Publications

Candida albicans is a critical priority fungal pathogen causing invasive fungal infections with high mortality rates in immunocompromised patients. The increasing fungal infection rate and resistance of fungal pathogens to existing antifungal treatments have emphasized the need for the development of novel antifungal medicine. The ergosterol biosynthesis pathway has been a successful target for antifungal compounds, but many enzymatic steps remain unexplored. 24C-sterol methyltransferase (24C-SMT) catalyzes a critical fungal-specific step in ergosterol biosynthesis. When 24C-SMT is disrupted, fungal pathogens are sensitized to temperature, various inhibitors, and antifungals, and a loss of virulence can be observed. In this study, five 24C-SMT variants with different lengths of N-termini were heterologously produced in Escherichia coli and three were purified to near-homogeneity with immobilized metal-affinity and size-exclusion chromatography. N-terminally truncated C. albicans 24C-SMT was utilized for crystallization trials due to its increased stability and higher purity compared to the full-length protein. 24C-SMT crystals were obtained in the presence of Sadenosyl-homocysteine, but diffracted to low resolution. Therefore, we established a starting point for 24C-SMT crystallization by providing an optimized protocol for heterologous 24C-SMT production, purification and initial crystallization conditions, which could be used for further downstream crystallographic studies. Less |Related Solutions: Formulator^®

Talin (herein referring collectively to talin 1 and 2) couples the actomyosin cytoskeleton to integrins and transmits tension to the extracellular matrix. Talin also interacts with numerous additional proteins capable of modulating the actin-integrin linkage and thus downstream mechanosignaling cascades. Here, we demonstrate that the scaffold protein Caskin2 interacts directly with the R8 domain of talin through its C-terminal LD motif. Caskin2 also associates with the WAVE regulatory complex to promote cell migration in an Abi1-dependent manner. Furthermore, we demonstrate that the Caskin2–Abi1 interaction is regulated by growth factor-induced phosphorylation of Caskin2 on serine 878. In MCF7 and UACC893 cells, which contain an amplification of CASKIN2, Caskin2 localizes in plasma membrane-associated plaques and around focal adhesions in cortical microtubule stabilization complexes. Taken together, our results identify Caskin2 as a novel talin-binding protein that might not only connect integrin-mediated adhesion to actin polymerization but could also play a role in crosstalk between integrins and microtubules. Less |Related Solutions: Formulator^®

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization. Less |Related Solutions: Formulator^®

Nowadays, the vastly increasing demand for novel biotechnological products is supported by the continuous development of biocatalytic applications that provide sustainable green alternatives to chemical processes. The success of a biocatalytic application is critically dependent on how quickly we can identify and characterize enzyme variants fitting the conditions of industrial processes. While miniaturization and parallelization have dramatically increased the throughput of next-generation sequencing systems, the subsequent characterization of the obtained candidates is still a limiting process in identifying the desired biocatalysts. Only a few commercial microfluidic systems for enzyme analysis are currently available, and the transformation of numerous published prototypes into commercial platforms is still to be streamlined. This review presents the state-of-the-art, recent trends, and perspectives in applying microfluidic tools in the functional and structural analysis of biocatalysts. We discuss the advantages and disadvantages of available technologies, their reproducibility and robustness, and readiness for routine laboratory use. We also highlight the unexplored potential of microfluidics to leverage the power of machine learning for biocatalyst development. Less |Related Solutions: Formulator^®

Diffraction-based structural methods contribute a large fraction of the biomolecular structural models available, providing a critical understanding of macromolecular architecture. These methods require crystallization of the target molecule, which remains a primary bottleneck in crystal-based structure determination. The National High-Throughput Crystallization Center at Hauptman–Woodward Medical Research Institute has focused on overcoming obstacles to crystallization through a combination of robotics-enabled high-throughput screening and advanced imaging to increase the success of finding crystallization conditions. This paper will describe the lessons learned from over 20 years of operation of our high-throughput crystallization services. The current experimental pipelines, instrumentation, imaging capabilities and software for image viewing and crystal scoring are detailed. New developments in the field and opportunities for further improvements in biomolecular crystallization are reflected on. Less |Related Solutions: Formulator^®

