The Impact of Redox, Hydrolysis and Dehydration Chemistry on the Structural and Magnetic Properties of Magnetoferritin Prepared in Variable Thermal Conditions

Ferritin, a spherically shaped protein complex, is responsible for iron storage in bacteria, plants, animals, and humans. Various ferritin iron core compositions in organisms are associated with specific living requirements, health state, and different biochemical roles of ferritin isomers. Magnetoferritin, a synthetic ferritin derivative, serves as an artificial model system of unusual iron phase structures found in humans. We present the results of a complex structural study of magnetoferritins prepared by controlled in vitro synthesis. Using various complementary methods, it was observed that manipulation of the synthesis technology can improve the physicochemical parameters of the system, which is useful in applications. Thus, a higher synthesis temperature leads to an increase in magnetization due to the formation of the magnetite phase. An increase in the iron loading factor has a more pronounced impact on the protein shell structure in comparison with the pH of the aqueous medium. On the other hand, a higher loading factor at physiological temperature enhances the formation of an amorphous phase instead of magnetite crystallization. It was confirmed that the iron-overloading effect alone (observed during pathological events) cannot contribute to the formation of magnetite.

Enhancing the Cell-Free Expression of Native Membrane Proteins by In Silico Optimization of the Coding Sequence—An Experimental Study of the Human Voltage-Dependent Anion Channel

Membrane proteins are involved in many aspects of cellular biology; for example, they regulate how cells interact with their environment, so such proteins are important drug targets. The rapid advancement in the field of immune effector cell therapy has been expanding the horizons of synthetic membrane receptors in the areas of cell-based immunotherapy and cellular medicine. However, the investigation of membrane proteins, which are key constituents of cells, is hampered by the difficulty and complexity of their in vitro synthesis, which is of unpredictable yield. Cell-free synthesis is herein employed to unravel the impact of the expression construct on gene transcription and translation, without the complex regulatory mechanisms of cellular systems. Through the systematic design of plasmids in the immediacy of the start of the target gene, it was possible to identify translation initiation and the conformation of mRNA as the main factors governing the cell-free expression efficiency of the human voltage-dependent anion channel (VDAC), which is a relevant membrane protein in drug-based therapy. A simple translation initiation model was developed to quantitatively assess the expression potential for the designed constructs. A scoring function that quantifies the feasibility of the formation of the translation initiation complex through the ribosome–mRNA hybridization energy and the accessibility of the mRNA segment binding to the ribosome is proposed. The scoring function enables one to optimize plasmid sequences and semi-quantitatively predict protein expression efficiencies. This scoring function is publicly available as webservice XenoExpressO at University of Vienna, Austria.

Enzymatic Synthesis of Cellulose in Space: Gravity Is a Crucial Factor for Building Cellulose II Gel Structure

We previously reported in vitro synthesis of highly ordered crystalline cellulose II by reverse reaction of cellodextrin phosphorylase from the cellulolytic bacterium Clostridium ( Hungateiclostridium ) thermocellum ( Ct CDP), but the formation mechanism of the cellulose crystals and highly ordered structure has long been unclear. Considering the specific density of cellulose versus water, the formation of crystalline and highly ordered structure in an aqueous solution should be affected by gravity. Thus, we synthesized cellulose with Ct CDP at the International Space Station, where sedimentation and convection due to gravity are negligible. Optical microscopic observation suggested that cellulose in space has a gel-like appearance without apparent aggregation, in contrast to cellulose synthesized on the ground. Small-angle Xray scattering (SAXS) and wide-angle X-ray scattering (WAXS) indicated that cellulose synthesized in space has a more uniform particle distribution in the ~100 nm scale region than cellulose synthesized on the ground. Scanning electron microscopy (SEM) showed that both celluloses have a micrometer scale network structure, whereas a fine fiber network was constructed only under microgravity. These results indicate that gravity plays a role in cellulose II crystal sedimentation and the building of network structure, and synthesis in space could play a role in the design of unique materials.

The “beauty in the beast”—the multiple uses of Priestia megaterium in biotechnology

Over 30 years, the Gram-positive bacterium Priestia megaterium (previously known as Bacillus megaterium) was systematically developed for biotechnological applications ranging from the production of small molecules like vitamin B12, over polymers like polyhydroxybutyrate (PHB) up to the in vivo and in vitro synthesis of multiple proteins and finally whole-cell applications. Here we describe the use of the natural vitamin B12 (cobalamin) producer P. megaterium for the elucidation of the biosynthetic pathway and the subsequent systematic knowledge-based development for production purposes. The formation of PHB, a natural product of P. megaterium and potential petro-plastic substitute, is covered and discussed. Further important biotechnological characteristics of P. megaterium for recombinant protein production including high protein secretion capacity and simple cultivation on value-added carbon sources are outlined. This includes the advanced system with almost 30 commercially available expression vectors for the intracellular and extracellular production of recombinant proteins at the g/L scale. We also revealed a novel P. megaterium transcription-translation system as a complementary and versatile biotechnological tool kit. As an impressive biotechnology application, the formation of various cytochrome P450 is also critically highlighted. Finally, whole cellular applications in plant protection are completing the overall picture of P. megaterium as a versatile giant cell factory. Key points • The use of Priestia megaterium for the biosynthesis of small molecules and recombinant proteins through to whole-cell applications is reviewed. • P. megaterium can act as a promising alternative host in biotechnological production processes.

