tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut.

Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G-U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G-C base pairs (G3:C70 and G5:C68).

Enzymes known as PylRS offer the remarkable ability to expand the natural genetic code of a living cell with unnatural amino acids. Currently, over 200 unnatural amino acids can be genetically encoded with the help of PylRS and its partner tRNAPyl, enabling us to endow proteins with novel properties, or regulate protein activity using light or inducible cross-linking. One intriguing feature of PylRS enzymes is their ability to avoid cross-reactivity when two PylRS homologs from different organisms-such as those from the archaea Methanosarcina mazei and Methanomethylophilus alvus-are co-expressed in a single cell. This makes it possible to simultaneously encode two unnatural amino acids in a single protein. This study illuminates the elusive mechanism of PylRS specificity by using cryo-electron microscopy, biochemistry and molecular simulations. The interaction of PylRS from M. alvus with its tRNAPyl is best described as two pieces of a jigsaw puzzle; in which PylRS recognizes the unique shape of its cognate tRNA instead of specific nucleotides in the tRNA sequence like other tRNA-binding enzymes. This finding may streamline the rational design of tools for simultaneous genetic incorporation of multiple unnatural amino acids, thereby facilitating the development of valuable proteins for research, medicine, and biotechnology.

The unique ORF8 protein from SARS-CoV-2 binds to human dendritic cells and induces a hyper-inflammatory cytokine storm.

The novel coronavirus pandemic, first reported in December 2019, was caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infection leads to a strong immune response and activation of antigen-presenting cells, which can elicit acute respiratory distress syndrome (ARDS) characterized by the rapid onset of widespread inflammation, the so-called cytokine storm. In response to viral infections, monocytes are recruited into the lung and subsequently differentiate into dendritic cells (DCs). DCs are critical players in the development of the acute lung inflammation that causes ARDS. Here we focus on the interaction of a specific SARS-CoV-2 open reading frame protein, ORF8, with DCs. We show that ORF8 binds to DCs, causes a pre-maturation of differentiating DCs, and induces the secretion of multiple proinflammatory cytokines by these cells. In addition, we identified DC-SIGN as a possible interaction partner of ORF8 on DCs. Blockade of ORF8 leads to reduced production of IL-1ß, IL-6, IL-12p70, TNF-α, MCP-1 (also named CCL2), and IL-10 by DCs. Therefore, a neutralizing antibody blocking the ORF8-mediated cytokine and chemokine response could be an improved therapeutical strategy against severe SARS-CoV-2.

The dynamic nature of netrin-1 and the structural basis for glycosaminoglycan fragment-induced filament formation.

Netrin-1 is a bifunctional chemotropic guidance cue that plays key roles in diverse cellular processes including axon pathfinding, cell migration, adhesion, differentiation, and survival. Here, we present a molecular understanding of netrin-1 mediated interactions with glycosaminoglycan chains of diverse heparan sulfate proteoglycans (HSPGs) and short heparin oligosaccharides. Whereas interactions with HSPGs act as platform to co-localise netrin-1 close to the cell surface, heparin oligosaccharides have a significant impact on the highly dynamic behaviour of netrin-1. Remarkably, the monomer-dimer equilibrium of netrin-1 in solution is abolished in the presence of heparin oligosaccharides and replaced with highly hierarchical and distinct super assemblies leading to unique, yet unknown netrin-1 filament formation. In our integrated approach we provide a molecular mechanism for the filament assembly which opens fresh paths towards a molecular understanding of netrin-1 functions.

Heparin Mimetics and Their Impact on Extracellular Matrix Protein Assemblies.

