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Showcasing research from the Federal Institute for Material Research and Testing Berlin and Fraunhofer Institute for Celltherapy and Immunology Branch Bioanalytics and Bioprocesses Potsdam.
Bio-SAXS of single-stranded DNA-binding proteins: Radiation protection by the compatible solute ectoine.
We aimed to increase the possible undisturbed exposure time during bio-SAXS measurements of single-stranded DNA-binding proteins. Therefore small angle X-ray scattering was performed on Gene-V Protein (G5P/GVP), which is involved in DNA repair processes. To achieve this, irradiations were performed in presence and absence of the hydroxyl-radical scavenger and osmolyte Ectoine, which showed efficient radiation protection and prevented protein aggregation, thus allows for a non-disturbing way to improve structure-determination of biomolecules.
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 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.
Dose enhancement by gold nanoparticles (AuNP) was shown to increase the biological effectiveness of radiation damage in biomolecules and tissue. Most of the current studies focus on external beam therapy on combination with AuNP. Here we present a Monte-Carlo study (Geant4) to characterise radioactive AuNP. Radioactive ¹⁹⁸Au emits beta and gamma rays and is considered for applications with solid tumours. To effectively apply ¹⁹⁸AuNP their energy deposit characteristics have to be determined in terms of intrinsic and extrinsic properties e.g. AuNP diameter, AuNP density, and their clustering behaviour. After each decay process, the energy deposit, inelastic scattering events, kinetic energy spectrum of secondary particles within the AuNP themselves and in a spherical target volume of water up to 1 μm radius were determined. Simulations were performed for AuNP radii ranging from 2.5 nm to 20 nm radius, different cluster sizes and densities. The results show an increase of the energy deposit in the vicinity of the AuNP up to 150 nm. This effect nearly vanishes for distances up to one micron. For the case of AuNP clusters and the same activity, the enhancement of the energy deposit increases with the relative gold mass percentage and therefore can be adjusted by changing AuNP radius or clustering behaviour.
Dose enhancement by gold nanoparticles (AuNP) increases the biological effectiveness of Radiation damage in biomolecules and tissue. To apply them effectively during cancer therapy their influence on the locally delivered dose has to be determined. Hereby, the AuNP locations strongly influence the energy deposit in the nucleus, mitochondria, membrane and the cytosol of the targeted cells. To estimate these effects, particle scattering simulations are applied. In general, different approaches for modeling the AuNP and their distribution within the cell are possible. In this work, two newly developed continuous and discrete-geometric models for simulations of AuNP in cells are presented.
These models are applicable to simulations of internal emitters and external radiation sources. Most of the current studies on AuNP focus on external beam therapy. In contrast, we apply the presented models in Monte-Carlo particle scattering simulations to characterize the energy deposit in cell organelles by radioactive 198AuNP. They emit beta and gamma rays and are therefore considered for applications with solid tumors. Differences in local dose enhancement between randomly distributed and nucleus targeted nanoparticles are compared. Hereby nucleus targeted nanoparticels showed a strong local dose enhancement in the radio sensitive nucleus. These results are the foundation for future experimental work which aims to obtain a mechanistic understanding of cell death induced by radioactive 198Au.
Ectoine is an important osmolyte, which allows microorganisms to survive in extreme environmental salinity. The hygroscopic effects of ectoine in pure water can be explained by a strong water binding behavior whereas a study on the effects of ectoine in salty solution is yet missing. We provide Raman spectroscopic evidence that the influence of ectoine and NaCl are opposing and completely independent of each other. The effect can be explained by the formation of strongly hydrogen-bonded water molecules around ectoine which compensate the influence of the salt on the water dynamics. The mechanism is corroborated by first principles calculations and broadens our understanding of zwitterionic osmolytes in aqueous solution. Our findings allow us to provide a possible explanation for the relatively high osmolyte concentrations in halotolerant bacteria.
Ectoine, a compatible solute and osmolyte, is known to be an effective protectant of biomolecules and whole cells against heating, freezing and extreme salinity. Protection of cells (human keratinocytes) by ectoine against ultraviolet radiation has also been reported by various authors, although the underlying mechanism is not yet understood. We present the first electron irradiation of DNA in a fully aqueous environment in the presence of ectoine and at high salt concentrations. The results demonstrate effective protection of DNA by ectoine against the induction of single-strand breaks by ionizing radiation. The effect is explained by an increase in low-energy electron scattering at the enhanced free-vibrational density of states of water due to ectoine, as well as the use of ectoine as an ˙OH-radical scavenger. This was demonstrated by Raman spectroscopy and electron paramagnetic resonance (EPR).
