Biology subjects

Groves, J. T.

Publications and source records attributed to Groves, J. T..

At least 19 recordsLinked to original sources

bioRxiv2026

Single molecule Grb2 dynamics reveal spatiomechanical modulation of RTK signaling across length scales

Extracellular matrix (ECM)-mediated cell adhesion and tension regulate development and disease through crosstalk with Receptor Tyrosine Kinases (RTKs). RTKs stimulate proliferation, survival, and metabolic reprogramming via Ras/ERK and PI3K/Akt signaling. Emerging evidence identify the biophysical and biochemical integration of integrin-mediated adhesions with RTK signaling, however, the molecular mechanisms regulating this phenotype remain poorly understood. Here, we demonstrate that ECM stiffness potentiates ligand-mediated epidermal growth factor receptor (EGFR) signaling at scales ranging from the single adaptor protein, Grb2 upward to multicellular spheroids and tissues. We show that ECM stiffness-driven RTK signaling is mediated by integrin adhesions that reshape the membrane to induce the formation of RTK signaling hotspots distributed globally across the cell membrane. A parsimonious computational model and biochemical membrane perturbations causally implicate membrane curvature and topography in tension-dependent integrin-EGFR crosstalk. The results demonstrate how the mechanical properties of the ECM direct molecular recruitment and confinement of signaling molecules to regulate growth factor-dependent RTK signaling. Given that many RTK-driven solid tumors develop a stiff stroma, the findings could explain why many therapeutic tyrosine kinase inhibitors develop clinical resistance. The findings suggest clarifying how tension regulates transmembrane receptor signaling could identify unique force-dependent molecular regulators towards which new antitumor therapies could be applied.

biophysics↗

bioRxiv2026

LAT condensation gates PLCγ1 activation via bimodal LAT phosphorylation

T cells can respond to even a single molecular binding event of antigen to a TCR. A key step in the TCR signaling pathway that definitively exhibits this single molecule response is the initiation of calcium influx by activation of PLC{gamma}1 in the LAT protein condensate. Here, we describe detailed kinetic measurements examining how protein condensation of LAT regulates activation of PLC{gamma}1 using a reconstituted membrane system. The results reveal that membrane recruitment of PLC{gamma}1 is tightly controlled by the LAT phosphorylation state, with no measurable independent recruitment to PIP2 or PIP3 lipids via the PLC{gamma}1 PH domains. We further observe PLC{gamma}1 is rapidly activated by membrane-associated kinase upon recruitment, irrespective of the LAT condensation state. These studies also revealed a crosstalk mechanism in which the TEC family kinases responsible for PLC{gamma}1 activation also phosphorylate LAT. This interaction establishes a positive feedback loop in LAT phosphorylation, mediated through LAT condensation, which drives a bimodal LAT phosphorylation response to TCR activation. Kinetic modeling reveals how this LAT phosphorylation response can cooperatively gate PLC{gamma}1 activation from a single TCR. These results suggest the LAT condensate facilitates both signal amplification and noise suppression in PLC{gamma}1 activation through a bimodal switch affecting LAT phosphorylation. Significance StatementT cells are sensitive sensors capable of detecting and responding to trace amounts of foreign antigen. Understanding how they achieve such sensitivity while maintaining accurate antigen discrimination remains a key challenge. Here, through detailed kinetic measurements of PLC{gamma}1 activation, we identify a cross reactivity in which kinases responsible for PLC{gamma}1 phosphorylation also phosphorylate LAT. This creates a bimodal switch controlling LAT phosphorylation levels, which gates PLC{gamma}1 activation from single TCR signals. We suggest this mechanism plays a key role in the signal amplification and noise suppression required for T cells to detect single antigen molecules.

