Biology subjects

Poolman, B.

Publications and source records attributed to Poolman, B..

At least 19 recordsLinked to original sources

bioRxiv2026

Single-protein diffusion in micrometer-size vesicles is governed by viscosity, surface charge, and membrane interactions

Giant unilamellar vesicles (GUVs) are a powerful model system for studying protein dynamics in cell-sized compartments with a precisely tunable physicochemical environment. Here, we apply Single-Molecule Displacement Mapping (SMdM) to quantify how luminal viscosity, ionic strength, membrane lipid composition, and protein surface charge of soluble proteins govern intraluminal single-molecule diffusion. Increasing viscosity led to a predictable decrease in the diffusion coefficient. However, spatially resolved diffusion measurements reveal reduced protein mobility near the membrane, suggesting additional membrane-proximal interactions not captured by particle-based simulations. We observe long-range (~1 um) effects of membrane charge on protein mobility that extend well beyond classical Debye screening, which may reflect shape fluctuations of the GUV membrane. For the membrane-associated protein LactC2, we resolve two distinct diffusing populations within a single vesicle, corresponding to free and membrane-bound states. Our workflow provides a quantitative framework for probing single-protein mobility in vesicle-based synthetic cells. These results advance our understanding of how molecular crowding and transient membrane interactions shape protein diffusion within cell-sized compartments, with possible implications for metabolic organization.

biophysics↗

bioRxiv2026

Accessible Gibbs energy at metabolic activation limits long-term cell growth

When exposed to a nutrient, cells activate metabolism by reorganizing metabolite pools and enzyme expression to approach the maximal growth rate permitted by physicochemical constraints. While these constraints define reachable steady states, here we propose that the Gibbs energy accessible at activation further limits which states are reached. Using minimal metabolic models, we find that limited accessible Gibbs energy can trap cells in low-growth states by constraining metabolic reorganization and imposing a proteomic burden on transport and phosphorylation reactions. To investigate this experimentally, we reconstituted the arginine deiminase pathway in vesicles, revealing that the size of a conserved pool of interconverting metabolites (arginine, citrulline, and ornithine) determines accessible Gibbs energy and constrains steady-state ATP production rate, a proxy for growth. Together, these results indicate that cellular metabolism retains memory of its initial energetic state, with accessible Gibbs energy at activation acting as a thermodynamic constraint on long-term growth.

systems biology↗

bioRxiv2025

Condition-dependent, amorphous protein agglomerates control cytoplasmic rheology

Molecular crowding in the bacterial cytoplasm restricts the diffusion of large molecules, impacting cellular processes. However, how nutrient availability influences cytoplasmic rheology is not well understood. With single-particle tracking in Escherichia coli, we observed a threefold variation in the diffusion of a 40-nm particle across exponential growth conditions. Previously suggested determinants of rheology did not account for this variation; instead, we found a strong anticorrelation between the diffusion coefficient and the abundance of amino acid metabolism proteins, persisting upon genetic perturbations and showing that lower diffusion is associated with increased viscoelasticity. Photoactivated light microscopy revealed that some amino acid metabolism proteins form clusters. Electron microscopy showed that these proteins could form amorphous agglomerates at physiological concentrations in vitro, likely driven by their low intrinsic disorder, high compactness and hydropathy score. These findings show that protein agglomerates regulate cytoplasmic rheology in a condition-dependent manner, suggesting an underappreciated level of cytoplasmic organization. HighlightsO_LIDiffusion of 40-nm particles varies threefold across growth conditions in E. coli C_LIO_LICytoplasmic diffusion inversely correlates with COG-E protein abundance COG-E proteins form agglomerates that increase cytoplasmic viscoelasticity C_LIO_LIProtein compactness and hydrophobicity predict condition-dependent crowding effects C_LI

biophysics↗

bioRxiv2023

Ergosterol mediates aggregation of natamycin in the yeast plasma membrane

Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability becomes compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and resulted in reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogs of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. We conclude that ergosterol-specific aggregation of natamycin in the yeast PM underlies its antifungal activity, which can be synergistically enhanced by inhibitors of sphingolipid synthesis. SignificanceErgosterol is the major sterol in the membranes of fungi and a major target for antifungal treatments. Polyene macrolides, such as natamycin, are known to target ergosterol but the underlying mechanisms for their preference for this yeast sterol compared to mammalian cholesterol is not understood. This study shows that natamycin forms aggregates when associated with yeast S. cerevisiae in an ergosterol-dependent manner. Cholesterol can only partially substitute for ergosterol with respect to natamycin binding and aggregation. Membrane-associated aggregation of natamycin is not the result of pre-formed sterol domains in the cell membrane, as we show by direct visualization of minimally modified ergosterol and cholesterol analogs. Inhibiting sphingolipid synthesis increased membrane association and antifungal activity of natamycin, suggesting that targeting sphingolipids in combination with polyene macrolides could lead to novel drug treatment approaches against fungal infections.

