Biomedical subjects

Ben de Kruijff

Publications and source records attributed to Ben de Kruijff.

At least 55 records · Page 3Linked to original sources

PubMed2003

NMR study of mersacidin and lipid II interaction in dodecylphosphocholine micelles. Conformational changes are a key to antimicrobial activity.

Mersacidin belongs to the type B lantibiotics (lanthionine-containing antibiotics) that contain post-translationally modified amino acids and cyclic ring structures. It targets the cell wall precursor lipid II and thereby inhibits cell wall synthesis. In light of the emerging antibiotics resistance problem, the understanding of the antibacterial activity on a structural basis provides a key to circumvent this issue. Here we present solution NMR studies of mersacidin-lipid II interaction in dodecylphosphocholine (DPC) micelles. Distinct solution structures of mersacidin were determined in three different states: in water/methanol solution and in DPC micelles with and without lipid II. The structures in various sample conditions reveal remarkable conformational changes in which the junction between Ala-12 and Abu-13 (where Abu is aminobutyric acid) effectively serves as the hinge for the opening and closure of the ring structures. The DPC micelle-bound form resembles the previously determined NMR and x-ray crystal structures of mersacidin in pure methanol but substantially deviates from the other two states in our current report. The structural changes delineate the large chemical shift perturbations observed during the course of a two-step (15)N-(1)H heteronuclear single quantum coherence titration. They also modulate the surface charge distribution of mersacidin suggesting that electrostatics play a central role in the mersacidin-lipid II interaction. The observed conformational adaptability of mersacidin might be a general feature of lipid II-interacting antibiotics/peptides.

Amino Acid Sequence↗

PubMed2003

Continuous equilibration of phosphatidylcholine and its precursors between endoplasmic reticulum and mitochondria in yeast.

In Saccharomyces cerevisiae phosphatidylcholine (PC) is synthesized in the ER and transported to mitochondria via an unknown mechanism. The transport of PC synthesized by the triple methylation of phosphatidylethanolamine was investigated by pulsing yeast spheroplasts with l-[methyl-3H]methionine, followed by a chase with unlabeled methionine and subcellular fractionation. During the pulse, increasing amounts of PC and its mono- and dimethylated precursors (PMME and PDME, respectively) appear in similar proportions in both microsomes and mitochondria, with the extent of incorporation in microsomes being twice that in mitochondria. During the chase, the [3H]-methyl label from the precursors accumulates into PC with similar kinetics in both organelles. The results demonstrate that transport of methylated phospholipids from ER to mitochondria is 1) coupled to synthesis, 2) not selective for PC, 3) at least as fast as the fastest step in the methylation of PE, and 4) bidirectional for PMME and PDME. The interorganellar equilibration of methylated phospholipids was reconstituted in vitro and did not depend on ongoing methylation, cytosolic factors, ATP, and energization of the mitochondria, although energization could accelerate the reaction. The exchange of methylated phospholipids was reduced after pretreating both microsomes and mitochondria with trypsin, indicating the involvement of membrane proteins from both organelles.

Endoplasmic Reticulum↗

PubMed2003

Phospholipid flop induced by transmembrane peptides in model membranes is modulated by lipid composition.

Since phospholipid synthesis is generally confined to one leaflet of a membrane, membrane growth requires phospholipid translocation (flip-flop). It is generally assumed that this process is protein-mediated; however, the mechanism of flip-flop remains elusive. Previously, we have demonstrated flop of 2-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]caproyl] (C6NBD) phospholipids, induced by the presence of membrane-spanning peptides in vesicles composed of an Escherichia coli phospholipid extract, supporting the hypothesis that the presence of transmembrane stretches of proteins in the bilayer is sufficient to allow phospholipid flip-flop in the inner membrane of E. coli [Kol et al. (2001) Biochemistry 40, 10500]. Here, we investigated whether the specific phospholipid composition of E. coli is a prerequisite for transmembrane helix-induced flop of phospholipids. This was tested by determining the amount of C6NBD-phospholipid that was translocated from the inner leaflet to the outer leaflet of a model membrane in time, using a dithionite reduction assay. The transmembrane peptides GWWL(AL)8WWA (WALP23) and GKKL(AL)8KKA (KALP23) induced phospholipid flop in model membranes composed of various lipid mixtures. The rate of peptide-induced flop was found to decrease with increasing dioleoylphosphatidylethanolamine (DOPE) content of vesicles composed of DOPE and dioleoylphosphatidylcholine (DOPC), and the rate of KALP23-induced flop was shown to be stimulated by higher dioleoylphosphatidylglycerol (DOPG) content in model membranes composed of DOPG and DOPC. Furthermore, the incorporation of cholesterol had an inhibitory effect on peptide-induced flop. Finally, flop efficiency was strongly dependent on the phospholipid headgroup of the NBD-phospholipid analogue. Possible implications for transmembrane helix-induced flop in biomembranes in general are discussed.

