Biology subjects

Conway, J. F.

Publications and source records attributed to Conway, J. F..

15 recordsLinked to original sources

bioRxiv2026

Computational Prediction of Recombinant Expression Efficiency for Industrial Enzymes: An Integrated Co-Translational Sequence Analysis Platform

Industrial biocatalysis relies on the cost effective production of active enzymes in heterologous hosts, yet a substantial fraction of candidate sequences fails during recombinant expression because sequence-level determinants of translational efficiency are not captured by conventional codon optimization alone. Here we present an integrated computational platform for pre-clinical assessment of industrial enzyme expression potential that combines baseline sequence metrics with co-translational modeling of ribosome stalling, translation rhythm, codon harmonization, folding bottlenecks, solubility proxies, and proteolysis risk. The platform accepts DNA, protein, FASTA, and PDB inputs and returns interpretable expression risk assessments, component-level diagnostics, variant design proposals, and actionable engineering recommendations. Analyses are parameterized for industrial expression hosts (Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, and mammalian cells) and for native enzyme sources including Streptomyces, Bacillus, filamentous fungi, actinomycetes, and extremophiles. All analyses execute locally without dependence on external databases, enabling rapid and secure screening of proprietary enzyme candidates prior to gene synthesis and fermenter scale-up. We report three layers of preliminary validation on small cohorts: (i) a retrospective public industrial-enzyme ranking benchmark (n = 20; rank subset n = 10); (ii) calibration of the solubility module on held-out eSOL enzymes with quantitative experimental solubility; and (iii) an independent collaborator wet-lab construct set retained as a true external-test holdout and never used for model fitting (n = 38 labeled constructs). On the external holdout, solubility calls achieved accuracy 68.4% (95% CI 52.5 - 80.9%), sensitivity 56.5% (36.8 - 74.4%), specificity 86.7% (62.1 - 96.3%), balanced accuracy 71.6% (bootstrap 95% CI 57.4 - 84.4%), and MCC 0.43 (0.15 - 0.69). Sensitivity remains close to chance on this small set, whereas specificity is higher; intervals are wide and the evidence should be read as directional rather than definitive. A directional arginine-surface redesign example (SrMilN insoluble parent to SrMilN_SASAR partially soluble variant; incomplete rescue with multiple soluble bands) was recovered. These findings support preliminary screening utility and transparent experimental agreement, but do not constitute high-accuracy prediction of industrial fermenter titre. Offline EC-class heuristics are retained only as optional provisional labels and are not presented as a validated platform capability (agreement 3/20 = 15%, 95% CI 5.2 - 36%; chance for six classes is 16.7%).

bioinformatics↗

bioRxiv2026

Structural and Mechanistic Basis of FapB Accessory Functional Amyloid Assembly in Pseudomonas

Functional amyloid formation in Pseudomonas biofilms is coordinated by the six-gene Fap operon (fapA-fapF), which governs the production, secretion, and assembly of extracellular fibrils that reinforce the biofilm matrix. FapC, FapB, and FapE constitute the extracellular amyloid. FapC is the major fibrillar component, whereas FapB and FapE occur at lower abundance and other accessory Fap proteins regulate fibril composition, processing, and secretion. We recently determined the structure of FapC, a cross-b-solenoid major Pseudomonas extracellular fibril. The assembly mechanism and architecture of FapB, however, have remained undefined. Here, we elucidate the structural and mechanistic basis of FapB accessory amyloid, from an intrinsically disordered monomer to a cross-b-solenoid fibril. Using well-defined monomeric preparations, controlled fibrillation assays, NMR spectroscopy, cryo-EM, and structure prediction, we show that FapB efficiently self-assembles into structurally homogeneous helical fibrils. FapB fibrillation is highly sensitive to its initial oligomeric state and purity, as well as to environmental factors such as pH. Monomeric FapC does not measurably perturb the disordered conformational ensemble of FapB, whereas preformed FapB and FapC fibrils reciprocally cross-seed each other, revealing direct kinetic coupling between the two amyloid components. Finally, we present a 3.4A resolution cryo-EM structure of the FapB accessory fibril, revealing a compact cross-b-solenoid architecture closely related to that of FapC. Together, these findings establish FapB as an independently amyloidogenic, structurally defined component of the Fap system, provide structural basis for the first of accessory cross-b-solenoid fibril, and reveal a mechanistic framework for the post-secretion assembly of multicomponent extracellular functional amyloids in Pseudomonas biofilms.

microbiology↗

bioRxiv2026

Structural dynamics insights into principles underlying the fitness of new broadly potent AAVs