biophysics↗

bioRxiv2026

Supported membrane assay probes PLCγ1 activity in LAT condensates

Phospholipase C-{gamma}1 (PLC{gamma}1) plays a critical role linking T cell receptor activation with downstream signaling pathways including calcium. PLC{gamma}1 activation in T Cells relies on phosphotyrosine-mediated recruitment to the membrane-bound scaffold LAT, which becomes crosslinked through a bond percolation network with Grb2 and other scaffold and signaling molecules to form a signaling condensate. PLC{gamma}1 in these LAT condensates becomes activated, leading to induction of extracellular calcium influx. While PLC{gamma}1-driven calcium signaling is clearly correlated with LAT condensation, it is less clear how--or if-- the LAT condensation state facilitates PLC{gamma}1 activity. Here we develop an image-based PLC{gamma}1 activity assay in supported bilayers that enables simultaneous measurement of both PLC{gamma}1 recruitment to phosphorylated LAT and PLC{gamma}1-catalyzed hydrolysis of PIP2 in the membrane. The condensation state of LAT is independently controlled by adjusting levels of co-condensation proteins such as Grb2, SOS, GADS, and SLP76. The hydrolysis product, diacylglycerol (DAG), remains in the membrane and is monitored as a readout of catalytic activity using a DAG sensor based on the C1b (DAG binding) domain of PKC{theta}. Assays are performed directly with mammalian cell lysate containing fluorescent PLC{gamma}1 fusion constructs. The results reveal that PLC{gamma}1 is highly active when recruited to dispersed LAT and that the condensed state does not promote activity. Overall, this assay platform reveals that despite the correlation between PLC{gamma}1 signal gating and LAT condensation, the physical environment of the condensate itself is not a key regulator of PLC{gamma}1 signaling. More broadly, this assay system offers a quantitative means of probing how PLC{gamma}1 activity is controlled at the membrane.

biophysics↗

bioRxiv2025

eIF2B Selectively Anchors and Activates Mutant KRAS

Much is known about how RAS oncoproteins regulate mRNA translation factors, but the reverse relationship, how translation factors influence RAS activity, has remained largely unexplored. At the plasma membrane (PM), Son of Sevenless (SOS) acts as the canonical guanine nucleotide exchange factor (GEF) for RAS proteins, yet mechanisms governing its specificity for individual RAS isoforms remain unknown. Here, we show that the translation initiation factor eIF2B, best known for its GEF function in translation initiation, forms a distinct complex with SOS and mutant KRAS at the PM, but not with other oncogenic RAS variants. Mechanistically, eIF2B acts as an allosteric regulator of SOS, selectively enhancing GDP-GTP exchange on mutant KRAS. This specificity arises from the translational activity of eIF2B, which upregulates glycosphingolipid (GSL) biosynthesis to remodel PM lipids and preferentially anchor mutant KRAS. Together, our results uncover an unexpected moonlighting function of eIF2B: acting both as a direct activator of SOS and as a regulator of GSL pathway that shapes the membrane landscape, both required for mutant KRAS activation. These insights redefine our understanding of eIF2B and mutant KRAS functions in cancer and have profound implications for KRAS-driven oncogenesis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/686860v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1f50934org.highwire.dtl.DTLVardef@2f55b9org.highwire.dtl.DTLVardef@1a51e39org.highwire.dtl.DTLVardef@1634aac_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIeIF2B interacts with mutant KRAS and SOS at the plasma membrane (PM). C_LIO_LIThe eIF2B:SOS complex promotes the GTP-bound active state of mutant KRAS. C_LIO_LIeIF2B enhances the translation of B4GALT5 mRNA, encoding a key enzyme of glycosphingolipid (GSL) biosynthesis. C_LIO_LIUpregulation of the GSL metabolites, ganglioside GM3 and sulfatide SM4, remodels PM lipid composition to facilitate eIF2B:SOS:KRAS complex formation and mutant KRAS nanoclustering. C_LIO_LIThrough its interaction with SOS and stimulation of GSL synthesis, eIF2B selectively activates mutant KRAS at the PM among RAS isoforms. C_LIO_LIeIF2B is required for the development of mKRAS-driven lung adenocarcinoma in mice. C_LIO_LIeIF2B is a marker of poor prognosis in mutant KRAS-driven cancers. C_LI

cancer biology↗

bioRxiv2025

T cell costimulatory and inhibitory signals differentially modulate LAT condensate nucleation propensity after TCR ligation

Linker for activation of T cells (LAT) is a membrane-surface scaffold protein that assembles key signaling molecules upon T cell receptor (TCR) activation. Recent observations have revealed LAT assembly occurs in discretized condensates traceable to single molecular TCR activation events. Moreover, the condensation process occurs abruptly, but after a distinct delay, indicative of a type of phase transition. Here we examine the effects of costimulatory (CD80, CD86) and inhibitory (PD-L1) ligands on the nucleation propensity of LAT condensation. We utilize single molecule mobility tracking to resolve the moment a pMHC molecule binds to TCR combined with intracellular imaging to measure the time delay between pMHC:TCR binding and nucleation of the corresponding LAT condensate. These delay time distributions reflect the propensity function for stochastic nucleation of the LAT condensate and provide a measure of the effective signal strength from a single, activated TCR. The results reveal that CD28 stimulation by CD80 or CD86 causes a distinct reduction in the mean delay time to LAT condensation while PD-L1 is mildly antagonistic to the process. Collectively, these observations strengthen evidence that probability and timing of the LAT nucleation process itself is tightly associated with signal propagation downstream from individual TCR activation events.

