter large costs for travel or workshops, we apply for using financial support by Hochschulkontor for laboratory material and equipment for key project aspects beyond the limit of 1500 Euros outlined in the project guidelines.

Direct and indirect target group of the project

Direct: The project will provide attractive applications for the neutron scattering group at the Institute of Physics in Tartu, Estonia, while the existing biophysical research at HU Berlin and TU Berlin will benefit from a new approach to characterize biomolecular structures and structural changes under nearly physiological conditions using small-angle scattering with contrast variation. In addition, one postdoc and four PhD students will be involved in the project at the Institute of Physics (Maksym Golub, Nicholas Croy, Mina Hajizadeh), HU Berlin (Jelena Boyka), and TU Berlin (Marcus Moldenhauer). Within Estonia, a community of about 20-30 scientists will benefit from the dissemination of the expected results.

Indirect: The results and methodological developments of this project are interesting for different scientific communities: the biophysics community generally interested in structural research on biomolecules (ca. 100 scientists in Europe), the community working on biotechnological applications of photosynthesis, and, finally, the neutron scattering community interested in the development of new neutron scattering methods and sample environment for biological applications – a major field of application for neutron scattering methods (ca. 800 scientists in Europe). An important aspect for Estonia is the build-up of a local neutron community of neutron scatterers who can take advantage of the novel experimental possibilities at the ESS (currently ca. 20 scientists in Estonia). In turn, Germany is one of the leading countries in neutron scattering in Europe and, thus, has a great interest in the further development of this method – also in relation to ESS ERIC. Finally, society will in perspective benefit from the development of cleaner and more sustainable energy sources.

The community of Estonian neutron scatterers is still rather small and amounts to about 20 scientists, but their scientific output in terms of publications has seen constant growth since about 2010 along with its involvement in ESS. Within Europe, the interested community consists of about 8000 scientists. The latter information is taken from a report of the BrightnESS ERC project compiled together with the European Neutron Scattering Association (ENSA) in 2020.