Dissociation Mechanics and Stability of Type a Botulinum Neurotoxin Complex by Means of Biophysical Evaluation

Biophysical characterization of type A botulinum neurotoxin (BoNT/A) complex along with its thermodynamic stability was assessed through a combination of various methods. BoNT/A exists as large complexes in association with neurotoxin associated proteins (NAPs). To evaluate its biophysical behavior, size-exclusion chromatography (SEC), multi-angled light scattering (MALS), enzyme linked immunosorbent assay (ELISA), and dynamic light scattering (DLS) were utilized. Initially, a single peak (peak 1) of SEC was observed at pH 6.0, and an additional peak (peak 2) appeared at pH 7.4 with a decrement of peak 1. Through MALS and ELISA, the peak 2 was determined to be BoNT/A dissociated from its complex. The dissociation was accelerated by time and temperature. At 37°C, dissociated BoNT/A self-associated at pH 7.4 in the presence of polysorbate 20. On the other hand, the dissociation was partly reversible when titrated back to pH 6.0. Overall, BoNT/A was more stable when associated with NAPs at pH 6.0 compared to its dissociated state at pH 7.4. The conventional analytical methods could be utilized to relatively quantify its amount in different formulations.

Efficient production of immunologically active Shigella invasion plasmid antigens IpaB and IpaH using a cell-free expression system

Shigella spp. invade the colonic epithelium and cause bacillary dysentery in humans. Individuals living in areas that lack access to clean water and sanitation are the most affected. Even though infection can be treated with antibiotics, Shigella antimicrobial drug resistance complicates clinical management. Despite decades of effort, there are no licensed vaccines to prevent shigellosis. The highly conserved invasion plasmid antigens (Ipa), which are components of the Shigella type III secretion system, participate in bacterial epithelial cell invasion and have been pursued as vaccine targets. However, expression and purification of these proteins in conventional cell-based systems have been challenging due to solubility issues and extremely low recovery yields. These difficulties have impeded manufacturing and clinical advancement. In this study, we describe a new method to express Ipa proteins using the Xpress+TM cell-free protein synthesis (CFPS) platform. Both IpaB and the C-terminal domain of IpaH1.4 (IpaH-CTD) were efficiently produced with this technology at yields > 200 mg/L. Furthermore, the expression was linearly scaled in a bioreactor under controlled conditions, and proteins were successfully purified using multimode column chromatography to > 95% purity as determined by SDS-PAGE. Biophysical characterization of the cell-free synthetized IpaB and IpaH-CTD using SEC-MALS analysis showed well-defined oligomeric states of the proteins in solution. Functional analysis revealed similar immunoreactivity as compared to antigens purified from E. coli. These results demonstrate the efficiency of CFPS for Shigella protein production; the practicality and scalability of this method will facilitate production of antigens for Shigella vaccine development and immunological analysis. Key points • First report of Shigella IpaB and IpaH produced at high purity and yield using CFPS • CFPS-IpaB and IpaH perform similarly to E. coli–produced proteins in immunoassays • CFPS-IpaB and IpaH react with Shigella-specific human antibodies and are immunogenic in mice. Graphical abstract

Biophysical characterization of the inactivation of E. coli transketolase by aqueous co-solvents

Transketolase (TK) has been previously engineered, using semi-rational directed evolution and substrate walking, to accept increasingly aliphatic, cyclic, and then aromatic substrates. This has ultimately led to the poor water solubility of new substrates, as a potential bottleneck to further exploitation of this enzyme in biocatalysis. Here we used a range of biophysical studies to characterise the response of both E. coli apo- and holo-TK activity and structure to a range of polar organic co-solvents: acetonitrile (AcCN), n-butanol (nBuOH), ethyl acetate (EtOAc), isopropanol (iPrOH), and tetrahydrofuran (THF). The mechanism of enzyme deactivation was found to be predominantly via solvent-induced local unfolding. Holo-TK is thermodynamically more stable than apo-TK and yet for four of the five co-solvents it retained less activity than apo-TK after exposure to organic solvents, indicating that solvent tolerance was not simply correlated to global conformational stability. The co-solvent concentrations required for complete enzyme inactivation was inversely proportional to co-solvent log(P), while the unfolding rate was directly proportional, indicating that the solvents interact with and partially unfold the enzyme through hydrophobic contacts. Small amounts of aggregate formed in some cases, but this was not sufficient to explain the enzyme inactivation. TK was found to be tolerant to 15% (v/v) iPrOH, 10% (v/v) AcCN, or 6% (v/v) nBuOH over 3 h. This work indicates that future attempts to engineer the enzyme to better tolerate co-solvents should focus on increasing the stability of the protein to local unfolding, particularly in and around the cofactor-binding loops.