biophysics↗

bioRxiv2025

A unifying model of LAT condensates in reconstitution experiments

The formation of condensates by the Linker for the Activation of T-cells (LAT) is a key signal gating and amplification step in the T-cell receptor signaling pathway. LAT condensation is challenging to study in-vivo and is therefore often investigated using reconstitution experiments. While these experiments recapitulate key aspects of LAT condensation, they also exhibit some puzzling features. Here, we describe the mechanisms underlying these observations using two complementary models. First, we employ a Smoluchowski aggregation model to show that the delay time before condensation is observed arises from a low effective binding probability between LAT monomers. Second, we propose a field-theoretic model that reproduces all condensate morphologies observed in experiments, showing that they can arise from common underlying dynamics modulated by variations in experimental conditions. This result unifies different experimental observations reported previously. While this article addresses open questions regarding the formation of LAT condensates, our results also provide a common framework for understanding condensation of other multivalent membrane proteins such as EGFR, FGFR2, and nephrin.

biophysics↗

bioRxiv2025

Engineering auxin degradation into root-associated bacteria promotes plant growth

Overproduction of indole-3-acetic acid (IAA) by rhizosphere bacteria disrupts plant auxin homeostasis and induces root growth inhibition (RGI). Variovorax reverses this effect by degrading IAA, but the underlying pathway remains incompletely resolved. Here, using genetics, metabolomics, and isotope tracing, we define a nine-gene region (iadCDEFGHIJK2) required for IAA catabolism in Variovorax paradoxus CL014, identify a previously uncharacterized intermediate (CHNO), and revise the early oxidative steps of the pathway. Although IadDE adopts a Rieske-type oxygenase architecture, our data are consistent with the IadCDE complex functioning as a monooxygenase during IAA degradation. Using these insights, we introduce iad genes into two rhizobacterial chassis, Polaromonas MF047 and Paraburkholderia MF376. These engineered strains degrade IAA and alleviate RGI induced by exogenous IAA, an auxin-producing strain, and a synthetic bacterial community. Engineered Paraburkholderia MF376 delivers the strongest performance, improving plant growth in natural soil. Together, these results establish a framework for engineering auxin-balancing root commensals.

microbiology↗

bioRxiv2025

Machine-learning strategy for high error tolerance in image-based digital molecular assays

There is a significant global health need to translate more in vitro diagnostic tests (IVDs) from clinical laboratories to field-based applications, including point-of-care (POC) and self-administered test formats. These applications typically require smaller sample sizes, limit the extent of sample processing and measurement capabilities, and introduce greater handling variability. Error tolerance is one of the most critical factors for successful field-based assay design. Here, we examine machine-learning (ML) strategies to enhance the error tolerance of image-based nanoparticle immunoassays. Random dispersions of nanoparticles were imaged in microliter sample volumes, and images were processed to determine analyte concentrations based on nanoparticle appearance. Assay performance was characterized using two common blood diagnostics: C-reactive protein (CRP) and S.CoV-2 IgG. We compare the results from a conventional image analysis, a hybrid ML-conventional approach based on pixel segmentation, and a full end-to-end image regression using a targeted regularization strategy. Training images for the full image regression approach required only a single label for training - the analyte concentration - eliminating the need for labor-intensive pixel-level labeling. Ultimately, the fully ML-based analysis significantly improved dynamic range, sensitivity, and reproducibility in high-error settings, including direct measurements performed in whole blood.