Coproporphyrin ferrochelatases (CpfCs) are enzymes catalyzing the penultimate step in the coproporphyrin-dependent (CPD) heme biosynthesis pathway, which is mainly utilized by monoderm bacteria. Ferrochelatases insert ferrous iron into a porphyrin macrocycle and have been studied for many decades, nevertheless many mechanistic questions remain unanswered to date. Especially CpfCs, which are found in the CPD pathway, are currently in the spotlight of research. This pathway was identified in 2015 and revealed that the correct substrate for these ferrochelatases is coproporphyrin III (cpIII) instead of protoporphyrin IX, as believed prior the discovery of the CPD pathway. The chemistry of cpIII, which has four propionates, differs significantly from protoporphyrin IX, which features two propionate and two vinyl groups. These findings let us to thoroughly describe the physiological cpIII-ferrochelatase complex in solution and in the crystal phase. Here, we present the first crystallographic structure of the CpfC from the representative monoderm pathogen Listeria monocytogenes bound to its physiological substrate, cpIII, together with the in-solution data obtained by resonance Raman and UV–vis spectroscopy, for wild-type ferrochelatase and variants, analyzing propionate interactions. The results allow us to evaluate the porphyrin distortion and provide an in-depth characterization of the catalytically-relevant binding mode of cpIII prior to iron insertion. Our findings are discussed in the light of the observed structural restraints and necessities for this porphyrin-enzyme complex to catalyze the iron insertion process. Knowledge about this initial situation is essential for understanding the preconditions for iron insertion in CpfCs and builds the basis for future studies. Less |Related Solutions: Formulator^®

Histone deacetylase 6 (HDAC6) is an atypical lysine deacetylase with tandem catalytic domains and an ubiquitin-binding zinc finger domain. HDAC6 is involved in various biological processes, such as cell motility or stress responses, and has been implicated in pathologies ranging from cancer to neurodegeneration. Due to this broad range of functions, there has been considerable interest in developing HDAC6-specific small molecule inhibitors, several of which are already available. The crystal structure of the tandem catalytic domains of zebrafish HDAC6 has revealed an arrangement with twofold symmetry and extensive surface interaction between the catalytic domains. Further dissection of the biochemical properties of HDAC6 and the development of novel inhibitors will benefit from being able to routinely express high-quality protein. We present here our optimized protocol for expression and crystallization of the zebrafish tandem catalytic domains. Less |Related Solutions: Formulator^®

As discussed in previous chapters, the methylation of specific arginine and lysine side chains is carried out by two families of histone methyltransferases, the Protein Arginine Methyltransferase (PRMT) family for arginine, and the SET domain family for lysine. The methylation of H3K79 by Dot1 is a notable outlier. In all cases, X-ray crystallography has been a powerful technique that has provided the framework for understanding the enzyme mechanism, kinetics, regulation and specificity of these enzymes and is now a platform for the design of compounds aimed to inhibit their activity either to further understand their function or in a therapeutic setting. Notably, in combination with the structures of the complementary recognition domains that recognize their products, these structures have provided an important insight into how integral the number of methyl groups added to the acceptor amine is to making histone methylation a key process in epigenetic regulation of gene transcription. Here the concepts applied to determine their structure by X-ray crystallography are outlined, with particular emphasis on lysine methylation by the SET domain. Less |Related Solutions: Formulator^®