Gliadin proteolytical resistant peptides: the interplay between structure and self-assembly in gluten-related disorders

In recent years, the evaluation of the structural properties of food has become of crucial importance in the understanding of food-related disorders. One of the most exciting systems is gliadin, a protein in wheat gluten, that plays a protagonist role in gluten-related disorders with a worldwide prevalence of 5%, including autoimmune celiac disease (CeD) (1%) and non-celiac wheat sensitivity (0.5–13%). It is accepted that gliadin is not fully digested by humans, producing large peptides that reach the gut mucosa. The gliadin peptides cross the lamina propria eliciting different immune responses in susceptible patients. Many clinical and biomedical efforts aim to diagnose and understand gluten-related disorders; meanwhile, the early stages of the inflammatory events remain elusive. Interestingly, although the primary sequence of many gliadin peptides is well known, it was only recently revealed the self-assembly capability of two pathogenic gliadin fragments and their connection to the early stage of diseases. This review is dedicated to the most relevant biophysical characterization of the complex gliadin digest and the two most studied gliadin fragments, the immunodominant 33-mer peptide and the toxic p31-43 in connection with inflammation and innate immune response. Here, we want to emphasize that combining different biophysical methods with cellular and in vivo models is of key importance to get an integrative understanding of a complex biological problem, as discussed here.

Site-Specific Synthesis of N4-Acetylcytidine in RNA Reveals Physiological Duplex Stabilization

N4-acetylcytidine (ac4C) is a post-transcriptional modification of RNA that is conserved across all domains of life. All characterized sites of ac4C in eukaryotic RNA occur in the central nucleotide of a CCG consensus sequence. However, the thermodynamic consequences of cytidine acetylation in this context have never been assessed due to its challenging synthesis. Here we report the synthesis and biophysical characterization of ac4C in its endogenous eukaryotic sequence context. First, we develop a synthetic route to homogenous RNAs containing electrophilic acetyl groups. Next, we use thermal denaturation to interrogate the effects of ac4C on duplex stability and mismatch discrimination in a native sequence found in human ribosomal RNA. Finally, we demonstrate the ability of this chemistry to incorporate ac4C into the complex modification landscape of human tRNA, and use duplex melting combined with sequence analysis to highlight a potentially unique enforcing role for ac4C in this setting. By enabling the analysis of nucleic acid acetylation in its physiological sequence context, these studies establish a chemical foundation for understanding the function of a universally-conserved nucleobase in biology and disease.

Structure Insight into Photosystem I Octamer from Cyanobacteria

Diversity of photosystem oligomers is essential to understand how photosynthetic organisms adopted to light conditions. Given by the structural and physiological significance, the assemblies of PSI supercomplex is of great interest in both chloroplast and cyanobacteria recently. In this study, two novel photosystem I supercomplexes were isolated for the first time from the low light incubated culture of filamentous cyanobacterium Anabaena sp. PCC 7120. These complexes were defined as PSI hexamers and octamers through biochemical and biophysical characterization. Their 77K emission spectra indicated that the red forms of chlorophylls seemed not to be affected during oligomerization. By cryo-EM single particle analysis, a near-atomic (7.0 Å) resolution structure of PSI octamer was resolved, and the molecular assemblies of stable PSI octamer was revealed.

Pyomelanin-Producing Brevundimonas vitisensis sp. nov., Isolated From Grape (Vitis vinifera L.)