4-Chloro-7-nitrobenzofurazan↗

PubMed2003

Crystal structure of SecB from Escherichia coli.

The chaperone SecB from Escherichia coli is primarily involved in passing precursor proteins into the Sec system via specific interactions with SecA. The crystal structure of SecB from E. coli has been solved to 2.35 A resolution. The structure shows flexibility in the crossover loop and the helix-connecting loop, regions that have been implicated to be part of the SecB substrate-binding site. Moreover conformational variability of Trp36 is observed as well as different loop conformations for the different monomers. Based on this, we speculate that SecB can regulate the access or extent of its hydrophobic substrate-binding site, by modulating the conformation of the crossover loop and the helix-connecting loop. The structure also clearly explains why the tetrameric equilibrium is shifted towards the dimeric state in the mutant SecBCys76Tyr. The buried cysteine residue is crucial for tight packing, and mutations are likely to disrupt the tetramer formation but not the dimer formation.

Bacterial Proteins↗

PubMed2003

Structural requirements of the fructan-lipid interaction.

Fructans are a group of fructose-based oligo- and polysaccharides. They are proposed to be involved in membrane protection of plants during dehydration. In accordance with this hypothesis, they show an interaction with hydrated lipid model systems. However, the structural requirements for this interaction are not known both with respect to the fructans as to the lipids. To get insight into this matter, the interaction of several inulins and levan with lipids was investigated using a monomolecular lipid system or the MC 540 probe in a bilayer system. MD was used to get conformational information concerning the polysaccharides. It was found that levan-type fructan interacted comparably with model membranes composed of glyco- or phospholipids but showed a preference for lipids with a small headgroup. Furthermore, it was found that there was an inulin chain-length-dependent interaction with lipids. The results also suggested that inulin-type fructan had a more profound interaction with the membrane than levan-type fructan. MD simulations indicated that the favorable conformation for levan is a helix, whereas inulin tends to form random coil structures. This suggests that flexibility is an important determinant for the fructan-lipid interaction.

Computer Simulation↗

PubMed2003

The effect of fructan on the phospholipid organization in the dry state.

Fructans, a family of oligo- and polyfructoses, are implicated to play a drought-protecting role in plants. Inulin-type fructan is able to preserve the membrane barrier during dehydration. However, whether other fructans would be able to perform this function is unknown. In addition, almost nothing is known about the organization of these systems, which could give insight into the protective mechanism. To get insight into these questions the effect of different fructans on phosphatidylcholine-based model systems under conditions of dehydration was analyzed. Using a vesicle leakage assay, it was found that both levan- and inulin-type fructans protected the membrane barrier. This suggests that fructans in general would be able to protect the membrane barrier function. Furthermore, both fructan-types inhibited vesicle fusion to a large extent as measured using a lipid-mixing assay. Using x-ray diffraction, it was found that in the presence of both inulin- and levan-type fructans the lamellar repeat distance increased considerably. From this it was concluded that fructans are present between the lipid bilayers during drying. Furthermore, they stabilize the L(alpha) phase. In contrast to fructans, dextran did not increase the lamellar repeat distance and it even promoted L(beta) phase formation. These data support the hypothesis that fructans can have a membrane-protecting role during dehydration, and give insight into the mechanism of protection.

Desiccation↗

PubMed2003

The effect of fructan on membrane lipid organization and dynamics in the dry state.

Fructans are a group of fructose-based oligo- and polysaccharides, which appear to be involved in membrane preservation during dehydration by interacting with the membrane lipids. To get further understanding of the protective mechanism, the consequences of the fructan-membrane lipid interaction for the molecular organization and dynamics in the dry state were studied. POPC and DMPC were investigated in the dry state by (2)H, (31)P NMR, and Fourier transform infrared spectroscopy using two types of fructan and dextran. The order-disorder transition temperature of dry POPC was reduced by 70 degrees C in the presence of fructan. Fructan increased the mobility of the acyl chains, but immobilized the lipid headgroup region. Most likely, fructans insert between the headgroups of lipids, thereby spacing the acyl chains. This results in a much lower phase transition temperature. The headgroup is immobilized by the interaction with fructan. The location of the interaction with the lipid headgroup is different for the inulin-type fructan compared to the levan-type fructan, since inulin shows interaction with the lipid phosphate group, whereas levan does not. Dextran did not influence the phase transition temperature of dry POPC showing that reduction of this temperature is not a general property of polysaccharides.