The cysteine of HCD (C286) in DYRK1A is involved in disulfide bridge formation with a cysteine (C312) in the DFGSSC sequence. The purpose of this project was to investigate how the state of the disulfide bridge would affect enzyme catalytic and ligand binding properties of the protein kinase. A mutant, DYRK1A C312A, was thus designed to eliminate the disulfide bridge. The mutant was expressed and purified following the same protocol as for DYRK1A wt, including HisTrap purification, TEV cleavage and size exclusion chromatography. Crystallization trials were performed for both the wt and the mutant with the kinase inhibitor Staurosporine. DYRK1A wt with STU crystallized and diffracted with at a resolution of 2.33 Å. The DYRK1A C312A mutant with STU crystallized and diffracted with a resolution of 2.59 Å. The structure was solved by molecular replacement in Molrep (CCP4) and refined by Refmac5 and Phenix. Molecular dynamics (MD) simulations (SCHRODINGER) were performed with the intent to compare diverse disulfide bridge states. Ligand binding and enzyme catalytic properties were analyzed using a combination of techniques, including activity assays, microscale thermophoresis, and isothermal calorimetry. The Thermofluor assay confirmed that both the wt and the mutant bind tightly to STU and AZ-191. It also showed that the mutant consistently has a slightly lower melting temperature than the wt, which would indicate that it is less stable. Solvent accessible surface area (SASA) analysis support the theory of accessibility to conserved cysteine residues. Less |Related Solutions: Formulator^®

Although monoclonal antibodies (mAbs) have been shown to be extremely effective in treating a number of diseases, they often suffer from poor developability attributes, such as high viscosity and low solubility at elevated concentrations. Since experimental candidate screening is often materials and labor intensive, there is substantial interest in developing in silico tools for expediting mAb design. Here, we present a strategy using machine learning-based QSAR models for the a priori estimation of mAb solubility. The extrapolated protein solubilities of a set of 111 antibodies in a histidine buffer were determined using a high throughput PEG precipitation assay. 3D homology models of the antibodies were determined, and a large set of in house and commercially available molecular descriptors were then calculated. The resulting experimental and descriptor data were then used for the development of QSAR models of mAb solubilities. After feature selection and training with different machine learning algorithms, the models were evaluated with external test sets. The resulting regression models were able to estimate the solubility values of external test set data with R2 of 0.81 and 0.85 for the two regression models developed. In addition, three class and binary classification models were developed and shown to be good estimators of mAb solubility behavior, with overall test set accuracies of 0.70 and 0.95, respectively. The analysis of the selected molecular descriptors in these models was also found to be informative and suggested that several charge-based descriptors and isotype may play important roles in mAb solubility. The combination of high throughput relative solubility experimental techniques in concert with efficient machine learning QSAR models offers an opportunity to rapidly screen potential mAb candidates and to design therapeutics with improved solubility characteristics. Less |Related Solutions: Formulator^®

Ubiquitination is a highly abundant post-translation modification that is involved in the control of a large number of cellular processes. Target ubiquitination is achieved through the action of three separate enzymes; the E1, ubiquitin-activating enzyme, the E2, ubiquitin-conjugating enzyme and the E3, ubiquitin ligase. TRIM E3 ligases are the largest family of RING-type E3 ligases and are classified by a N-terminal tripartite motif consisting of the catalytic RING domain, one or two B-box domains, B1 and B2, and a coiled-coil domain. In addition, most TRIMs possess a C-terminal substrate-binding domain, which classifies them into one of eleven TRIM classes. The PRYSPRY domain is the most common substrate-binding domain in humans and links class IV TRIMs to roles in cellular innate immunity. TRIM22 and TRIM6, are Class IV TRIMs that share high sequence identity with the well-studied HIV restriction factor, TRIM5, and have also been implicated in the anti-viral response. TRIM22 is reported to function directly as a viral restriction factor against RNA viruses such as, IAV, HCV and EMCV. While TRIM6 functions to activate the innate immune signalling pathway through activation of the immune signalling factor, IKK. Aspects of TRIM22 and TRIM6 function remain understudied, including their biochemical and biophysical properties and this is the focus of this study. The results described herein outline key differences in the self-association properties of these proteins in comparison to TRIM5. Furthermore, they highlight discrepancies between the ubiquitination profiles of TRIM22 and TRIM6 presented in the literature and the activity observed in this study. Overall, this emphasizes the need for further study of the roles of TRIM22 and TRIM6, to verify current proposed functions, as well as identify potential additional functions within the cell. Less |Related Solutions: Formulator^®