Heparan sulfate is a crucial extracellular matrix component that organizes structural features and functional protein processes. This occurs through the formation of protein-heparan sulfate assemblies around cell surfaces, which allow for the deliberate local and temporal control of cellular signaling. As such, heparin-mimicking drugs can directly affect these processes by competing with naturally occurring heparan sulfate and heparin chains that then disturb protein assemblies and decrease regulatory capacities. The high number of heparan-sulfate-binding proteins that are present in the extracellular matrix can cause obscure pathological effects that should be considered and examined in more detail, especially when developing novel mimetics for clinical use. The objective of this article is to investigate recent studies that present heparan-sulfate-mediated protein assemblies and the impact of heparin mimetics on the assembly and function of these protein complexes.

A Structural Analysis of Proteinaceous Nanotube Cavities and Their Applications in Nanotechnology.

Protein nanotubes offer unique properties to the materials science field that allow them to fulfill various functions in drug delivery, biosensors and energy storage. Protein nanotubes are chemically diverse, modular, biodegradable and nontoxic. Furthermore, although the initial design or repurposing of such nanotubes is highly complex, the field has matured to understand underlying chemical and physical properties to a point where applications are successfully being developed. An important feature of a nanotube is its ability to bind ligands via its internal cavities. As ligands of interest vary in size, shape and chemical properties, cavities have to be able to accommodate very specific features. As such, understanding cavities on a structural level is essential for their effective application. The objective of this review is to present the chemical and physical diversity of protein nanotube cavities and highlight their potential applications in materials science, specifically in biotechnology.

Amyloidogenicity assessment of transthyretin gene variants.

OBJECTIVE: Hereditary transthyretin-mediated amyloidosis is a treatable condition caused by amyloidogenic variants in the transthyretin-gene resulting in severe peripheral neuropathy or cardiomyopathy. Only about a third of over 130 known variants are clearly pathogenic, most are classified as variants of uncertain significance. A clear delineation of these into pathogenic or non-pathogenic is highly desirable but hampered by low frequency and penetrance. We thus sought to characterize their amylogenic potential by an unbiased in vitro approach. METHODS: Thioflavin T and turbidity assays were used to compare the potential of mammalian cell expressed wt-transthyretin and 12 variant proteins (either variants of uncertain significance, benign, pathogenic) to aggregate and produce amyloid fibrils in vitro. As proof of principle, the assays were applied to transthyretin-Ala65Val, a variant that was newly detected in a family with peripheral neuropathy and amyloid deposits in biopsies. In silico analysis was performed to compare the position of the benign and pathogenic variants. RESULTS: Transthyretin-Ala65Val showed a significantly higher amyloidogenic potential than wt-transthyretin, in both turbidity- and Thioflavin T-assays, comparable to known pathogenic variants. The other eight tested variants did not show an increased amyloidogenic potential. In silico structural analysis further confirmed differences between pathogenic and benign variants in position and interactions. INTERPRETATION: We propose a biochemical approach to assess amyloidogenic potential of transthyretin variants. As exemplified by transthyretin-Ala65Val, data of three assays together with histopathology clearly demonstrates its amyloidogenicity.

Improved SARS-CoV-2 main protease high-throughput screening assay using a 5-carboxyfluorescein substrate.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a global threat to human health has highlighted the need for the development of novel therapies targeting current and emerging coronaviruses with pandemic potential. The coronavirus main protease (Mpro, also called 3CLpro) is a validated drug target against coronaviruses and has been heavily studied since the emergence of SARS-CoV-2 in late 2019. Here, we report the biophysical and enzymatic characterization of native Mpro, then characterize the steady-state kinetics of several commonly used FRET substrates, fluorogenic substrates, and six of the 11 reported SARS-CoV-2 polyprotein cleavage sequences. We then assessed the suitability of these substrates for high-throughput screening. Guided by our assessment of these substrates, we developed an improved 5-carboxyfluorescein-based FRET substrate, which is better suited for high-throughput screening and is less susceptible to interference and false positives than existing substrates. This study provides a useful framework for the design of coronavirus Mpro enzyme assays to facilitate the discovery and development of therapies targeting Mpro.

A C-Terminally Truncated Variant of Neurospora crassa VDAC Assembles Into a Partially Functional Form in the Mitochondrial Outer Membrane and Forms Multimers in vitro.