biophysics↗

bioRxiv2023

Fitness landscape of substrate-adaptive mutations in evolved APC transporters

The emergence of new protein functions is crucial for the evolution of organisms. This process has been extensively researched for soluble enzymes, but it is largely unexplored for membrane transporters, even though the ability to acquire new nutrients from a changing environment requires evolvability of transport functions. Here, we demonstrate the importance of environmental pressure in obtaining a new activity or altering a promiscuous activity in members of the Amino acid-Polyamine-organoCation (APC)-type yeast amino acid transporters family. We identify APC members that have broader substrate spectra than previously described. Using in vivo experimental evolution, we evolve two of these transporter genes, AGP1 and PUT4, towards new substrate specificities. Single mutations on these transporters are found to be sufficient for expanding the substrate range of the proteins, while retaining the capacity to transport all original substrates. Nonetheless, each adaptive mutation comes with a distinct effect on the fitness for each of the original substrates, illustrating a trade-off between the ancestral and evolved functions. Collectively, our findings reveal how substrate-adaptive mutations in membrane transporters contribute to fitness and provide insights into how organisms can use transporter evolution to explore new ecological niches.

biochemistry↗

bioRxiv2023

Probing the stability and interdomain interactions in the ABC transporter OpuA, using single-molecule optical tweezers

Transmembrane transporter proteins are essential for the maintenance of cellular homeostasis and, as such, are key drug targets. Many transmembrane transporter proteins are known to undergo large structural rearrangements during their functional cycles. Despite the large amount of detailed structural and functional data available for these systems, our understanding of their dynamics and therefore how they function is generally limited. We introduce an innovative approach which enables us to directly measure the dynamics and stabilities of inter-domain interactions of transmembrane proteins using optical tweezers. Focusing on the osmoregulatory ABC transporter OpuA from Lactococcus lactis, we examine the mechanical properties and potential interactions of its substrate-binding domains. Our measurements are performed in lipid nanodiscs, providing a native-mimicking environment for the transmembrane protein. The technique provides high spatial and temporal resolution and allows us to study the functionally-relevant motions and inter-domain interactions of individual transmembrane transporter proteins in real-time in a lipid bilayer.

biophysics↗

bioRxiv2023

The substrate-binding domains of the osmoregulatory ABC importer OpuA physically interact

Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs transiently interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that the physical interactions between SBDs and cooperativity in substrate delivery are part of the transport mechanism.

molecular biology↗

bioRxiv2023

Simulation-based Reconstructed Diffusion unveils the effect of aging on protein diffusion in Escherichia coli

We have developed Simulation-based Reconstructed Diffusion (SbRD) to determine diffusion coefficients corrected for confinement effects and for the bias introduced by two-dimensional models describing a three-dimensional motion. We validate the method on simulated diffusion data in three-dimensional cell-shaped compartments. We use SbRD, combined with a new cell detection method, to infer the diffusion coefficients of a set of native proteins in Escherichia coli. We observe slower diffusion at the cell poles than in the nucleoid region of exponentially growing cells. We find that this observation is independent of the presence of polysomes. Furthermore, we show that the newly formed pole of dividing cells exhibits a faster diffusion than the old one. We hypothesize that the observed slowdown at the cell poles is caused by the accumulation of aggregated or damaged proteins, and that the effect is asymmetric due to cell aging.

biophysics↗

bioRxiv2021

Membrane thickness, lipid phase and sterol type are determining factors in the permeability of membranes to small solutes

Cell membranes provide a selective semi-permeable barrier to the passive transport of molecules. This property differs greatly between organisms. While the cytoplasmic membrane of bacterial cells is highly permeable for weak acids and glycerol, yeasts can maintain large concentration gradients. Here we show that such differences can arise from the physical state of the plasma membrane. By combining stopped-flow kinetic measurements with molecular dynamics simulations, we performed a systematic analysis of the permeability through synthetic lipid membranes to obtain detailed molecular insight into the permeation mechanisms. While membrane thickness is an important parameter for the permeability through fluid membranes, the largest differences occur when the membranes transit from the liquid-disordered to liquid-ordered and/or to gel state. By comparing our results with in vivo measurements from yeast, we conclude that the yeast membrane exists in a highly ordered and rigid state, which is comparable to synthetic saturated DPPC-sterol membranes.

biochemistry↗

bioRxiv2021

Stability of ligand-induced protein conformation influences affinity in maltose-binding protein