Enhancing the Cell-Free Expression of Native Membrane Proteins by in-Silico Optimisation of the Coding Sequence - An Experimental Study of the Human Voltage-Dependent Anion Channel

The investigation of membrane proteins, key constituents of cells, is hampered by the difficulty and complexity of their in vitro synthesis, of unpredictable yield. Cell-free synthesis is herein employed to unravel the impact of the expression construct on gene transcription and translation, without the complex regulatory mechanisms of cellular systems. Through the systematic design of plasmids in the immediacy of the start of the target gene, it was possible to identify translation initiation and the conformation of mRNA as the main factors governing the cell-free expression efficiency of the human voltage dependent anion channel (VDAC), a relevant membrane protein in drug-based therapy. A simple translation initiation model was developed to quantitatively assess the expression potential for the designed constructs. A scoring function is proposed that quantifies the feasibility of formation of the translation initiation complex through the ribosome-mRNA hybridization energy and the accessibility of the mRNA segment binding to the ribosome. The scoring function enables to optimize plasmid sequences and semi-quantitatively predict protein expression efficiencies. This scoring function is publicly available as webservice XenoExpressO at rna.tbi.univie.ac.at.

Supervised Learning Model Predicts Protein Adsorption to Nanotubes

Engineered nanoparticles are advantageous for numerous biotechnology applications, including biomolecular sensing and delivery. However, testing the compatibility and function of nanotechnologies in biological systems requires a heuristic approach, where unpredictable biofouling often prevents effective implementation. Such biofouling is the result of spontaneous protein adsorption to the nanoparticle surface, forming the "protein corona" and altering the physicochemical properties, and thus intended function, of the nanotechnology. To better apply engineered nanoparticles in biological systems, herein, we develop a random forest classifier (RFC) trained with proteomic mass spectrometry data that identifies which proteins adsorb to nanoparticles. We model proteins that populate the corona of a single-walled carbon nanotube (SWCNT)-based optical nanosensor. We optimize the classifier and characterize the classifier performance against other models. To evaluate the predictive power of our model, we then apply the classifier to rapidly identify and experimentally validate proteins with high binding affinity to SWCNTs. Using protein properties based solely on amino acid sequence, we further determine protein features associated with increased likelihood of SWCNT binding: proteins with high content of solvent-exposed glycine residues and non-secondary structure-associated amino acids. Furthermore, proteins with high leucine residue content and beta-sheet-associated amino acids are less likely to form the SWCNT protein corona. The classifier presented herein provides an important tool to undertake the otherwise intractable problem of predicting protein-nanoparticle interactions, which is needed for more rapid and effective translation of nanobiotechnologies from in vitro synthesis to in vivo use.

Antimicrobial Hexaaquacopper(II) Complexes with Novel Polyiodide Chains

The non-toxic inorganic antimicrobial agents iodine (I2) and copper (Cu) are interesting alternatives for biocidal applications. Iodine is broad-spectrum antimicrobial agent but its use is overshadowed by compound instability, uncontrolled iodine release and short-term effectiveness. These disadvantages can be reduced by forming complex-stabilized, polymeric polyiodides. In a facile, in-vitro synthesis we prepared the copper-pentaiodide complex [Cu(H2O)6(12-crown-4)5]I6 ´ 2I2, investigated its structure and antimicrobial properties. The chemical structure of the compound has been verified. We used agar well and disc-diffusion method assays against nine microbial reference strains in comparison to common antibiotics. The stable complex revealed excellent inhibition zones against C. albicans WDCM 00054, and strong antibacterial activities against several pathogens. [Cu(H2O)6(12-crown-4)5]I6 ´ 2I2 is a strong antimicrobial agent with an interesting crystal structure consisting of complexes located on an inversion center and surrounded by six 12-crown-4 molecules forming a cationic substructure. The six 12-crown-4 molecules form hydrogen bonds with the central Cu(H2O)6 . The anionic substructure is a halogen bonded polymer which is formed by formal I5− repetition units. The topology of this chain-type polyiodide is unique. The I5− repetition units can be understood as a triodide anion connected to two iodine molecules.

In Vitro Synthesis, Structure elucidation and its antioxidant properties of Platinum(IV)-hydrazide complexes: Molecular modeling of free-hydrazides suggested as potent lipoxygenase inhibitor

Background: Combination of biologically active ligand and metal in one molecule may increase the activity and reduce the toxicity. Objectives: In this study synthesis and characterization of platinum(IV) complexes with bioactive hydrazide ligands is discussed. Method: Elemental analysis, conductivity measurements and spectroscopic studies were used to elucidate the structure of complexes. Results: Our study suggests that hydrazide ligands coordinate with Pt(IV) in bidenate fashion. The platinum(IV) complexes have octahedral geometry with metal to ligand ratio of 1:2. Hydrazide ligands coordinated with central metal platinum(IV) by oxygen of carbonyl group and nitrogen of primary amine. Synthesized complexes exhibited variable DPPH radical scavenging and lipoxygenase inhibition activity. Furthermore, it is also found that Pt(IV)-hydrazide complexes are more potent superoxide and nitric oxide radical scavengers than their uncoordinated hydrazide ligands while in case of lipoxygenase enzyme inhibition, some of the free hydrazide ligands are more active than their respective Pt(IV) complexes. In silico docking technique explores molecular interactions of synthesized ligands in the active site of lipoxygenase enzyme. Predicted docking energies are in good agreement with experimental data suggesting that in silico studies might be useful for discovery of therapeutic candidates. Conclusion: Structure function relationship demonstrates that the radical scavenging and enzyme inhibition activities of the Pt(IV) compounds are effected by nature of ligand, position of substituent, electronic and steric effects. However, electronic factor seem to play more important role than other factors.