The voltage-dependent anion-selective channel (VDAC) is a porin in the mitochondrial outer membrane (MOM). Unlike bacterial porins, several mitochondrial ß-barrels comprise an odd number of ß-strands, as is the case for the 19-ß-stranded VDAC. Previously, a variant of a VDAC from Neurospora crassa, VDAC-ΔC, lacking the predicted 19th ß-strand, was found to form gated, anion-selective channels in artificial membranes. In vivo, the two C-terminal ß-strands (ß18 and ß19) in VDAC form a ß-hairpin necessary for import from the cytoplasm into mitochondria and the ß-signal required for assembly in the mitochondrial outer membrane resides in ß19. The current study demonstrated that the putative 18-stranded ß-barrel formed by VDAC-ΔC can be imported and assembled in the MOM in vivo and can also partially rescue the phenotype associated with the deletion of VDAC from a strain of N. crassa. Furthermore, when expressed and purified from Escherichia coli, VDAC-ΔC can be folded into a ß-strand-rich form in decyl-maltoside. Size exclusion chromatography (SEC) alone or combined with multi-angle light scattering (SEC-MALS) and analytical ultracentrifugation revealed that, unlike full-length VDACs, VDAC-ΔC can self-organize into dimers and higher order oligomers in the absence of sterol.

Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor.

The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. KEY POINTS: • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality.

Boron rich nanotube drug carrier system is suited for boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is a two-step therapeutic process that utilizes Boron-10 in combination with low energy neutrons to effectively eliminate targeted cells. This therapy is primarily used for difficult to treat head and neck carcinomas; recent advances have expanded this method to cover a broader range of carcinomas. However, it still remains an unconventional therapy where one of the barriers for widespread adoption is the adequate delivery of Boron-10 to target cells. In an effort to address this issue, we examined a unique nanoparticle drug delivery system based on a highly stable and modular proteinaceous nanotube. Initially, we confirmed and structurally analyzed ortho-carborane binding into the cavities of the nanotube. The high ratio of Boron to proteinaceous mass and excellent thermal stability suggest the nanotube system as a suitable candidate for drug delivery into cancer cells. The full physicochemical characterization of the nanotube then allowed for further mechanistic molecular dynamic studies of the ortho-carborane uptake and calculations of corresponding energy profiles. Visualization of the binding event highlighted the protein dynamics and the importance of the interhelical channel formation to allow movement of the boron cluster into the nanotube. Additionally, cell assays showed that the nanotube can penetrate outer membranes of cancer cells followed by localization around the cells' nuclei. This work uses an integrative approach combining experimental data from structural, molecular dynamics simulations and biological experiments to thoroughly present an alternative drug delivery device for BNCT which offers additional benefits over current delivery methods.

The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra.

BACKGROUND: The Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report a proteomic analysis of Hydra head tissue leading to the identification of an astacin family proteinase as a Wnt processing factor. RESULTS: Hydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt activity as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3-induced double axes in Xenopus embryos could be rescued by coinjection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model. CONCLUSIONS: We show the astacin family protease HAS-7 maintains a single head organizer through proteolysis of HyWnt3. Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra.

New approaches to reduce sample processing times for the determination of polycyclic aromatic compounds in environmental samples.