Our understanding of what determines ligand affinity of proteins is poor, even with high-resolution structures available. Both the non-covalent ligand-protein interactions and the relative free energies of available conformations contribute to the affinity of a protein for a ligand. Distant, non-binding site residues can influence the ligand affinity by altering the free energy difference between a ligand-free and ligand-bound conformation. Our hypothesis is that when different ligands induce distinct ligand-bound conformations, it should be possible to tweak their affinities by changing the free energies of the available conformations. We tested this idea for the maltose-binding protein (MPB) from Escherichia coli. We used singlemolecule Forster resonance energy transfer (smFRET) to distinguish several unique ligand-bound conformations of MBP. We engineered mutations, distant from the binding site, to affect the stabilities of different ligand-bound conformations. We show that ligand affinity can indeed be altered in a conformation-dependent manner. Our studies provide a framework for the tuning of ligand affinity, apart from modifying binding site residues.

biochemistry↗

bioRxiv2020

Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid, and asparagine in Gram-positive bacteria. It features two extracytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from L. lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Forster-resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE-FRET to monitor protein-protein interactions and conformational states simultaneously. HIGHLIGHTSO_LIResolved crystal structure of tandem SBD1-2 of GlnPQ from Lactococcus lactis C_LIO_LIConformational states and ligand binding affinities of individual domains SBD1 and SBD2 are similar to tandem SBD1-2 C_LIO_LINo cooperative effects are seen for different ligands for SBDs in the tandem C_LIO_LIProof of concept experiments show that PIFE-FRET can monitor SBD conformations and protein-protein interaction simultaneously C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/423572v1_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@1fa36f7org.highwire.dtl.DTLVardef@2e973forg.highwire.dtl.DTLVardef@cbae51org.highwire.dtl.DTLVardef@2185cc_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Oxygen-independent chemogenetic protein tags for live-cell fluorescence microscopy

Fluorescent proteins enable targeted visualization of biomolecules in living cells, but their maturation is oxygen-dependent and they are susceptible to aggregation and/or suffer from poor photophysical properties. Organic fluorophores are oxygen-independent with superior photophysical properties, but targeting biomolecules in vivo is challenging. Here, we introduce two oxygen-independent chemogenetic protein (OICP) tags that impart fluorogenicity and fluorescence lifetime enhancement to bound organic dyes. We present a photo- and physicochemical characterization of thirty fluorophores interacting with two OICPs and conclude that aromatic planar structures bind with high specificity to the hydrophobic pockets of the proteins. The binding specificity of the tags and the superior photophysical properties of organic fluorophores enable microscopy of living bacterial and eukaryotic cells. The exchange of photobleached dye for unbleached fluorophore enables prolonged live-cell imaging. Our protein tags provide a general tool for investigating (sub)cellular protein localization and dynamics, protein-protein interactions, and microscopy applications under strictly oxygen-free conditions.

biochemistry↗

bioRxiv2020

Periprotein lipidomes of Saccharomyces cerevisiae provide a flexible environment for conformational changes of membrane proteins

Yeast tolerates a low pH and high solvent concentrations. The permeability of the plasma membrane (PM) for small molecules is low and lateral diffusion of proteins is slow. These findings suggest a high degree of lipid order, which raises the question of how membrane proteins function in such an environment. The yeast PM is segregated into the Micro-Compartment-of-Can1 (MCC) and Pma1 (MCP), which have different lipid compositions. We extracted proteins from these microdomains via stoichiometric capture of lipids and proteins in styrene-maleic-acid-lipid-particles (SMALPs). We purified SMALP-lipid-protein complexes by chromatography and quantitatively analyzed periprotein lipids located within the diameter defined by one SMALP. Phospholipid and sterol concentrations are similar for MCC and MCP, but sphingolipids are enriched in MCP. Ergosterol is depleted from this periprotein lipidome, whereas phosphatidylserine is enriched relative to the bulk of the plasma membrane. Direct detection of PM lipids in the periprotein space supports the conclusion that proteins function in the presence of a locally disordered lipid state.

biochemistry↗

bioRxiv2020

Membrane lipid requirements of the lysine transporter Lyp1 from Saccharomyces cerevisiae