bioengineering↗

bioRxiv2025

Kinetic Proofreading through Parallel Reactions on a Single T Cell Receptor

T cells can recognize a few molecules of cognate antigen amongst vastly outnumbering non-cognate ligands. The T cell receptor (TCR) differentiates antigens based on antigen-TCR binding dwell time through a kinetic proofreading process. Historically, this has been modeled as the ligated receptor undergoing a series of reactions before producing a signal. In such a sequential mechanism, the number of steps is a key determinant of discrimination fidelity. Here, we consider two features of the molecular mechanism of TCR activation that diverge from a sequential process and suggest that an alternative kinetic proofreading mechanism may be at play. First, activation processes of multiple ITAM domains of the TCR represent parallel reaction sequences taking place on a single TCR molecule. Second, the states of the parallel proofreading reactions are integrated to produce a binary output from each TCR in the form of a discrete LAT condensation event, which may or may not occur. We examine a revised kinetic proofreading scheme based on parallel reactions followed by an integration step (multi-thread scheme) and compare its performance with the sequential scheme in a stochastic setting. A distinct difference in a multi-thread scheme is that multiplicity of the parallel reaction threads provides an additional means to increase discrimination fidelity. This relieves the need for fine-tuned kinetics among chemically distinct reaction steps, which is a major hurdle for physical implementation of a sequential mechanism. Lastly, we reinterpret previously reported experimental observations and find that various proofreading behaviors are well described as proofreading through parallel reaction threads. Significance StatementThe kinetic proofreading mechanism by which T cells discriminate antigen has attracted interest from both physical and immunological perspectives. The proofreading process has generally been modeled as a multi-step sequence of reactions, and many experimental observations have been interpreted based on this presumed mechanism. However, these models do not capture key molecular features of the TCR signaling mechanism, including ITAM multiplicity and LAT condensation. Here, we find distinct consequences of these molecular features by examining kinetic models and reinterpreting published experimental data. ITAM multiplicity offers multiple parallel reaction threads, re-integrated by the LAT condensation step, which readily improves discrimination fidelity up to the observed levels. Multi-thread reactions are suggested to play a central role in amplifying TCR kinetic proofreading performance.

biophysics↗

bioRxiv2025

BTK Autoinhibition Analyzed by High-Throughput SH2 Domain Swaps

BTK, a Tec-family tyrosine kinase, resembles the Src and Abl kinases in that an SH2-SH3 module regulates the activity of the kinase domain, principally through an inhibitory interaction between the SH3 and kinase domains. In Src kinases, phosphorylation of a C-terminal tail latches the SH2 domain onto the kinase domain, positioning the SH3 domain in an inhibitory conformation; in Abl, interaction between the kinase domain and a myristoyl group on the N- terminal segment provides the same latching function. The structure of autoinhibited BTK resembles that of the Src and Abl kinases, but BTK lacks an SH2-kinase latch. To assess autoinhibition in BTK, we generated hundreds of chimeric BTK molecules and measured their fitness using a high-throughput assay in T and B cells. Surprisingly, many SH2 domains increased fitness when substituted into BTK. Analysis of one set of chimeric proteins demonstrated that the increase in fitness stems from the ability of the substituted SH2 domains to disrupt BTK autoinhibition while maintaining phosphotyrosine targeting. These results reveal the importance of distributed interactions between the SH2 and kinase domains of BTK in stabilizing the inhibitory conformation, and suggest that the specialized latching mechanisms in Src and Abl kinases may be later evolutionary refinements.

biochemistry↗

bioRxiv2024

Autophosphorylation of the oncogenic protein TEL-ABL confers resistance to the allosteric ABL inhibitor asciminib

Chromosomal translocations that fuse the ABL1 gene to BCR and TEL cause human leukemias. Oligomerization and the loss of an inhibitory myristoylation modification lead to unregulated kinase activity of the BCR-ABL and TEL-ABL fusion proteins. ATP-competitive ABL inhibitors, such as imatinib and ponatinib, are effective against both fusion proteins. We discovered that asciminib, an allosteric inhibitor of BCR-ABL that binds to the myristoyl binding site in the ABL kinase domain, is >2000-fold less potent against TEL-ABL than BCR-ABL in cell-growth assays. This is surprising because the ABL components of the two fusion proteins, including the asciminib binding sites, have identical sequence. We deleted a short helical segment in the ABL kinase domain that closes over asciminib when it is bound. This deletion results in asciminib resistance in BCR-ABL, but has no effect on TEL-ABL, suggesting that the native autoinhibitory mechanism that asciminib engages in BCR-ABL is disrupted in TEL-ABL. We show, using mammalian cell expression and single-molecule microscopy, that BCR-ABL is mainly dimeric while TEL-ABL forms higher-order oligomers. Oligomerization can promote trans-autophosphorylation of ABL, and we find that a regulatory phosphorylation site in the SH3 domain of ABL (Tyr 89) is highly phosphorylated in TEL-ABL. This phosphorylation is expected to disassemble the autoinhibited conformation of ABL, thereby preventing asciminib binding. We show that TEL-ABL is intrinsically susceptible to inhibition by asciminib, but that increased phosphorylation results in resistance. Our results demonstrate that different ABL fusion proteins can have dramatically different responses to allosteric inhibitors due to differential phosphorylation. One Sentence SummaryWhen TEL-ABL is phosphorylated, it is insensitive to asciminib. However, when TEL-ABL is dephosphorylated by a phosphatase, asciminib sensitivity is restored.