Immunoglobulin G-based monoclonal antibodies (mAbs) have become a dominant class of biotherapeutics in recent decades. Approved antibodies are mainly of the subclasses IgG1, IgG2, and IgG4, as well as their derivatives. Over the decades, the selection of IgG subclass has frequently been based on the needs of Fc gamma receptor engagement and effector functions for the desired mechanism of action, while the effect on drug product developability has been less thoroughly characterized. One of the major reasons is the lack of systematic understanding of the impact of IgG subclass on the molecular properties. Several efforts have been made recently to compare molecular property differences among these IgG subclasses, but the conclusions from these studies are sometimes obscured by the interference from variable regions. To further establish mechanistic understandings, we conducted a systematic study by grafting three independent variable regions onto human IgG1, an IgG1 variant, IgG2, and an IgG4 variant constant domains and evaluating the impact of subclass and variable regions on their molecular properties. Structural and computational analysis revealed specific molecular features that potentially account for the differential behavior of the IgG subclasses observed experimentally. Our data indicate that IgG subclass plays a significant role on molecular properties, either through direct effects or via the interplay with the variable region, the IgG1 mAbs tend to have higher solubility than either IgG2 or IgG4 mAbs in a common pH 6 buffer matrix, and solution behavior relies heavily on the charge status of the antibody at the desirable pH. Less |Related Solutions: Formulator^®

BceF is a bacterial tyrosine kinase (BY-kinase) from Burkholderia cepacia, a Gram-negative bacterium accountable for respiratory infections in immunocompromised and cystic fibrosis patients. BceF is involved in the production of exopolysaccharides secreted to the biofilm matrix and promotes resistant and aggressive infections. BY-kinases share no homology with mammalian kinases, and thereby offer a means to develop novel and specific antivirulence drugs. Here, we report the crystal structure of the BceF kinase domain at 1.85 Å resolution. The isolated BceF kinase domain is assembled as a dimer in solution and crystallized as a dimer in the asymmetric unit with endogenous adenosine-diphosphate bound at the active sites. The low enzymatic efficiency measured in solution may be explained by the partial obstruction of the active sites at the crystallographic dimer interface. This study provides insights into self-assembly and the specific activity of isolated catalytic domains. Several unique variations around the active site compared to other BY-kinases may allow for structure-based design of specific inhibitors to target Burkholderia cepacia virulence. Less |Related Solutions: Formulator^®

The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19. Less |Related Solutions: Formulator^®

EMBL Grenoble operates the High Throughput Crystallization Laboratory (HTX Lab), a large-scale user facility offering high throughput crystallography services to users worldwide. The HTX lab has a strong focus in the development of new methods in macromolecular crystallography. Through the combination of a high throughput crystallization platform, the CrystalDirect technology for fully automated crystal mounting and cryocooling and the CRIMS software we have developed fully automated pipelines for macromolecular crystallography that can be remotely operated over the internet. These include a protein-to-structure pipeline for the determination of new structures, a pipeline for the rapid characterization of protein-ligand complexes in support of medicinal chemistry, and a large-scale, automated fragment screening pipeline enabling evaluation of libraries of over 1000 fragments. Here we describe how to access and use these resources. Less |Related Solutions: Formulator^®

In this thesis, the chloride transport mechanism of the light-driven microbial chloride pump Nonlabens marinus halorhodopsin (NmHR) is presented. Members of the rhodopsin family, such as NmHR, are integral membrane proteins comprising seven α helices and a retinal chromophore, which is covalently bound to the protein via a protonated Schiff base and renders the proteins photoactive. Through photoactivation, the retinal chromophore undergoes an isomerization reaction, which then drives conformational changes in the protein. Through variations in residue composition, rhodopsin can catalyze diverse chemical reactions, including pumping protons, sodium, or chloride ions. Chloride transport is a fundamental process in biology and crucial for maintaining the electrochemical balance of the cell.