This study validates two approaches to streamlining the processing of sediment and biota for a suite of polycyclic aromatic compounds (PACs) with a wide range of chemical properties, including polycyclic aromatic hydrocarbons (PAHs) and alkyl-PAHs (APAHs), and a new class of environmental contaminants, halogenated PAHs (HPAHs). One method is based on one-step in situ extraction/cleanup using accelerated solvent extraction (ASE) in which a mixture of copper, deactivated alumina and silica gel were added directly to the ASE cell along with sample; the second technique is based on dispersive solid phase extraction (dSPE) using alumina/silica for cleanup of biota samples to augment conventional ASE extraction combined with gel permeation chromatography. Validation protocols were performed in accordance with the ISO/IEC 17025 guidelines, whereby method performance characteristics, i.e., accuracy, precision, linearity, limits of detection and ruggedness, were evaluated. Accuracies generally ranged from 70 to 120% for the in situ ASE method and 70-100% for the dSPE technique. Limits of detection/quantitation for the 45 target analytes for in situ ASE and dSPE methods were determined to be < 2.5/8 pg µL-1, and < 20/60 pg µL-1, respectively. Intra- and inter-day repeatability for both methods were < 25% except for 1 APAH which had an inter-day precision of 35% using the dSPE method. Neither method was affected by any of the purposeful changes attempted which implies that both methods are robust. Results of our validation studies showed excellent data quality for both methods in addition to achieving a reduction in sample processing times.

Molecular characterization of the RNA-protein complex directing -2/-1 programmed ribosomal frameshifting during arterivirus replicase expression.

Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs -1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical -1 and -2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1ß (nsp1ß) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1ß and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate -1 and -2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1ß and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1ß:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1ß within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1ß and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1ß and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism.

Affinity-Enhanced Multimeric VEGF (Vascular Endothelial Growth Factor) and PlGF (Placental Growth Factor) Variants for Specific Adsorption of sFlt-1 to Restore Angiogenic Balance in Preeclampsia.

Preeclampsia is a potentially life-threatening multisystem disease affecting 4% to 8% of pregnant women after the 20th week of gestation. An excess of placental expressed antiangiogenic soluble VEGF (vascular endothelial growth factor)-receptor 1 (soluble FMS-like tyrosine kinase 1) scavenges VEGF and PlGF (placental growth factor), causing generalized endothelial dysfunction. Interventions to restore the angiogenic balance in preeclamptic pregnancies are intensively studied and improve maternal and neonatal outcomes. Especially extracorporeal strategies to remove sFlt-1 are promising in human pregnancy. However, available apheresis systems adsorb sFlt-1 unspecifically and with low efficiency. Affinity-enhanced ligands are needed to improve performance and compatibility of apheresis treatments. Using computerized molecular modeling, we developed multimeric VEGF molecules comprised of single-chain VEGF165 dimers (scVEGF165). A short peptide linker hampers intrachain dimerization to induce assembly preferably as tetrameric molecules as visualized in negative staining electron microscopy. scVEGF165 multimers possess 1.2-fold higher affinity for sFlt-1 as compared to the available antibodies or monomeric VEGF. Consequently, scVEGF multimers have the ability to competitively release sFlt-1 bound PlGF and, in particular, VEGF. In ex vivo adsorption experiments using serum samples from patients with preeclampsia, scVEGF multimers reduce sFlt-1 levels by 85% and increase PlGF and VEGF levels by 20- and 9-fold, respectively. Finally, performance and stability of sFlt-1 capturing scVEGF165 multimers were scrutinized on different matrices of which biocompatible agarose matrix yielded optimal results. We introduce the first VEGF-based highly efficient sFlt-1 apheresis system that is directly applicable in vivo due to utilization of inert agarose matrix, using a homomultimeric form of VEGF165 to restore the angiogenic balance in preeclampsia.

Structural and Hydrodynamic Characterization of Dimeric Human Oligoadenylate Synthetase 2.