Adeno-associated virus (AAV) is a leading platform for gene therapy, but current clinical-stage vectors require high doses associated with adverse events. Engineering of AAVs has produced more efficient vectors, although the mechanism underlying these improvements often remains poorly understood, limiting further development and raising potential safety concerns. Here, we leveraged a new workflow for AAV engineering with single-cell resolution, called scAAVengr-Hunt, to create best-in-class AAVs for gene delivery. ATX002, the top-performing vector, demonstrates broad potency across species, including nonhuman primate, mouse, and human, as well as across retina and brain. To understand the mechanism underlying this broad potency, we performed molecular dynamics simulations comparing AAV variants spanning a range of fitness levels. Structural dynamics analysis revealed a bifunctional molecular mechanism that confers potency through increased affinity of the capsid to the AAV receptor and regulation of heparan sulfate binding. This work provides critical insights relating structural mechanism to the fitness of engineered AAVs and establishes rich new avenues for AAV engineering through the integration of sequence-level analysis with computational biophysics.

bioengineering↗

bioRxiv2025

Structural insights into scaffold-guided assembly of the Pseudomonas phage D3 capsid

Tailed bacteriophages comprise the largest structural family of viruses with close relatives in archaea and the eukaryotic herpesviruses. The common assembly pathway produces an icosahedrally symmetric protein shell, called capsid, into which the double-stranded DNA genome is packaged. While capsid sizes and amino acid sequences vary considerably, the major capsid protein (MCP) folds are remarkably similar throughout the family. To investigate the mechanisms governing capsid size, we characterized the procapsid and mature capsid of phage D3, which expresses an icosahedral lattice with Triangulation number T=9. We found that the MCP scaffold domain binds to the interior capsid surface, acting as a clamp to constrain subunit interactions. Following scaffold digestion, the MCP capsid domains form strong interactions that maintain capsid structure throughout maturation. The scaffold constraints appear critical for capsid size determination and provide important understanding of the factors governing capsid assembly in general and expands our understanding of these ecologically and biomedically important viruses.

molecular biology↗

bioRxiv2025

Deletion of the Envelope gene attenuates SARS-CoV-2 infection by altered Spike localization and increased cell-to-cell transmission

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a highly transmissible acute respiratory infection that can result in severe pneumonia and death. Many details of SARS-CoV-2 infection are not fully understood, including the cell biology and host-virus interactions involved in coronavirus assembly and release, in which the Envelope (E) structural protein is instrumental. Deletion of E in other coronaviruses has been shown previously to either attenuate or abrogate infection. To determine the role of E on SARS-CoV-2 virus production and infectivity, we produced reporter SARS-CoV-2 with or without the E gene deleted using a bacterial artificial chromosome. Replication of {delta}E SARS-CoV-2 was attenuated in Vero E6 cells expressing human ACE2 and TMPRSS2 and in human epithelial cell lines. Electron and immunofluorescence microscopy and virology assays showed that {delta}E SARS-CoV-2 increased cell surface expression of Spike (S) glycoprotein, leading to reduced S incorporation into {delta}E SARS-CoV-2 particles and promotion of increased cell-to-cell transmission that evades neutralizing antibody inhibition. Trans-complementation of E partially rescued {delta}E SARS-CoV-2 S incorporation and restored cell-free transmission. In addition to validating the role of E in retention of S in the ER-Golgi intermediate complex (ERGIC), our results showed that a lack of E led to reorganization of the ERGIC and Golgi during SARS-CoV-2 infection. Improved understanding of E in SARS-CoV-2 replication and host pathogenesis may help development of novel therapeutics.

microbiology↗

bioRxiv2025

Structural Basis of Pseudomonas FapC Biofilm-Forming Functional Amyloid Formation

Biofilm-protected Pseudomonas aeruginosa causes chronic infections that are difficult to treat. FapC, the major biofilm forming functional-amyloid in Pseudomonas, is essential for biofilm integrity, yet its structural details remain unresolved. Using an integrative structural biology approach, we combine solution NMR-based structural ensemble of unfolded monomeric FapC, a [~]3.3 [A] resolution CryoEM density map of FapC fibril, and all-atom MD simulations to capture transition from unfolded to folded monomer to fibrillar fold, providing a complete structural view of FapC biogenesis. CryoEM reveals a unique triple-layer {beta}-solenoid cross-{beta} fibril composed of a single protofilament. MD simulations initiated from monomeric and fibrillar FapC mapped structural transitions, offering mechanistic insights into amyloid assembly and disassembly. Understanding FapC reveals how Pseudomonas exploits functional amyloids for biofilm formation and establishes a structural and mechanistic foundation for developing therapeutics targeting biofilm-related infection and antimicrobial resistance.

microbiology↗

bioRxiv2023

Structures of kinetic intermediate states of HIV-1 reverse transcriptase DNA synthesis