biochemistry↗

bioRxiv2024

Grb2 Phosphorylation Antagonizes EGFR-driven Ras Activation by Interfering with Condensate Assembly

Upon ligand binding, the kinase domain of EGFR phosphorylates multiple tyrosine residues on the receptor cytoplasmic tail through a trans-autophosphorylation process. Phosphotyrosine sites on activated receptors recruit Grb2, which further recruits SOS to initiate downstream signaling by activating Ras. Multivalent binding between SOS and Grb2, as well as direct Grb2:Grb2 interactions, contribute to formation of a protein condensate of activated EGFR. The condensed state of EGFR facilitates autoinhibition release in SOS and exerts regulatory control over signal propagation from activated EGFR to Ras. While kinase activity of EGFR is an essential driver of this signaling process, phosphorylation at residue Y160 on Grb2 blocks Grb2:Grb2 binding and can interfere with EGFR condensation. Here, using a reconstituted system, we examine how titrating kinase activity in the EGFR system can both promote and inhibit signal output to Ras. The results reveal how effects of tyrosine kinase inhibition can, under some circumstances, promote Ras activation by inhibiting negative feedback through Grb2 phosphorylation and disruption of a Grb2 SH2/SH3 dimer interface. Statement of SignificanceActivated EGFR forms a biomolecular condensate, via linkage of multiple EGFR through Grb2 and SOS, and the condensation state of EGFR influences signal propagation to Ras. While tyrosine phosphorylation is a critical step in EGFR activation, phosphorylation of Grb2 can have an inhibitory effect on EGFR condensation and subsequent Ras activation. Under some conditions, kinase inhibition can promote signaling from EGFR to Ras.

biophysics↗

bioRxiv2024

Discrete protein condensation events govern calcium signal dynamics in T cells

Calcium level variations, which occur downstream of T cell receptor (TCR) signaling, are an essential aspect of T cell antigen recognition. Although coordinated ion channel activities are known to drive calcium oscillations in other cell types, observations of nonperiodic and heterogeneous calcium patterns in T cells are inconsistent with this mechanism. Here, we track the complete ensemble of individual molecular peptide-major histocompatibility complex (pMHC) binding events to TCR, while simultaneously imaging LAT condensation events and calcium level. Individual LAT condensates induce a rapid and additive calcium response, which quickly attenuates upon condensate dissolution. No evidence of cooperativity between LAT condensates or oscillatory calcium response was detected. These results reveal stochastic LAT protein condensation events as a primary driver of calcium signal dynamics in T cells. One-Sentence SummaryCa2+ fluctuations in T cells reflect stochastic protein condensation events triggered by single molecular antigen-TCR binding.

biophysics↗

bioRxiv2024

Differential Roles of Kinetic On- and Off-Rates in T-Cell Receptor Signal Integration Revealed with a Modified Fab'-DNA Ligand

Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly-binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide-MHC (pMHC) ligands bind their cognate TCRs. Because ligand dwell time is a critical parameter that tightly correlates with physiological function of the TCR signaling system, there is a general need, both in research and therapeutics, for universal TCR ligands with controlled kinetic binding parameters. To this end, we have introduced point mutations into recombinantly expressed -TCR{beta} H57 Fab to modulate the dwell time of monovalent Fab binding to TCR. When tethered to a supported lipid bilayer via DNA complementation, these monovalent Fab-DNA ligands activate T cells with potencies well-correlated with their TCR binding dwell time. Single-molecule tracking studies in live T cells reveal that individual binding events between Fab-DNA ligands and TCRs elicit local signaling responses closely resembling native pMHC. The unique combination of high on- and off-rate of the H57 R97L mutant enables direct observations of cooperative interplay between ligand binding and TCR-proximal condensation of the linker for activation of T cells (LAT), which is not readily visualized with pMHC. This work provides insights into how T cells integrate kinetic information from synthetic ligands and introduces a method to develop affinity panels for polyclonal T cells, such as cells from a human patient. STATEMENT OF SIGNIFICANCET cells read kinetic information from ligands binding to T-cell receptors (TCRs) to make cell fate decisions. Unique kinetic features of a modified Fab-DNA ligand enable direct visualization multiple TCR signal coordination through a nascent LAT condensation event. We further observed positive feedback through a kinetic on-rate enhancement in the growing LAT condensate. These observations help unify several seemingly disparate aspects of TCR signaling that have been debated in the literature. Furthermore, calibration of the Fab-DNA ligand against native agonist pMHC establishes a basis for quantitative analysis of TCR signaling in polyclonal T cell populations.