Microorganisms accumulate molar concentrations of compatible solutes like ectoine to prevent proteins from denaturation. Direct structural or spectroscopic information on the mechanism and about the hydration shell around ectoine are scarce. We combined surface plasmon resonance (SPR), confocal Raman spectroscopy, molecular dynamics simulations, and density functional theory (DFT) calculations to study the local hydration shell around ectoine and its influence on the binding of a gene-S-protein (G5P) to a single-stranded DNA (dT(25)). Due to the very high hygroscopicity of ectoine, it was possible to analyze the highly stable hydration shell by confocal Raman spectroscopy. Corresponding molecular dynamics simulation results revealed a significant change of the water dielectric constant in the presence of a high molar ectoine concentration as compared to pure water. The SPR data showed that the amount of protein bound to DNA decreases in the presence of ectoine, and hence, the protein-DNA dissociation constant increases in a concentration-dependent manner. Concomitantly, the Raman spectra in terms of the amide I region revealed large changes in the protein secondary structure. Our results indicate that ectoine strongly affects the molecular recognition between the protein and the oligonudeotide, which has important consequences for osmotic regulation mechanisms.
The damage caused by ionizing radiation to DNA and proteins is the reason to treat cancer by radiation therapy. A better understanding of the molecular processes and quantification of the different damaging mechanisms is the prerequisite to develop more efficient therapies. Hereby the understanding of the processes involved in the damage to DNA are of key interest due to its central role in reproduction and mutation.
For radiation with low linear energy transfer (LET), most of the damage is caused by the secondary particles produced by scattering of the ionizing radiation with water. Thereby a multitude of species are produced, whereby especially kinetic low energy electrons, prehydrated electrons, OH-radicals and ions are of importance. With higher LET the relative amount of the direct damaging effects increases. This is especially important considering the increased usage of high LET nucleons in radiation therapy. Therefore, the quantification of the contribution to DNA damage of direct and indirect effects and the different secondary species is of high interest due to the increase of radio biological efficiency when applying high LET radiation.
Here we present an approach to investigate the relative contributions to DNA strand break yield for radiation of different LET within a single electron microscope in combination with electron scattering simulations.
In dosimetry the determination of the effectiveness of the damaging processes is standardized and accounted for by the radiation and tissue weighting factor. For the underlying constituents of the tissue, that is the various biomolecules, such a systematic approach doesn't exist. This makes it difficult to compare results obtained under different experimental conditions. In the following work, we will describe a method to obtain comparable values for the radiation-biomolecule interaction, measured under different conditions. This approach can lead to standardization of dosedamage relationship at the molecular level. Such approach is necessary for a better understanding of the relations between the damage of the single constituents of biological tissue and the whole – finally gaining a more complete picture of irradiation damage.
Ectoine is a small zwitterionic osmolyte and compatible solute, which does not interfere with cell metabolism even at molar concentrations. Plasmid DNA (pUC19) was irradiated with ultraviolet radiation (UV-C at 266 nm) under quasi physiological conditions (PBS) and in pure water in the presence and absence of ectoine (THP(B)) and hydroxyectoine (THP(A)). Different types of UV induced DNA damage were analysed: DNA single-strand breaks (SSBs), abasic sites and cyclobutane pyrimidine dimers (CPDs). A complex interplay between these factors was observed with respect to the nature and occurrence of DNA damage with 266 nm photons. In PBS, the cosolutes showed efficient protection against base damage, whilst in pure water, a dramatic shift from SSB damage to base damage was observed when cosolutes were added. To test whether these effects are caused by ectoine binding to DNA, further experiments were conducted: small-angle X-ray scattering (SAXS), surface-plasmon resonance (SPR) measurements and Raman spectroscopy. The results show, for the first time, a close interaction between ectoine and DNA. This is in stark contrast to the assumption made by preferential exclusion models, which are often used to interpret the behaviour of compatible solutes within cells and with biomolecules. It is tentatively proposed that the alterations of UV damage to DNA are attributed to ectoine influence on nucleobases through the direct interaction between ectoine and DNA.