Dextrans↗

PubMed2003

Modulation of membrane curvature by phosphatidic acid and lysophosphatidic acid.

The local generation of phosphatidic acid plays a key role in the regulation of intracellular membrane transport through mechanisms which are largely unknown. Phosphatidic acid may recruit and activate downstream effectors, or change the biophysical properties of the membrane and directly induce membrane bending and/or destabilization. To evaluate these possibilities, we determined the phase properties of phosphatidic acid and lysophosphatidic acid at physiological conditions of pH and ion concentrations. In single-lipid systems, unsaturated phosphatidic acid behaved as a cylindrical, bilayer-preferring lipid at cytosolic conditions (37 degrees C, pH 7.2, 0.5 mM free Mg2+), but acquired a type-II shape at typical intra-Golgi conditions, a mildly acidic pH and submillimolar free Ca2+ (pH 6.6-5.9, 0.3 mM Ca2+). Lysophosphatidic acid formed type-I lipid micelles in the absence of divalent cations, but anhydrous cation-lysophosphatidic acid bilayer complexes in their presence. These data suggest a similar molecular shape for phosphatidic acid and lysophosphatidic acid at cytosolic conditions; however, experiments in mixed-lipid systems indicate that their shape is not identical. Lysophosphatidic acid stabilized the bilayer phase of unsaturated phosphatidylethanolamine, while the opposite effect was observed in the presence of phosphatidic acid. These results support the hypothesis that a conversion of lysophosphatidic acid into phosphatidic acid by endophilin or BARS (50 kDa brefeldin A ribosylated substrate) may induce negative spontaneous monolayer curvature and regulate endocytic and Golgi membrane fission. Alternative models for the regulation of membrane fission based on the strong dependence of the molecular shape of (lyso)phosphatidic acid on pH and divalent cations are also discussed.

Calcium Chloride↗

PubMed2003

Membrane interaction of the glycosyltransferase MurG: a special role for cardiolipin.

MurG is a peripheral membrane protein that is one of the key enzymes in peptidoglycan biosynthesis. The crystal structure of Escherichia coli MurG (S. Ha, D. Walker, Y. Shi, and S. Walker, Protein Sci. 9:1045-1052, 2000) contains a hydrophobic patch surrounded by basic residues that may represent a membrane association site. To allow investigation of the membrane interaction of MurG on a molecular level, we expressed and purified MurG from E. coli in the absence of detergent. Surprisingly, we found that lipid vesicles copurify with MurG. Freeze fracture electron microscopy of whole cells and lysates suggested that these vesicles are derived from vesicular intracellular membranes that are formed during overexpression. This is the first study which shows that overexpression of a peripheral membrane protein results in formation of additional membranes within the cell. The cardiolipin content of cells overexpressing MurG was increased from 1 +/- 1 to 7 +/- 1 mol% compared to nonoverexpressing cells. The lipids that copurify with MurG were even further enriched in cardiolipin (13 +/- 4 mol%). MurG activity measurements of lipid I, its natural substrate, incorporated in pure lipid vesicles showed that the MurG activity is higher for vesicles containing cardiolipin than for vesicles with phosphatidylglycerol. These findings support the suggestion that MurG interacts with phospholipids of the bacterial membrane. In addition, the results show a special role for cardiolipin in the MurG-membrane interaction.

Bacterial Outer Membrane Proteins↗

PubMed2002

Characterization and membrane assembly of the TatA component of the Escherichia coli twin-arginine protein transport system.

Proteins bearing a signal peptide with a consensus twin-arginine motif are translocated via the Tat pathway, a multiprotein system consisting minimally of the integral inner membrane proteins TatA, TatB, and TatC. On a molar basis, TatA is the major pathway component. Here we show that TatA can be purified independently of the other Tat proteins as a 460 kDa homooligomeric complex. Homooligomer formation requires the amino-terminal membrane-anchoring domain of TatA. According to circular dichroism spectroscopy, approximately half of the TatA polypeptide forms alpha-helical secondary structure in both detergent solution and proteoliposomes. An expressed construct without the transmembrane segment is largely unstructured in aqueous solution but is able to insert into phospholipid monolayers and interacts with membrane bilayers. Protease accessibility experiments indicate that the extramembranous region of TatA is located at the cytoplasmic face of the cell membrane.

