Bert Poolman

University of Groningen | RUG · Department of Biochemistry

The human pathogen Listeria monocytogenes can cope with severe environmental challenges, for which the high molecular weight stressosome complex acts as the sensing hub in a complicated signal transduction pathway. Here, we show the dynamics and functional roles of the stressosome protein RsbR1 and its paralogue, the blue-light receptor RsbL, using...

The passive permeability of cell membranes is of key importance in biology, biomedical research and biotechnology as it determines the extent to which various molecules such as drugs, products of metabolism, and toxins can enter or leave the cell unaided by dedicated transport proteins. The quantification of passive solute permeation is possible wi...

We analyze the structure of the cytoplasm by performing single-molecule displacement mapping on a diverse set of native cytoplasmic proteins in exponentially growing Escherichia coli. We evaluate the method for application in small compartments and find that confining effects of the cell membrane affect the diffusion maps. Our analysis reveals that...

The human pathogen Listeria monocytogenes is a model microorganism in infection biology. The organism can transform from a saprophyte to an intracellular pathogen and cope with severe environmental challenges, for which the high molecular weight stressosome complex acts as the sensing hub in a complex signal transduction pathway. Yet, little is kno...

Natamycin is a polyene macrolide, widely employed to treat fungal keratitis and other yeast infections as well as to protect food products against fungal molds. In contrast to other polyene macrolides, such as nystatin or amphotericin B, natamycin does not form pores in yeast membranes, and its mode of action is not well understood. Here, we have e...

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...

Effective metabolic pathways are essential for the construction of in vitro systems mimicking the biochemical complexity of living cells. Such pathways require the inclusion of a metabolic branch that ensures the availability of reducing equivalents. Here, we built a minimal enzymatic pathway confinable in the lumen of liposomes, in which the redox...

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...

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...

Many proteins have a multimeric structure and are composed of two or more identical subunits. While this can be advantageous for the host organism, it can be a challenge when targeting specific residues in biochemical analyses. In vitro splitting and re-dimerization to circumvent this problem is a tedious process that requires stable proteins. We p...

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 affini...

Fluorogenic protein tagging systems have been less developed for prokaryotes than for eukaryotic cell systems. Here, we extend the concept of noncovalent fluorogenic protein tags in bacteria by introducing transcription factor-based tags, namely, LmrR and RamR, for probe binding and fluorescence readout under aerobic and anaerobic conditions. We de...

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 extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, bi...

Obtaining (dynamic) structure related information on proteins is key for understanding their function. Methods as single-molecule Förster Resonance Energy Transfer (smFRET) and Electron Paramagnetic Resonance (EPR) that measure distances between labeled residues to obtain dynamic information rely on selection of suitable residue pairs for chemical...

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 affini...

Protein mobility in the cytoplasm is essential for cellular functions, and slow diffusion may limit the rates of biochemical reactions in the living cell. Here, we determined the apparent lateral diffusion coefficient (D L ) of GFP in Listeria monocytogenes as a function of osmotic stress, temperature, and media composition. We find that D L is muc...

Fluorescence-based sensors play a fundamental role in biological research. These sensors can be based on fluorescent proteins, fluorescent probes or they can be hybrid systems. The availability of a very large dataset of fluorescent molecules, both genetically encoded and synthetically produced, together with the structural insights on many sensing...

Amino acids are essential metabolites but can also be toxic when present at high levels intracellularly. Substrate-induced downregulation 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...

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, bi...

Proton coupled transport of α-glucosides via Mal11 into Saccharomyces cerevisiae costs one ATP per imported molecule. Targeted mutation of all three acidic residues in the active site resulted in sugar uniport, but expression of these mutant transporters in yeast did not enable growth on sucrose. We then isolated six unique transporter variants of...

(Micro)organisms are exposed to fluctuating environmental conditions, and adaptation to stress is essential for survival. Increased osmolality (hypertonicity) causes outflow of water and loss of turgor and is dangerous if the cell is not capable of rapidly restoring its volume. The osmoregulatory adenosine triphosphate–binding cassette transporter...

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....

Prokaryotic ATP-binding cassette (ABC) importers require a substrate-binding protein (SBP) for the capture and delivery of the cognate substrate to the transmembrane domain (TMD) of the transporter. Various biochemical compounds have been identified that bind to the SBP but are not transported. The mechanistic basis for the "non-cognate" substrates...

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 hav...

The structure of a trifunctional linker is shown. The linker contains a fluorescent dye (green), two palmitoyl moieties (light blue) and is covalently coupled to a protein (schematically shown in dark blue). The tripartite is used to study the effect palmitoylation on the localization of membrane proteins or peptides in giant‐unilamellar vesicles (...

In cells the breakdown of arginine to ornithine, ammonium ion plus carbon dioxide is coupled to the generation of metabolic energy in the form of ATP. The arginine breakdown pathway is minimally composed of arginine deiminase, ornithine transcarbamoylase, carbamate kinase and an arginine/ornithine antiporter; ammonia and carbon dioxide most likely...

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 Micr...