The advent of bright X-ray light sources such as third-generation synchrotrons and X ray free electron lasers has resulted in the emergence of time-resolved serial crystallography. These novel serial crystallography methods were combined with time resolved spectroscopy and hybrid quantum mechanics/molecular mechanics simulations to study conformational changes and chloride translocation in NmHR after photoactivation. Five active state structural intermediates, determined in the picosecond to microsecond time domain, have been determined at the X-ray free electron laser. Structural insight into the late photocycle of NmHR was provided by time-resolved serial crystallography at the synchrotron, resulting in ten additional active state intermediates in the millisecond time domain. In addition, a new method was developed that allowed tracing of the anomalous substructure in the photostationary state, providing critical clues on the anion transport pathway in NmHR.

Together with resolving the position of four new transient chloride binding sites in time, the mechanism driving chloride transport is proposed based on the observed conformational changes of the protein after photoactivation. In summary, in the resting state chloride interacts with the protonated Schiff base of the retinal chromophore. Upon absorption of a photon, the retinal chromophore then isomerizes from the all trans to 13-cis configuration, which flips the protonated Schiff base and disrupts the interaction with the chloride ion. In the following step, the chloride translocation is initiated as the anion is pulled over the retinal chromophore to reestablish the interaction with the positive charge on the protonated Schiff base. After chloride is released into the exit tunnel to further diffuse towards the cytoplasm, a steric gate prevents chloride from flowing back into the dark state binding site. At the same time as the release of the chloride ion into the cytoplasm, a new anion is taken up from the extracellular space. In the uptake tunnel, the anion encounters a bottleneck formed by a salt bridge between an arginine and aspartate residue which forms an electrostatic gate. Upon opening of this electrostatic gate, chloride can enter the retinal binding pocket, a hydrophilic cavity in which the dark state binding site is located. Together with the closure of the electrostatic gate, the retinal chromophore isomerizes back to the all-trans-configuration, and the dark state chloride binding site is regenerated.

This thesis thereby presents the first detailed structural dynamics of ion transport by a chloride pumping rhodopsin and demonstrates the capabilities of novel serial crystallography methods. Less |Related Solutions: Formulator^®

Monoheme c-type cytochromes are important electron transporters in all domains of life. They possess a common fold hallmarked by three α-helices that surround a covalently attached heme. An intriguing feature of many monoheme c-type cytochromes is their capacity to form oligomers by exchanging at least one of their α-helices, which is often referred to as 3D domain swapping. Here, the crystal structure of NirC, a c-type cytochrome co-encoded with other proteins involved in nitrite reduction by the opportunistic pathogen Pseudomonas aeruginosa, has been determined. The crystals diffracted anisotropically to a maximum resolution of 2.12 Å (spherical resolution of 2.83 Å) and initial phases were obtained by Fe-SAD phasing, revealing the presence of 11 NirC chains in the asymmetric unit. Surprisingly, these protomers arrange into one monomer and two different types of 3D domain-swapped dimers, one of which shows pronounced asymmetry. While the simultaneous observation of monomers and dimers probably reflects the interplay between the high protein concentration required for crystallization and the structural plasticity of monoheme c-type cytochromes, the identification of conserved structural motifs in the monomer together with a comparison with similar proteins may offer new leads to unravel the unknown function of NirC. Less |Related Solutions: Formulator^®

Hit-to-lead optimization is a critical phase in drug discovery. Herein, we report on the fragment-based discovery and optimization of 2-aminopyridine derivatives as a novel lead-like structure for the treatment of the dangerous opportunistic pathogen Pseudomonas aeruginosa. We pursue an innovative treatment strategy by interfering with the Pseudomonas quinolone signal (PQS) quorum sensing (QS) system leading to an abolishment of bacterial pathogenicity. Our compounds act on the PQS receptor (PqsR), a key transcription factor controlling the expression of various pathogenicity determinants. In this target-driven approach, we made use of biophysical screening via surface plasmon resonance (SPR) followed by isothermal titration calorimetry (ITC)-enabled enthalpic efficiency (EE) evaluation. Hit optimization then involved growth vector identification and exploitation. Astonishingly, the latter was successfully achieved by introducing flexible linkers rather than rigid motifs leading to a boost in activity on the target receptor and anti-virulence potency. Less |Related Solutions: Formulator^®