Membrane lipids act as solvents and functional cofactors for integral membrane proteins. The yeast plasma membrane is unusual in that it may have a high lipid order, which coincides with low passive permeability for small molecules and a slow lateral diffusion of proteins. Yet, membrane proteins whose functions require altered conformation must have flexibility within membranes. We have determined the molecular composition of yeast plasma membrane lipids located within a defined diameter of model proteins, including the APC-superfamily lysine transporter Lyp1. We now use the composition of lipids that naturally surround Lyp1 to guide testing of lipids that support the normal functioning of the transporter, when reconstituted in vesicles of defined lipid composition. We find that phosphatidylserine and ergosterol are essential for Lyp1 function, and the transport activity displays a sigmoidal relationship with the concentration of these lipids. Non-bilayer lipids stimulate transport activity, but different types are interchangeable. Remarkably, Lyp1 requires a relatively high fraction of lipids with one or more unsaturated acyl chains. The transport data and predictions of the periprotein lipidome of Lyp1, support a new model in which a narrow band of lipids immediately surrounding the transmembrane stalk of a model protein allows conformational changes in the protein.

biochemistry↗

bioRxiv2020

Periprotein membrane lipidomics and the role of lipids in transporter function in yeast

The yeast plasma membrane is segregated into domains: the Micro-Compartment-of-Can1 (MCC) and Pma1 (MCP) have a different protein composition, but their lipid composition is largely unknown. We extracted proteins residing in these microdomains via stoichiometric capture of lipids and proteins in styrene-maleic-acid-lipid-particles (SMALPs). We purified SMALPs by affinity chromatography and quantitatively analyzed the lipids by mass spectrometry and their role in transporter function. We found that phospholipid and sterol concentrations are similar for MCC and MCP, but sphingolipids are enriched in MCP. Ergosterol is depleted from the periprotein lipidome, whereas phosphatidylserine is enriched relative to the bulk of the plasma membrane. Phosphatidylserine, non-bilayer lipids and ergosterol are essential for activity of Lyp1; the transporter also requires a balance of saturated/unsaturated fatty acids. We propose that proteins can function in the yeast plasma membrane by the disordered state of surrounded lipids and diffuse slowly in domains of high lipid order. Impact statementMembrane protein-specific lipidomics provides information on the organization of the yeast plasma membrane and the functioning of solute transporters

biochemistry↗

bioRxiv2019

A synthetic metabolic network for physicochemical homeostasis

One of the grand challenges in chemistry is the construction of functional out-of-equilibrium networks, which are typical of living cells. Building such a system from molecular components requires control over the formation and degradation of the interacting chemicals and homeostasis of the internal physical-chemical conditions. The provision and consumption of ATP lies at the heart of this challenge. We report the in vitro construction in vesicles of a pathway for sustained ATP production that is maintained away from equilibrium by control of energy dissipation. We maintain a constant level of ATP with varying load on the system. The pathway enables us to control the transmembrane fluxes of osmolytes and to demonstrate basic physicochemical homeostasis. Our work demonstrates metabolic energy conservation and cell volume regulatory mechanisms in a cell-like system at a level of complexity minimally needed for life.

synthetic biology↗

bioRxiv2019

Conformational and dynamical plasticity in substrate-binding proteins underlies selective transport in ABC importers

Substrate-binding proteins (SBPs) are associated with ATP-binding cassette importers and switch from an open-to a closed-conformation upon substrate binding providing specificity for transport. We investigated the effect of substrates on the conformational dynamics of six SBPs and the impact on transport. Using single-molecule FRET, we reveal an unrecognized diversity of plasticity in SBPs. We show that a unique closed SBP conformation does not exist for transported substrates. Instead, SBPs sample a range of conformations that activate transport. Certain non-transported ligands leave the structure largely unaltered or trigger a conformation distinct from that of transported substrates. Intriguingly, in some cases similar SBP conformations are formed by both transported and non-transported ligands. In this case, the inability for transport arises from slow opening of the SBP or the selectivity provided by the translocator. Our results reveal the complex interplay between ligand-SBP interactions, SBP conformational dynamics and substrate transport.

biophysics↗

bioRxiv2017

Growth inhibition by amino acids in Saccharomyces cerevisiae

Amino acids are essential metabolites but can also be toxic when present at high levels intracellularly. Substrate-induced down-regulation of amino acid transporters in Saccharomyces cerevisiae is thought to be a mechanism to avoid this toxicity. It has been shown that unregulated uptake by the general amino acid permease Gap1 causes cells to become sensitive to amino acids. Here, we show that overexpression of eight other amino acid transporters (Agp1, Bap2, Can1, Dip5, Gnp1, Lyp1, Put4 or Tat2) also induces a growth defect when specific single amino acids are present at concentrations of 0.5-5 mM. We can now state that all proteinogenic amino acids, as well as the important metabolite ornithine, are growth inhibitory to S. cerevisiae when transported into the cell at high enough levels. Measurements of initial transport rates and cytosolic pH show that toxicity is due to amino acid accumulation and not to the influx of co-transported protons. The amino acid sensitivity phenotype is a useful tool that reports on the in vivo activity of transporters and has allowed us to identify new transporter-specific substrates.

cell biology↗