biophysics↗

bioRxiv2024

Proofreading and single-molecule sensitivity in T-cellreceptor signaling by condensate nucleation

T-cells display the remarkable ability to detect single foreign peptides displayed on target cells, while ignoring highly abundant self peptides. This selectivity has been explained by kinetic proofreading in the T-cell receptor (TCR) signaling pathway, which prevents responses to short-lived binding events regardless of their abundance. However, the biochemical mechanisms that drive kinetic proofreading have remained unclear. Here, using computational modeling, we show that these key signaling properties of the TCR pathway can emerge from the dynamics of LAT phosphorylation, diffusion, and condensation following TCR-pMHC binding. In this model, time delays in LAT condensate nucleation underlie kinetic proofreading, enabling selective signaling responses to high-affinity pMHC ligands. The cooperativity in the nucleation and growth of LAT condensates also provides a mechanism to amplify weak signals from single foreign peptides and for condensates to grow with increasing antigen numbers. In contrast to other models, condensate-nucleation proofreading predicts a dependence of signal strength on pMHC spacing at fixed number, a prediction we validated experimentally using a protein scaffold to present pMHCs at defined intervals. Our results suggest that nucleation-condensation proofreading underlies the remarkable antigen detection capabilities of the TCR signaling pathway. SignificanceTo fight infections and cancer, T-cells must selectively recognize low levels of foreign peptides from pathogens or cancer cells, but the mechanisms that enable these properties have remained unclear.Using mathematical modeling and experiments, we find that T-cells can selectively detect single foreign peptides through a clustering process where a key protein downstream of the T-cell receptor forms condensates containing hundreds of signaling proteins. This condensate nucleation process can explain experimentally observed features of T-cell signaling, including our finding that the size of signaling clusters depends on the distance between foreign peptides. Our work reveals a key role for the condensation of signaling molecules in setting the spatial and temporal thresholds that control the sensitivity and selectivity of T-cells.

biophysics↗

bioRxiv2023

Utilization of 5-deoxy-nucleosides as Growth Substrates by Extraintestinal Pathogenic E. coli via the Dihydroxyacetone Phosphate Shunt

All organisms utilize S-adenosyl-L-methionine (SAM) as a key co-substrate for methylation of biological molecules, synthesis of polyamines, and radical SAM reactions. When these processes occur, 5-deoxy-nucleosides are formed as byproducts such as S-adenosyl-L-homocysteine (SAH), 5-methylthioadenosine (MTA), and 5-deoxyadenosine (5dAdo). One of the most prevalent pathways found in bacteria for the metabolism of MTA and 5dAdo is the DHAP shunt, which converts these compounds into dihydroxyacetone phosphate (DHAP) and 2-methylthioacetaldehyde or acetaldehyde, respectively. Previous work has shown that the DHAP shunt can enable methionine synthesis from MTA or serve as an MTA and 5dAdo detoxification pathway. Here we show that in Extraintestinal Pathogenic E. coil (ExPEC), the DHAP shunt serves none of these roles in any significant capacity, but rather physiologically functions as an assimilation pathway for use of MTA and 5dAdo as growth substrates. This is further supported by the observation that when MTA is the substrate for the ExPEC DHAP shunt, the sulfur components is not significantly recycled back to methionine, but rather accumulates as 2-methylthioethanol, which is slowly oxidized non-enzymatically under aerobic conditions. While the pathway is active both aerobically and anaerobically, it only supports aerobic ExPEC growth, suggesting that it primarily functions in oxygenic extraintestinal environments like blood and urine versus the predominantly anoxic gut. This reveals a heretofore overlooked role of the DHAP shunt in carbon assimilation and energy metabolism from ubiquitous SAM utilization byproducts and suggests a similar role may occur in other pathogenic and non-pathogenic bacteria with the DHAP shunt. ImportanceAcquisition and utilization of organic compounds that can serve as growth substrates is essential for pathogenic E. coli to survive and multiply. Ubiquitous enzymatic reactions involving S-adenosyl-L-methionine as a co-substrate result in the formation of the 5-deoxy-nucleoside byproducts, 5-methylthioadenosine and 5-deoxyadenosine. All E. coli possess a conserved nucleosidase that cleaves these 5-deoxy-nucleosides into 5-deoxy-pentose sugars for adenine salvage. The DHAP shunt pathway, which is found in ExPEC strains but neither in intestinal pathogenic nor commensal E. coli, enables utilization of 5-deoxy-nucleosides and 5-deoxy-pentose sugars as growth substrates by ExPEC strains. This provides insight into the diversity of sugar compounds accessible by ExPEC strains in recalcitrant and nutrient-poor environments such as the urinary tract during infection. Furthermore, given the dihydroxyacetone phosphate shunt pathway appears to only support aerobic E. coli growth, this suggests an explanation as to why intestinal strains that primarily exist in anoxic environments lack this pathway.