HheG from Ilumatobacter coccineus is a halohydrin dehalogenase with synthetically useful activity in the ring opening of cyclic epoxides with various small anionic nucleophiles. This enzyme provides access to chiral β-substituted alcohols that serve as building blocks in the pharmaceutical industry. Wild-type HheG suffers from low thermostability, which poses a significant drawback for potential applications. In an attempt to thermostabilize HheG by protein engineering, several single mutants at position 123 were identified which displayed up to 14 °C increased apparent melting temperatures and up to three-fold higher activity. Aromatic amino acids at position 123 resulted even in a slightly higher enantioselectivity. Crystal structures of variants T123W and T123G revealed a flexible loop opposite to amino acid 123. In variant T123G, this loop adopted two different positions resulting in an open or partially closed active site. Classical molecular dynamics simulations confirmed a high mobility of this loop. Moreover, in variant T123G this loop adopted a position much closer to residue 123 resulting in denser packing and increased buried surface area. Our results indicate an important role for position 123 in HheG and give first structural and mechanistic insight into the thermostabilizing effect of mutations T123W and T123G. Less |Related Solutions: Formulator^®

Inteins remove themselves from a precursor protein by protein splicing. Due to the concomitant structural changes of the host protein, this self-processing reaction has enabled many applications in protein biotechnology and chemical biology. We show that the evolved M86 mutant of the Ssp DnaB intein displays a significantly improved tolerance towards non-native amino acids at the N-terminally flanking (−1) extein position compared to the parent intein, in the form of both an artificially trans-splicing split intein and the cis-splicing mini-intein. Surprisingly, side chains with increased steric bulk compared to the native Gly(−1) residue, including D-amino acids, were found to compensate for the essential block B histidine in His73Ala mutants in the initial N–S acyl shift of the protein splicing pathway. In the case of the M86 intein, large (−1) side chains can even rescue protein splicing activity as a whole. With the comparison of three crystal structures, namely of the M86 intein as well as of its Gly(−1)Phe and Gly(−1)Phe/His73Ala mutants, our data supports a model in which the intein's active site can exert a strain by varying mechanisms on the different angles of the scissile bond at the extein–intein junction to effect a ground-state destabilization. The compensatory mechanism of the block B histidine is the first example for the direct functional role of an extein residue in protein splicing. It sheds new light on the extein–intein interplay and on possible consequences of their co-evolution as well as on the laboratory engineering of improved inteins. Less |Related Solutions: Formulator^®

Pseudomonas aeruginosa, a prevalent pathogen in nosocomial infections and a major burden in cystic fibrosis, uses three interconnected quorum-sensing systems to coordinate virulence processes. At variance with other Gram-negative bacteria, one of these systems relies on 2-alkyl-4(1H)-quinolones (Pseudomonas quinolone signal, PQS) and might hence be an attractive target for new anti-infective agents. Here we report crystal structures of the N-terminal domain of anthranilate-CoA ligase PqsA, the first enzyme of PQS biosynthesis, in complex with anthraniloyl-AMP and with 6-fluoroanthraniloyl-AMP (6FABA-AMP) at 1.4 and 1.7 Å resolution. We find that PqsA belongs to an unrecognized subfamily of anthranilate-CoA ligases that recognize the amino group of anthranilate through a water-mediated hydrogen bond. The complex with 6FABA-AMP explains why 6FABA, an inhibitor of PQS biosynthesis, is a good substrate of PqsA. Together, our data might pave a way to new pathoblockers in P. aeruginosa infections. Less |Related Solutions: Formulator^®