Oligoadenylate synthetases (OASs) are a family of interferon-inducible enzymes that require double-stranded RNA (dsRNA) as a cofactor. Upon binding dsRNA, OAS undergoes a conformational change and is activated to polymerize ATP into 2'-5'-oligoadenylate chains. The OAS family consists of several isozymes, with unique domain organizations to potentially interact with dsRNA of variable length, providing diversity in viral RNA recognition. In addition, oligomerization of OAS isozymes, potentially OAS1 and OAS2, is hypothesized to be important for 2'-5'-oligoadenylate chain building. In this study, we present the solution conformation of dimeric human OAS2 using an integrated approach involving small-angle x-ray scattering, analytical ultracentrifugation, and dynamic light scattering techniques. We also demonstrate OAS2 dimerization using immunoprecipitation approaches in human cells. Whereas mutation of a key active-site aspartic acid residue prevents OAS2 activity, a C-terminal mutation previously hypothesized to disrupt OAS self-association had only a minor effect on OAS2 activity. Finally, we also present the solution structure of OAS1 monomer and dimer, comparing their hydrodynamic properties with OAS2. In summary, our work presents the first, to our knowledge, dimeric structural models of OAS2 that enhance our understanding of the oligomerization and catalytic function of OAS enzymes.

Solution structure of the cytoplasmic domain of NhaP2 a K+/H+ antiporter from Vibrio cholera.

NhaP2 is a K+/H+ antiporter from Vibrio cholerae which consists of a transmembrane domain and a cytoplasmic domain of approximately 200 amino acids, both of which are required for cholera infectivity. Here we present the solution structure for a 165 amino acid minimal cytoplasmic domain (P2MIN) form of the protein. The structure reveals a compact N-terminal domain which resembles a Regulator of Conductance of K+ channels (RCK) domain connected to a more open C-terminal domain via a flexible 20 amino acid linker. NMR titration experiments showed that the protein binds ATP through its N-terminal domain, which was further supported by waterLOGSY and Saturation Transfer Difference NMR experiments. The two-domain organisation of the protein was confirmed by BIOSAXS, which also revealed that there are no detectable-ATP-induced conformational changes in the protein structure. Finally, in contrast to all known RCK domain structures solved to date, the current work shows that the protein is a monomer.

Solution structure and oligomeric state of the E. coliglycerol facilitator.

Protein dynamics at atomic resolution can provide deep insights into the biological activities of proteins and enzymes but they can also make structure and dynamics studies challenging. Despite their well-known biological and pharmaceutical importance, integral membrane protein structure and dynamics studies lag behind those of water-soluble proteins mainly owing to solubility problems that result upon their removal from the membrane. Escherichia coli glycerol facilitator (GF) is a member of the aquaglyceroporin family that allows for the highly selective passive diffusion of its substrate glycerol across the inner membrane of the bacterium. Previous molecular dynamics simulations and hydrogen-deuterium exchange studies suggested that protein dynamics play an important role in the passage of glycerol through the protein pore. With the aim of studying GF dynamics by solution and solid-state nuclear magnetic resonance (NMR) spectroscopy we optimized the expression of isotope-labelled GF and explored various solubilizing agents including detergents, osmolytes, amphipols, random heteropolymers, lipid nanodiscs, bicelles and other buffer additives to optimize the solubility and polydispersity of the protein. The GF protein is most stable and soluble in lauryl maltose neopentyl glycol (LMNG), where it exists in a tetramer-octamer equilibrium. The solution structures of the GF tetramer and octamer were determined by negative-stain transmission electron microscopy (TEM), size-exclusion chromatography small-angle X-ray scattering (SEC-SAXS) and solid-state magic-angle spinning NMR spectroscopy. Although NMR sample preparation still needs optimization for full structure and dynamics studies, negative stain TEM and SEC-SAXS revealed low-resolution structures of the detergent-solubilized tetramer and octamer particles. The non-native octamer appears to form from the association of the cytoplasmic faces of two tetramers, the interaction apparently mediated by their disordered N- and C-termini. This information may be useful in future studies directed at reducing the heterogeneity and self-association of the protein.

Validated quantitative cannabis profiling for Canadian regulatory compliance - Cannabinoids, aflatoxins, and terpenes.