Reverse transcription of the retroviral single-stranded RNA into double-stranded DNA is an integral step during HIV-1 replication, and reverse transcriptase (RT) is a primary target for antiviral therapy. Despite a wealth of structural information on RT, we lack critical insight into the intermediate kinetic states of DNA synthesis. Using catalytically active substrates, and a novel blot/diffusion cryo-electron microscopy approach, we captured 11 structures that define the substrate binding, reactant, transition and product states of dATP addition by RT at 1.9 to 2.4 [A] resolution in the active site. Initial dATP binding to RT-template/primer complex involves a single Mg2+ (site B), and promotes partial closure of the active site pocket by a large conformational change in the {beta}3-{beta}4 loop in the Fingers domain, and formation of a negatively charged pocket where a second "drifting" Mg2+ can bind (site A). During the transition state, the -phosphate oxygen from a previously unobserved dATP conformer aligns with the site A Mg2+ and the primer 3'-OH for nucleophilic attack. In the product state, we captured two substrate conformations in the active site: 1) dATP that had yet to be incorporated into the nascent DNA, and 2) an incorporated dAMP with the pyrophosphate leaving group coordinated by metal B and stabilized through H- bonds in the active site of RT. This study provides insights into a fundamental chemical reaction that impacts polymerase fidelity, nucleoside inhibitor drug design, and mechanisms of drug resistance.

biophysics↗

bioRxiv2023

Pneumococcal Extracellular Vesicles Mediate Horizontal Gene Transfer via the Transformation Machinery

Bacterial cells secrete extracellular vesicles (EVs), the function of which is a matter of intense investigation. Here, we show that the EVs secreted by the human pathogen Streptococcus pneumoniae (pneumococcus) are associated with bacterial DNA on their surface and can deliver this DNA to the transformation machinery of competent cells. These findings suggest that EVs contribute to gene transfer in Gram-positive bacteria, and in doing so, may promote the spread of drug resistance genes in the population. SignificanceThis work extends our understanding of horizontal gene transfer and the roles of extracellular vesicles in pneumococcus. This bacterium serves as the model for transformation, a process by which bacteria can take up naked DNA from the environment. Here we show that extracellular vesicles secreted by the pneumococcus have DNA on their surface, and that this DNA can be imported by the transformation machinery facilitating gene transfer. Understanding EV-mediated gene transfer may provide new avenues to manage the spread of antibiotic drug resistance.

microbiology↗

bioRxiv2022

A structural dendrogram of the actinobacteriophage major capsid proteins provides important structural insights into the evolution of capsid stability

Many double-stranded DNA viruses, including tailed bacteriophages (phages) and herpesviruses, use the HK97-fold in their major capsid protein to make the capsomers of the icosahedral viral capsid. Following the genome packaging at near-crystalline densities, the capsid is subjected to a major expansion and stabilization step that allows it to withstand environmental stresses and internal high pressure. Several different mechanisms for stabilizing the capsid have been structurally characterized, but how these mechanisms have evolved is still not understood. Using cryo-EM structure determination, structural comparisons, phylogenetic analyses, and Alphafold predictions, we have constructed a detailed structural dendrogram describing the evolution of capsid structural stability within the actinobacteriophages. The cryo-EM reconstructions of ten capsids solved to resolutions between 2.2 and 4 [A]ngstroms revealed that eight of them exhibit major capsid proteins that are linked by a covalent cross-linking (isopeptide bond) between subunits that was first described in the HK97 phage. Those covalent interactions ultimately lead to the formation of mutually interlinked capsomers that has been compared to the structure of chain mail. However, three of the closely related phages do not exhibit such an isopeptide bond as demonstrated by both our cryo-EM maps and the lack of the required residue. This work raises questions about the importance of previously described capsid stabilization mechanisms.

biochemistry↗

bioRxiv2022

Structural basis of DNA packaging by a ring-type ATPase from an archetypal viral system

Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos. Here we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for both this phage and a cos system, reveal unexpected mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.