A novel endophytic bacterial strain, designated GR-TSA-9T, was isolated from surface-sterilized grape (Vitis vinifera L.). 16S rRNA gene sequence analyses showed that the isolate was grouped within the genus Brevundimonas, displaying the highest similarity with Brevundimonas lenta DS-18T (97.9%) and Brevundimonas kwangchunensis KSL-102T (97.8%) and less than 97.5% similarity with other members of Brevundimonas. The strain GR-TSA-9T was a gram negative, rod shaped, facultatively anaerobic, catalase and oxidase positive, and motile bacterium. Its growth occurred at 10–37°C (optimally 25–30°C), at pH 7.0–8.0, and in NaCl 0–1% (optimally 0%). It contained ubiquinone-10 as a respiratory quinone, and the major cellular fatty acids (>10% of the total) were C16:0 (14.2%) and summed feature 8 (C18:1ω7c and/or C18:1ω6c, 65.6%). The polar lipids present in the strain were phosphoglycolipids, phosphatidylglycerol, 1,2-di-O-acyl-3-O-[d-glucopyranosyl-(1→4)-α-d-glucopyranuronosyl]glycerol, and unidentified lipids (L1, L2, and L4). The strain had one 2,976,716bp circular chromosome with a G+C content of 66.4%. The digital DNA–DNA hybridization value between strain GR-TSA-9T and B. lenta DS-18T was 20.9%, while the average nucleotide identity value was 76.7%. In addition, the dDDH and ANI values to other members in this genus, whose genome sequences are available, are less than 21.1 and 77.6%. Genome annotation predicted the presence of some gene clusters related to tyrosine degradation and pyomelanin formation. Strain GR-TSA-9T produced a brown melanin-like pigment in the presence of L-tyrosine-containing media. The highest pigment production (0.19g/L) was observed in tryptic soy broth with 1.0mg/ml L-tyrosine at 25°C for 6days of culture. Biophysical characterization by ultraviolet (UV)–visible spectroscopy, Fourier-transform infrared spectroscopy, and electrospray ionization mass spectrometry confirmed that the pigment was pyomelanin. Additionally, melanized GR-TSA-9T cells could protect the cells against UVC exposure. The phylogenetic, genomic, phenotypic, and chemotaxonomic features indicated that strain GR-TSA-9T represents a novel melanin-producing species of Brevundimonas. The type strain was GR-TSA-9T (KCTC 82386T=CGMCC 1.18820T).

Characterization of the tracrARN-DRARN genetic complex associated with the CRISPR-Cas9 system of the phytosymbiont Acholeplasma palmae: biotechnological interest

The CRISPR-Cas9 technology used in plant biotechnology is based on the use of Cas9 endonucleases to generate precise cuts in the genome, and a duplex consisting of a trans-activating CRISPR RNA (tracrRNA) and a CRISPR RNA (DRRNA) which are precursors of guide RNA (sgRNA) commercially redesigned (sgRNA-Cas9) to guide gene cleavage. Most of these tools come from clinical bacteria. However, there are several CRISPR-Cas9 systems in environmental microorganisms such as phytoendosymbionts of plants of the genus Acholeplasma. But the exploitation of these systems more compatible with plants requires using bioinformatics tools for prediction and study. We identified and characterized the elements associated with the duplex in the genome of A. palmae. For this, the protein information was obtained from the Protein Data Bank and the genomics from GenBank/NCBI. The CRISPR system was studied with the CRISPRfinder software. Alignment algorithms and NUPACK software were used to identify the tracrRNA and DRRNA modules, together with various computational software for genetic, structural and biophysical characterization. A CRISPR-Cas system was found in A. palmae with type II-C characteristics, as well as a thermodynamically very stable duplex, with flexible regions, exhibiting a docking power with Cas9 thermodynamically favored. These results are desirable in programmable gene editing systems and show the possibility of exploring native molecular tools in environmental microorganisms applicable to the genetic manipulation of plants, as more research is carried out. This study represents the first report on the thermodynamic stability and molecular docking of elements associated with the tracrRNA-DRRNA duplex in the phytosymbiont A. palmae.