Eukaryotes contain a diverse tapestry of specialized metabolites, many of which are of significant pharmaceutical and industrial importance to humans. Nevertheless, exploration of specialized metabolic pathways underlying specific chemical traits in nonmodel eukaryotic organisms has been technically challenging and historically lagged behind that of the bacterial systems. Recent advances in genomics, metabolomics, phylogenomics, and synthetic biology now enable a new workflow for interrogating unknown specialized metabolic systems in nonmodel eukaryotic hosts with greater efficiency and mechanistic depth. This chapter delineates such workflow by providing a collection of state-of-the-art approaches and tools, ranging from multiomics-guided candidate gene identification to in vitro and in vivo functional and structural characterization of specialized metabolic enzymes. As already demonstrated by several recent studies, this new workflow opens up a gateway into the largely untapped world of natural product biochemistry in eukaryotes. Less |Related Solutions: Formulator^®

Currently, macromolecular crystallography projects often require the use of highly automated facilities for crystallization and X-ray data collection. However, crystal harvesting and processing largely depend on manual operations. Here, a series of new methods are presented based on the use of a low X-ray-background film as a crystallization support and a photoablation laser that enable the automation of major operations required for the preparation of crystals for X-ray diffraction experiments. In this approach, the controlled removal of the mother liquor before crystal mounting simplifies the cryocooling process, in many cases eliminating the use of cryoprotectant agents, while crystal-soaking experiments are performed through diffusion, precluding the need for repeated sample-recovery and transfer operations. Moreover, the high-precision laser enables new mounting strategies that are not accessible through other methods. This approach bridges an important gap in automation and can contribute to expanding the capabilities of modern macromolecular crystallography facilities. Less |Related Solutions: Formulator^®

We must reliably map the interactomes of cellular macromolecular complexes in order to fully explore and understand biological systems. However, there are no methods to accurately predict how to capture a given macromolecular complex with its physiological binding partners. Here, we present a screen that comprehensively explores the parameters affecting the stability of interactions in affinity-captured complexes, enabling the discovery of physiological binding partners and the elucidation of their functional interactions in unparalleled detail. We have implemented this screen on several macromolecular complexes from a variety of organisms, revealing novel profiles even for well-studied proteins. Our approach is robust, economical and automatable, providing an inroad to the rigorous, systematic dissection of cellular interactomes. Less |Related Solutions: Formulator^®

Membrane transporters that clear the neurotransmitter glutamate from synapses are driven by symport of sodium ions and counter-transport of a potassium ion. Previous crystal structures of a homologous archaeal sodium and aspartate symporter showed that a dedicated transport domain carries the substrate and ions across the membrane. Here, we report new crystal structures of this homologue in ligand-free and ions-only bound outward- and inward-facing conformations. We show that after ligand release, the apo transport domain adopts a compact and occluded conformation that can traverse the membrane, completing the transport cycle. Sodium binding primes the transport domain to accept its substrate and triggers extracellular gate opening, which prevents inward domain translocation until substrate binding takes place. Furthermore, we describe a new cation-binding site ideally suited to bind a counter-transported ion. We suggest that potassium binding at this site stabilizes the translocation-competent conformation of the unloaded transport domain in mammalian homologues. Less |Related Solutions: Formulator^®

The use of design of experiments (DOE) in assay development (AD) has the potential to speed up assay optimisation (ie reduce assay development bottlenecks) and to facilitate a more thorough evaluation of assay variables. Only one liquid handling vendor currently offers application specific software and support for investigating DOE in biological assays. Although standalone DOE software packages are available, these were not written specifically for biological applications and they vary in their suitability for AD. DOE needs to be simpler to implement to make a major impact on AD. A market opportunity exists for a turnkey solution that directly links statistical design with automated liquid handler programming and also feeds the assay readout directly into the statistical analysis, to suggest and facilitate further iterative retesting. Until new tools or more encompassing solutions emerge, the full impact of DOE on AD is unlikely to be realised. Less |Related Solutions: Formulator^®