microbiology↗

bioRxiv2023

Bimodality in Ras signaling originates from processivity of the Ras activator SOS without classic kinetic bistability

Ras is a small GTPase that is central to important functional decisions in diverse cell types. An important aspect of Ras signaling is its ability to exhibit bimodal, or switch-like activity. We describe the total reconstitution of a receptor-mediated Ras activation-deactivation reaction catalyzed by SOS and p120-RasGAP on supported lipid membrane microarrays. The results reveal a bimodal Ras activation response, which is not a result of classic kinetic bistability, but is rather driven by the distinct processivity of the Ras activator, SOS. Furthermore, the bimodal response is controlled by the condensation state of the scaffold protein, LAT, to which SOS is recruited. Processivity-driven bimodality leads to stochastic bursts of Ras activation even under strongly deactivating conditions. This behavior contrasts classic kinetic bistability and is distinctly more resistant to pharmacological inhibition.

biophysics↗

bioRxiv2022

A membrane-associated light harvesting model is enabled by functionalized assemblies of gene-doubled TMV proteins

Photosynthetic light harvesting requires efficient energy transfer within dynamic networks of light harvesting complexes embedded within phospholipid membranes. Artificial light harvesting models are valuable tools for understanding the structural features underpinning energy absorption and transfer within chromophore arrays. Most artificial light harvesting complexes are static or in the solution phase, rather than in a two-dimensional fluid environment as in natural photosynthesis. We have developed a method for attaching a protein-based light harvesting model to a supported lipid bilayer (SLB), which provides an extended fluid membrane surface stably associated with a solid substrate. The protein model consisted of the tobacco mosaic viral capsid proteins (TMV) that were gene-doubled to create a tandem dimer (dTMV). Assemblies of dTMV were shown to break the facial symmetry of the double disk to allow for differentiation between the disk faces. Single reactive lysine and cysteine residues were incorporated into opposing surfaces of each monomer of the dTMV assemblies. This allowed for the site-selective attachment of both chromophores for light absorption and a peptide for attachment to the SLB. A cysteine modification strategy using the enzyme tyrosinase was employed for the bioconjugation of a peptide containing a polyhistidine tag for association with SLBs. The dual-modified dTMV complexes showed significant association with SLBs and exhibited mobility on the bilayer. The techniques used herein offer a new method for protein-surface attachment and provide a platform for evaluating excited state energy transfer events in a dynamic, fully synthetic artificial light harvesting system. Significance StatementHere we have constructed a model photosynthetic membrane containing proteins, chromophores, lipids, and aqueous components, all of which can be modified in their composition. This model is based on an asymmetric disk assembly consisting of engineered tandem dimers of the tobacco mosaic viral capsid protein (dTMV). We have developed methods to achieve dye conjugation and attachment of a supported lipid bilayers (SLB) site selectively on distinct protein surfaces. These dye-labeled protein complexes exhibit mobility on the SLB, resulting in a dynamic model of light harvesting membranes using entirely synthetic components. Additionally, this unique asymmetric assembly of TMV and the facile methods for protein functionalization are expected to expand the tunability of model light harvesting systems.

bioengineering↗