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 Micr...

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 Micr...

The permeability of the plasma membrane (PM) of yeast 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 MicroCompartment-of-Can1 (MCC) and Pma1 (MCP), which...

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 hav...

Yeast amino acid transporters of the APC superfamily are responsible for the proton motive force-driven uptake of amino acids into the cell, which for most secondary transporters is a reversible process. The l-lysine proton symporter Lyp1 of Saccharomyces cerevisiae is special in that the Michaelis constant from out-to-in transport ( K m out → i...

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 puri...

We modeled the relaxation dynamics of (lipid) vesicles upon osmotic upshift, taking into account volume variation, chemical reaction kinetics, and passive transport across the membrane. We focused on the relaxation kinetics upon addition of impermeable osmolytes such as KCl and membrane-permeable solutes such as weak acids. We studied the effect of...

Attachment of lipophilic groups is an important post‐translational modification of proteins, which involves the coupling of one or more anchors such as fatty acids, isoprenoids, phospholipids or glycosylphosphatidyl inositols. To study its impact on the membrane partitioning of hydrophobic peptides or proteins, we designed a tyrosine‐based trifunct...

Background A central theme in (micro)biology is understanding the molecular basis of fitness i.e. which strategies are successful under which conditions; how do organisms implement such strategies at the molecular level; and which constraints shape the trade-offs between alternative strategies. Highly standardized microbial laboratory evolution exp...

The nuclear pore complex (NPC) is embedded in the nuclear envelope and forms the main gateway to the nuclear interior including the inner nuclear membrane (INM). Two INM proteins in yeast are selectively imported. Their sorting signals consist of a nuclear localization signal, separated from the transmembrane domain by a long intrinsically disorder...

We present a fluorescence-based approach for determination of the permeability of small molecules across the membranes of lipid vesicles and living cells. With properly designed experiments, the method allows us to assess the membrane physical properties both in vitro and in vivo. We find that the permeability of weak acids increases in the order o...

We review the mechanisms responsible for amino acid homeostasis in Saccharomyces cerevisiae and other fungi. Amino acid homeostasis is essential for cell growth and survival. Hence, the de novo synthesis reactions, metabolic conversions, and transport of amino acids are tightly regulated. Regulation varies from nitrogen pool sensing to control by i...

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 c...

The bottom-up construction of synthetic cells from molecular components is arguably one of the most challenging areas of research in the life sciences. We review the impact of confining biological systems in synthetic vesicles. Complex cell-like systems require control of the internal pH, ionic strength, (macro)molecular crowding, redox state and m...

Secondary active transporters are fundamental to a myriad of biological processes. They use the electrochemical gradient of one solute to drive transport of another solute against its concentration gradient. Central to this mechanism is that the transport of one does not occur in the absence of the other. However, like in most of biology, imperfect...

We aim for a blue print for synthesizing complex subcellular systems from molecular components and ultimately for constructing life. Without comprehensive instructions and design principles we rely on simple reaction routes to operate the essential functions of life. The first forms of synthetic life will not make every building block for polymers...

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 c...

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 u...

Bacteria adapt to ever-changing environmental conditions such as osmotic stress and energy limitation. It is not well understood how biomolecules reorganize themselves inside Escherichia coli under these conditions. An altered biochemical organization would affect macromolecular crowding, which could influence reaction rates and diffusion of macrom...

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 u...

That diffusion is important for the proper functioning of cells is without question. The extent to which the diffusion coefficient is important is explored here for prokaryotic cells. We discuss the principles of diffusion focusing on diffusion-limited reactions, summarize the known values for diffusion coefficients in prokaryotes and in in vitro m...

Super-resolution imaging and single-particle tracking require cells to be immobile as any movement reduces the resolution of the measurements. Here, we present a method based on APTES-glutaraldehyde coating of glass surfaces to immobilize cells without compromising their growth. Our method of immobilization is compatible with Saccharomyces cerevisi...

Great progress has been made in elucidating the structural and mechanistic basis of (membrane) protein production. Here, we attempt to look ahead and indicate four directions in which our understanding of the protein production process can grow: (i) determine how the molecular mechanisms influence higher-level processes, such as the distribution of...

Förster resonance energy transfer (FRET)-based sensors are a valuable tool to quantify cell biology, yet identifying and preventing potential artifacts are needed to exploit their full potential. We show here that artifacts arising from slow donor mCerulean3 maturation can be substantially diminished by constitutive expression both in prokaryotic a...

Niosomes are used in studies for drug delivery or gene transfer. However, their physical properties and features relative to liposomes are not well documented. To characterize and more rationally optimize niosome formulations, the properties of these vesicle systems are compared to those of liposomes composed of phosphatidylcholine and phosphatidyl...

Encapsulation efficiency of the various vesicle types. Encapsulation efficiency was determined by calcein fluorescence as described by [1]. Briefly, vesicles were formed and subjected to five freeze-thaw cycles before extrusion (blue bars) or extruded without the freeze-thaw step (black bars). Vesicles were diluted 1000x and fluorescence was measur...