In response to the Canadian federal government's Cannabis Tracking and Licensing System compliance standards, a quantitative method was created for cannabis analysis, and validated using Eurachem V.2 (2014) guidelines. Cannabinol, cannabidiol, cannabigerol, cannabichromene, cannabidiolic acid, cannabigerolic acid, Δ-9-tetrahydrocannabinol, and Δ-9-tetrahydrocannabinolic acid A were all analysed by scheduled multiple reaction monitoring (MRM) via LC-MS/MS and isotope dilution. In addition, aflatoxins B1, B2, G1, and G2 were also analysed by scheduled MRM via LC-MS/MS and matrix matched calibration curves in order to achieve the reporting limits (≤2 µg kg-1) set out by the European Pharmacopoeia. The LODs/LOQs were 0.50/1.7, 2.0/6.7, 0.59/2.0, and 0.53/1.8 µg kg-1, for B1, B2, G1, and G2 respectively. Thirty one terpenes were analysed by selected reaction monitoring via GC-MS/MS and isotope dilution using ß-myrcene-d6 as a surrogate. All quantitative analyses can be accomplished using less than 1 g of material, with minimal solvent and consumable use, on low resolution instruments in less than 30 min of instrument time. Of important note is this method's power of selectivity, working ranges, and lack of need for extraction consumables such as SPE or QuEChERS, thereby minimising analytical costs and time.

Isolation of a Complex Formed Between Acinetobacter baumannii HemA and HemL, Key Enzymes of Tetrapyrroles Biosynthesis.

Plants, algae and most bacteria synthesize 5-aminolevulinic acid (ALA), the universal precursor of tetrapyrroles such as heme, chlorophyll and coenzyme B12, by a two-step transformation involving the NADPH-dependent glutamyl-tRNA reductase (HemA), which reduces tRNA-bound glutamate to glutamate-1-semialdehyde (GSA), and the pyridoxamine 5'-phosphate-dependent glutamate-1-semialdehyde-2,1-aminomutase (HemL), responsible for the isomerization of GSA into ALA. Since GSA is a very unstable compound at pH values around neutrality, the formation of a HemA-HemL complex has been proposed to occur, allowing for direct channeling of this intermediate from HemA to HemL. Experimental evidence of the formation of this complex has been obtained with the enzymes from Escherichia coli and Chlamydomonas reinhardtii. However, its isolation has never been attained, probably because HemA is degraded when intracellular heme accumulates. In this work, we devised a co-expression and co-purification strategy of HemA and HemL from Acinetobacter baumannii, which allowed the isolation of the HemA-HemL complex. Our results indicate that HemA is stabilized when co-expressed with HemL. The addition of citrate throughout the expression and purification procedure further promotes the formation of the HemA-HemL complex, which can be isolated in fair amount for functional and structural studies. This work lays the bases for a rational design of HemA-HemA inhibitors to be developed as antibacterial agents against A. baumannii, a multidrug resistant opportunistic pathogen responsible for a broad range of severe nosocomial infections.

A Cholesterol Analog Induces an Oligomeric Reorganization of VDAC.

The oligomeric organization of the voltage-dependent anion-selective channel (VDAC) and its interactions with hexokinase play integral roles in mitochondrially mediated apoptotic signaling. Various small to large assemblies of VDAC are observed in mitochondrial outer membranes, but they do not predominate in detergent-solubilized VDAC samples. In this study, a cholesterol analog, cholesteryl-hemisuccinate (CHS), was shown to induce the formation of detergent-soluble VDAC multimers. The various oligomeric states of VDAC induced by the addition of CHS were deciphered through an integrated biophysics approach using microscale thermophoresis, analytical ultracentrifugation, and size-exclusion chromatography small angle x-ray scattering. Furthermore, CHS stabilizes the interaction between VDAC and hexokinase (Kd of 27 ± 6 µM), confirming the biological relevance of oligomers generated. Thus, sterols such as cholesterol in higher eukaryotes or ergosterol in fungi may regulate the VDAC oligomeric state and may provide a potential target for the modulation of apoptotic signaling by effecting VDAC-VDAC and VDAC-hexokinase interactions. In addition, the integrated biophysical approach described provides a powerful platform for the study of membrane protein complexes in solution.