Sequence, Structure, and Function of DNA-Binding Protein in Deinococcus Wulumuqiensis R12

Deinococcus wulumuqiensis R12, which was isolated from arid irradiated soil in Xinjiang province of China, belongs to a genus Deinococcus that is well-known for its extreme resistance to ionizing radiation and oxidative stress. The DNA-binding protein Dps has been studied for its great contribution to oxidative resistance. To explore the role of Dps in D. wulumuqiensis R12, the Dps sequence and homologous structure were analyzed. In addition, the dps gene was knocked out and proteomics was used to verify the functions of Dps in D. wulumuqiensis R12. Docking data and DNA binding experiments in vitro showed that the R12 Dps has a better DNA binding ability with the N-terminal than the R1 Dps1. When the dps gene was deleted in D. wulumuqiensis R12, its resistance to H2O2 and UV rays was greatly reduced, and the cell envelope was destroyed by H2O2 treatment. Additionally, the qRT-PCR and proteomics data suggested that when the dps gene was deleted, the catalase gene was significantly down-regulated in cells. And the proteomics data indicated the metabolism, transport and oxidation-reduction processes in D. wulumuqiensis R12 were down-regulated after the deletion of dps gene. Dps protein might play an important role in Deinococcus wulumuqiensis R12.

Dps protein localization studies in nanostructured silicon matrix by scanning electron microscopy

The present work is related to the microscopic studies of the morphology of the planar and inner part of silicon nanowires arrays before and after immobilization with a natural nanomaterial, Dps protein of bacterial origin. Silicon nanowires were formed by metal-assisted wet chemical etching. To obtain the recombinant protein, Escherichia coli cells were used as excretion strain and purification were carried out using chromatography. The combination of silicon nanowires with protein molecules was carried out by layering at laboratory conditions followed by drying under air. The resulting hybrid material was studied by high-resolution scanning electron microscopy. Studies of the developed surface of the nanowires array were carried out before and after combining with the bioculture. The initial arrays of silicon wireshave a sharp boundaries in the planar part and in the depth of the array, transition layers are not observed. The diameter of the silicon nanowires is about 100 nm, the height is over a micrometer, while the distances between the nanowires are several hundred of nanometers. The pores formed in this way are available for filling with protein during the immobilization of protein.The effectiveness of using the scanning electron microscopy to study the surface morphology of the hybrid material “silicon wires – bacterial protein Dps” has been demonstrated. It is shown that the pores with an extremely developed surface can be combined with a bio-material by deposition deep into cavities. The protein molecules can easily penetrate through whole porous wires matrix array. The obtained results demonstrate the possibility of efficient immobilization of nanoscaled Dps protein molecules into an accessible and controllably developed surface of silicon nanowires.

Molecular dynamics of DNA-binding protein and its 2D-crystals

In this work the dodecamers and the two-dimensional crystals of DNA-binding protein from starved cells (DPS) of Escherichia coli bacteria were investigated. The DPS monomer contains 167 amino acids residues. It can form dimers, trimers, and dodecamers. The versatility of the DPS protein structure can be used to design nanomaterials with structures and functions not found in living nature. The ability of this protein to self-assemble into complex shapes and structures defined on the nanometer scale can make them highly demanded for various technological applications. It was used all-atom classical molecular dynamics simulation on 0.1 microsecond scale to obtain the spatial and energy characteristics of the proteins and the components of the simulation box. The fluctuation mobility of DPS protein at various temperatures was discussed. The diffusion of ions in the presence of dodecamers and 2D crystals was compared. It has been shown that this protein retains its ability to accumulate ions in a wide range of biological temperatures from 277 to 369K. It also retains the mobility of key amino acid residues involved in the formation of nanocrystals and the transport of ions into the cavity, even at low physiological temperatures.

Novel DNA-protection Protein (Dps) Originated From Deinococcus Geothermalis

We characterized two interrelated proteins from the Deinococcus geothermalis strain with regard to the role of DNA-binding and protection, such as a novel Dps, Dgeo_0257, and a Dps orthologous protein, Dgeo_0281. Despite the lack of conserved amino acid sequence for ferroxidase activity, Dgeo_0257 exhibited high DNA-binding affinity and formed a dodecameric conformation. In contrast, Dgeo_0281 protein showed less effective DNA-binding and was mainly observed monomeric or dimeric forms. Electrophoretic mobility shift assay showed that both purified proteins are non-specifically bound to DNA to protect against DNA degradation and the properties of Dps responding to specific metal ions. In the presence of ferrous and ferric ions, Dgeo_0257 or Dgeo_0281 protein does not readily bind to DNA. However, in Zn and Mn ions are present, both proteins had more stable DNA-binding. From the Dps gene disrupted mutant test, each showed a higher sensitivity to oxidative stress than the wild-type strain. In addition, the expression level of each gene was correlated with the presence of each other. In this study, we characterized and confirmed the functional roles of Dgeo_0257 for sensing metal ions and binding to DNAs along with the Dps orthologous Dgeo_0281. Based on various observational evidence, we propose that Dgeo_0257 is a novel Dps protein with no ferroxidase activity.