biochemistry↗

bioRxiv2022

Lipidoid nanoparticles increase ATP uptake into hypoxic brain endothelial cells

Lipidoid nanoparticles (LNPs) are clinically successful carriers for nucleic acid delivery to liver and muscle targets. Their ability to load and deliver small molecule drugs has not been reported yet. We propose that the delivery of adenosine triphosphate (ATP) to brain endothelial cells (BECs) lining the blood-brain barrier may increase cellular energetics of the injured BECs. We formulated and studied the physicochemical characteristics of ATP-loaded LNPs using the C12-200 ionizable cationic lipid and other helper lipids. Polyethylene glycol-dimyristoyl glycerol (PEG-DMG), one of the helper lipids, played a crucial role in maintaining colloidal stability of LNPs over time whereas the inclusion of both ATP and PEG-DMG maintained the colloidal stability of LNPs in the presence of serum proteins. ATP-LNPs formulated with PEG-DMG resulted in a 7.7- and 6.6-fold increased uptake of ATP into normoxic and hypoxic BECs, respectively. Altogether, our results demonstrate the potential of LNPs as a novel carrier for the delivery of small molecular mass actives to BECs--a CNS target. HighlightsO_LILNPs were formulated with ATP, a small molecule drug C_LIO_LIPEG-DMG plays a critical role in maintaining particle stability over tim C_LIO_LIATP and PEG-DMG play a critical role in maintaining particle stability in 10% serum C_LIO_LIATP-LNPs were internalized by normoxic and hypoxic brain endothelial cells (BECs) C_LIO_LILNP delivery to BECs broadens its applicability to CNS targets C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/487513v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1a315eaorg.highwire.dtl.DTLVardef@1f3d649org.highwire.dtl.DTLVardef@17989fdorg.highwire.dtl.DTLVardef@526ce2_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Potent neutralizing nanobodies resist convergent circulating variants of SARS-CoV-2 by targeting novel and conserved epitopes

There is an urgent need to develop effective interventions resistant to the evolving variants of SARS-CoV-2. Nanobodies (Nbs) are stable and cost-effective agents that can be delivered by novel aerosolization route to treat SARS-CoV-2 infections efficiently. However, it remains unknown if they possess broadly neutralizing activities against the prevalent circulating strains. We found that potent neutralizing Nbs are highly resistant to the convergent variants of concern that evade a large panel of neutralizing antibodies (Abs) and significantly reduce the activities of convalescent or vaccine-elicited sera. Subsequent determination of 9 high-resolution structures involving 6 potent neutralizing Nbs by cryoelectron microscopy reveals conserved and novel epitopes on virus spike inaccessible to Abs. Systematic structural comparison of neutralizing Abs and Nbs provides critical insights into how Nbs uniquely target the spike to achieve high-affinity and broadly neutralizing activity against the evolving virus. Our study will inform the rational design of novel pan-coronavirus vaccines and therapeutics.

biophysics↗

bioRxiv2020

Mechanisms of antibody binding revealed by asymmetric Fab-virus complexes

Overlap on the surface of parvovirus capsids between the antigenic epitope and the receptor binding site contributes to species jumping. Mab 14 strongly binds and neutralizes canine, but not feline parvovirus. The high resolution map of the canine parvovirus capsid complexed with Fab 14 was used to solve local structures of the Fab-bound and -unbound antigenic sites extracted from the same complex. The subsequent analysis includes a new method for using cryo EM to investigate complementarity of antibody binding.

microbiology↗

bioRxiv2020

The Mitochondrial Permeability Transition Phenomenon Elucidated by Cryo-EM Reveals the Genuine Impact of Calcium Overload on Mitochondrial Structure and Function

Mitochondria have a remarkable ability to uptake and store massive amounts of calcium. However, the consequences of massive calcium accumulation remain enigmatic. In the present study, we analyzed a series of time-course experiments to identify the sequence of events that occur in a population of guinea pig cardiac mitochondria exposed to excessive calcium overload that cause mitochondrial permeability transition (MPT). By analyzing coincident structural and functional data, we determined that excessive calcium overload is associated with large calcium phosphate granules and inner membrane fragmentation, which explains the extent of mitochondrial dysfunction. This data also reveals a novel mechanism for cyclosporin A, an inhibitor of MPT, in which it preserves inner membrane architecture despite the presence of massive calcium phosphate granules in the matrix. Overall, these findings establish a mechanism of calcium-induced mitochondrial dysfunction and the impact of calcium regulation on mitochondrial structure and function.

cell biology↗

bioRxiv2018

The apical region of the herpes simplex virus major capsid protein promotes capsid maturation

The herpesvirus capsid assembles in the nucleus as an immature procapsid precursor built around viral scaffold proteins. The event that initiates procapsid maturation is unknown, but it is dependent upon activation of the VP24 internal protease. Scaffold cleavage triggers angularization of the shell and its decoration with the VP26 and pUL25 capsid-surface proteins. In both the procapsid and mature angularized capsid, the apical region of the major capsid protein (VP5) is surface exposed. We investigated whether the VP5 apical region contributes to intracellular transport dynamics following entry into primary sensory neurons and also tested the hypothesis that conserved negatively-charged amino acids in the apical region contribute to VP26 acquisition. To our surprise neither hypothesis proved true. Instead, mutation of glutamic acid residues in the apical region delayed viral propagation and induced focal capsid accumulations in nuclei. Examination of capsid morphogenesis based on epitope unmasking, capsid composition, and ultrastructural analysis indicated that these clusters consisted of procapsids. The results demonstrate that, in addition to established events that occur inside the capsid, the exterior capsid shell promotes capsid morphogenesis and maturation.

microbiology↗