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Bio-SAXS of single-stranded DNA-binding proteins: Radiation protection by the compatible solute ectoine

  • Small-angle X-ray scattering (SAXS) can be used for structural determination of biological macromolecules and polymers in their native states (e.g. liquid phase). This means that the structural changes of (bio-)polymers, such as proteins and DNA, can be monitored in situ to understand their sensitivity to changes in chemical environments. In an attempt to improve the reliability of such experiments, the reduction of radiation damage occurring from exposure to X-rays is required. One such method, is to use scavenger molecules to protect macromolecules against radicals produced during radiation exposure, such as reactive oxygen species (ROS). In this study we investigate the feasibility of applying the compatible solute, osmolyte and radiation protector Ectoine (THP(B)), as a scavenger molecule during SAXS measurements of the single-stranded DNA-binding protein Gene-V Protein (G5P/GVP). In this case, we monitor the radiation induced changes of G5P during bio-SAXS measurments and theSmall-angle X-ray scattering (SAXS) can be used for structural determination of biological macromolecules and polymers in their native states (e.g. liquid phase). This means that the structural changes of (bio-)polymers, such as proteins and DNA, can be monitored in situ to understand their sensitivity to changes in chemical environments. In an attempt to improve the reliability of such experiments, the reduction of radiation damage occurring from exposure to X-rays is required. One such method, is to use scavenger molecules to protect macromolecules against radicals produced during radiation exposure, such as reactive oxygen species (ROS). In this study we investigate the feasibility of applying the compatible solute, osmolyte and radiation protector Ectoine (THP(B)), as a scavenger molecule during SAXS measurements of the single-stranded DNA-binding protein Gene-V Protein (G5P/GVP). In this case, we monitor the radiation induced changes of G5P during bio-SAXS measurments and the resulting microscopic energy-damage relation was determined from microdosimetric calculations by Monte-Carlo based particle scattering simulations with TOPAS/Geant4 and a custom target-model. This resulted in a median-lethal energy deposit of pure G5P at 4 mg mL−1 of E1/2 = 7 ± 5 eV, whereas a threefold increase of energy-deposit was needed under the presence of Ectoine to reach the same level of damage. This indicates that Ectoine increases the possible exposure time before radiation-damage to G5P is observed. Furthermore, the dominant type of damage shifted from aggregation in pure solutions towards a fragmentation for solutions containing Ectoine as a cosolute. These results are interpreted in terms of indirect radiation damage by reactive secondary species, as well as post-irradiation effects, related to preferential-exclusion of the cosolute from the protein surface. Hence, Ectoine is shown to provide a non-disturbing way to improve structure-determination of proteins via bio-SAXS in future studies.zeige mehrzeige weniger

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Metadaten
Autor*innen:Marc Benjamin HahnORCiD
Koautor*innen:Dorothea C. Hallier, H. Seitz, Glen Jacob Smales
Dokumenttyp:Posterpräsentation
Veröffentlichungsform:Präsentation
Sprache:Englisch
Jahr der Erstveröffentlichung:2023
Organisationseinheit der BAM:6 Materialchemie
6 Materialchemie / 6.6 Physik und chemische Analytik der Polymere
DDC-Klassifikation:Naturwissenschaften und Mathematik / Chemie / Analytische Chemie
Technik, Medizin, angewandte Wissenschaften / Ingenieurwissenschaften / Sanitär- und Kommunaltechnik; Umwelttechnik
Freie Schlagwörter:Bio-SAXS; BioSAXS; Compatible solute; Cosolute; DNA; Dosimetry; Ectoin; Ectoine; G5P; GVP; Geant4; Geant4-DNA; Hydroxyectoine; Ionizing radiation damage; LEE; McSAS3; Microdosimetry; Monte-Carlo simulations; OH Radical; OH radical scavenger; Osmolyte; Particle scattering simulations; Protein; Protein unfolding; ROS; Radiation damage; Radical Scavenger; SAXS; Single-stranded DNA-binding proteins; Small-angle xray scattering; Topas; Topas-MC; Topas-nBio; X-ray scattering; radical scavenger; ssDNA
Themenfelder/Aktivitätsfelder der BAM:Chemie und Prozesstechnik
Umwelt
Umwelt / Umwelt-Material-Interaktionen
Veranstaltung:#RSCposter 2023
Veranstaltungsort:Online meeting
Beginndatum der Veranstaltung:28.02.2023
Enddatum der Veranstaltung:01.03.2023
URL:https://pubs.rsc.org/en/content/articlehtml/2023/cp/d2cp05053f
Verfügbarkeit des Dokuments:Datei im Netzwerk der BAM verfügbar ("Closed Access")
Datum der Freischaltung:02.03.2023
Referierte Publikation:Nein
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