Time-Resolved Studies of Ytterbium Distribution at Interfacial Surfaces of Ferritin-Like Dps Protein Demonstrate Metal Uptake and Storage Pathways

Cage-shaped protein (CSP) complexes are frequently used in bionanotechnology, and they have a variety of different architectures and sizes. The smallest cage-shaped protein, Dps (DNA binding protein from starved cells), can naturally form iron oxide biominerals in a multistep process of ion attraction, translocation, oxidation, and nucleation. The structural basis of this biomineralization mechanism is still unclear. The aim of this paper is to further develop understanding of this topic. Time-resolved metal translocation of Yb3+ ions has been investigated on Dps surfaces using X-ray crystallography. The results reveal that the soak time of protein crystals with Yb3+ ions strongly affects metal positions during metal translocation, in particular, around and inside the ion translocation pore. We have trapped a dynamic state with ongoing translocation events and compared this to a static state, which is reached when the cavity of Dps is entirely filled by metal ions and translocation is therefore blocked. By comparison with La3+ and Co2+ datasets, the time-dependence together with the coordination sphere chemistry primarily determine metalprotein interactions. Our data can allow structure-based protein engineering to generate CSPs for the production of tailored nanoparticles.

Structural Rearrangement of Dps-DNA Complex Caused by Divalent Mg and Fe Cations

Two independent, complementary methods of structural analysis were used to elucidate the effect of divalent magnesium and iron cations on the structure of the protective Dps-DNA complex. Small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) demonstrate that Mg2+ ions block the N-terminals of the Dps protein preventing its interaction with DNA. Non-interacting macromolecules of Dps and DNA remain in the solution in this case. The subsequent addition of the chelating agent (EDTA) leads to a complete restoration of the structure of the complex. Different effect was observed when Fe cations were added to the Dps-DNA complex; the presence of Fe2+ in solution leads to the total complex destruction and aggregation without possibility of the complex restoration with the chelating agent. Here, we discuss these different responses of the Dps-DNA complex on the presence of additional free metal cations, investigating the structure of the Dps protein with and without cations using SAXS and cryo-EM. Additionally, the single particle analysis of Dps with accumulated iron performed by cryo-EM shows localization of iron nanoparticles inside the Dps cavity next to the acidic (hydrophobic) pore, near three glutamate residues.

Abstract OR-2: The Formation of Dps-DNA Complexes under Different Conditions According to Cryo-EM and SAXS

Background: The effect of Dps-DNA co-crystals formation, which occurs in stressed Escherichia coli cells exposed to extreme conditions, is well described in the literature. However, the exact mechanisms of co-crystals formation are yet to be postulated remaining largely unknown. Here we summarize the results obtained by our group over the last few years using cryo-Electron Microscopy (cryo-EM) and Small Angle X-ray Scattering (SAXS). Methods: Samples for cryo-EM were plunge frozen in liquid ethane with Vitrobot Mark IV and studied with Titan Krios (ThermoFisher Scientific, US) cryo-EM, equipped with Falcon 2 direct electron detector, Image corrector (CEOS, Germany), and Volta phase plate. Single Particle Analysis (SPA) and cryo-Electron Tomography (cryo-ET) studies were conducted with 300 kV accelerating voltage in low dose mode using EPU and Tomography software (ThermoFisher Scientific, US). Cryo-EM data processing was conducted using Warp, CryoSPARC, IMOD, EMAN, and Relion software packages. SAXS measurements were performed at the EMBL on the P12 BioSAXS beam line at the PETRAIII storage ring (DESY, Hamburg). Results: In this work, Dps-DNA complex formation is thoroughly studied using complementary cryo-EM (including SPA, cryo-ET, and subtomogram averaging) and SAXS methods. The formation of individual complexes of Dps with small linear DNA fragments and the Dps-Dps interaction was visualized using cryo-EM. It was found that Dps-DNA complex remains stable under various conditions and while the addition of different ions leads to the disruption of co-crystals, the process is completely or partially reversible. Conclusion: Recent studies conducted by our group showed that Dps-DNA co-crystals adopt triclinic or cubic crystal lattice (FEBS Lett., 2019; Biomolecules, 2020). Here we present the results on the studies of Dps interaction with small linear DNA fragments, demonstrate the effects of MgCl2, FeSO4, and EDTA on the Dps-DNA complex and individual Dps protein structure, discuss the influence of the temperature and time on the co-crystals.

Single-dose immunisation with a multimerised SARS-CoV-2 receptor binding domain (RBD) induces an enhanced and protective response in mice

The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, has triggered a worldwide health emergency. So far, several different types of vaccines have shown strong efficacy. However, both the emergence of new SARS-CoV-2 variants and the need to vaccinate a large fraction of the world's population necessitate the development of alternative vaccines, especially those that are simple and easy to store, transport and administer. Here, we showed that ferritin-like Dps protein from hyperthermophilic Sulfolobus islandicus can be covalently coupled with different SARS-CoV-2 antigens via the SpyCatcher system, to form extremely stable and defined multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS-CoV-2 receptor binding domain (RBD) coupled to Dps (RBD-S-Dps) shows particular promise as it elicited a higher antibody titre and an enhanced neutralising antibody response compared to the monomeric RBD. Furthermore, we showed that a single immunisation with the multivalent RBD-S-Dps completely protected hACE2-expressing mice from serious illness and led to efficient viral clearance from the lungs upon SARS-CoV-2 infection. Our data highlight that multimerised SARS-CoV-2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultra-stable scaffold.

Cytochrome C with Peroxidase-like Activity Encapsulated Inside the Small DPS Protein Nanocage

Nature utilizes self-assembled protein-based structures as subcellular compartments in prokaryotes to sequester catalysts for specialized biochemical reactions. These protein cage structures provide unique isolated environments for the encapsulated enzymes. Understanding...