Cell Membrane↗

PubMed2002

Lipid II induces a transmembrane orientation of the pore-forming peptide lantibiotic nisin.

Nisin is an antimicrobial peptide produced by Lactococcus lactis and used as a food preservative in dairy products. The peptide kills Gram-positive bacteria via the permeabilization of the membrane, most probably via pore formation using the cell wall precursor Lipid II as its docking molecule. In this study, site-directed tryptophan spectroscopy was used to determine the topology of nisin in the Lipid II containing membrane, as a start to elucidate the mechanism of targeted pore formation. Three single tryptophan mutants were used, which are viable representatives of the wild-type peptide. The emission spectra of tryptophans located at the N-terminus, the center, and the C-terminus as well as quenching by acrylamide and spin-labeled lipids were investigated using model membrane vesicles composed of DOPC containing 1 mol % Lipid II. Nisin was shown to adopt an orientation where the most probable position of the N-terminus was found to be near the Lipid II headgroup at the bilayer surface, the position of the center of nisin was in the middle of the phospholipid bilayer, and the C-terminus was located near the interface between the headgroups and acyl chain region. These results were used to propose a model for the orientation of nisin in Lipid II containing membranes. Our findings demonstrated that Lipid II changes the overall orientation of nisin in membranes from parallel to perpendicular with respect to the membrane surface. The stable transmembrane orientation of nisin in the presence of Lipid II might allow us to determine the structure of the nisin-Lipid II pores in the lipid bilayer.

Acrylamide↗

PubMed2002

Monte Carlo simulations of voltage-driven translocation of a signal sequence.

Transmembrane potentials play important but poorly understood roles in many biological processes, including signal sequence-mediated protein translocation across bacterial membranes. In this study we applied Monte Carlo techniques to simulate the way the potential acts on a signal sequence. The simulations demonstrate that in the absence of a potential the signal sequence prefers insertion in both helical hairpin and transmembrane alpha-helical conformations. However, in the presence of a potential only the transmembrane alpha-helical conformation is the state of lowest energy for the signal sequence. From these results it is concluded that the membrane potential stabilizes the transmembrane orientation of a signal sequence, explaining the membrane potential dependence of preprotein translocation.

Amino Acid Sequence↗

PubMed2002

Influence of lipids on membrane assembly and stability of the potassium channel KcsA.

Recently we observed in an in vitro system that newly synthesized KcsA assembles efficiently into a tetramer in lipid vesicles [van Dalen et al. (2002) FEBS Lett. 511, 51-58]. Here we used this system to get insight into the importance of the lipid composition for KcsA membrane association and tetramerization and we compared this to the lipid dependency of the thermo-stability of the KcsA tetramer. It was found that a large amount of phosphatidylethanolamine (>40 mol%) and a lower amount of phosphatidylglycerol (approximately 20-30 mol%) were optimal for efficient KcsA membrane association and tetramerization. Strikingly, vesicles of the abundant and commonly used membrane lipid phosphatidylcholine did not support assembly, further demonstrating the importance of membrane lipid composition for KcsA assembly. The in vitro assembled KcsA tetramer showed similar thermo-stability in biological and pure lipid membranes, demonstrating that both tetramers are alike. In addition, we show that solubilization of the membrane with detergent reduces the thermo-stability of the tetramer. The highest KcsA tetramer stability was observed in intact bilayers in the presence of anionic lipids.

Bacterial Proteins↗

PubMed2002

Influence of hydrophobic mismatch and palmitoylation on the association of transmembrane alpha-helical peptides with detergent-resistant membranes.

The aim of this study was to gain insight into the mechanism through which transmembrane proteins are targeted to liquid ordered (L(o)) phase domains or rafts. This was investigated by analyzing the Triton X-100 resistance of designed transmembrane peptides in model membranes of 1,2-dioleoyl-sn-glycero-3-phosphocholine, sphingomyelin and cholesterol (1/1/1, molar ratio), which contain both L(o) phase domains and fluid bilayers. By using peptides with one or two palmitate chains covalently linked to their N-terminus or with variable hydrophobic lengths, the roles of protein palmitoylation and of mismatch between the transmembrane segment of the protein and the bilayer thickness, respectively, were investigated. The results show that neither hydrophobic matching nor palmitoylation is sufficient for partitioning of peptides into L(o) phase domains. It is concluded that the L(o) phase itself, due to the tight packing of the lipids, constitutes an unfavorable environment for accommodation of protein transmembrane segments.

Binding Sites↗

PubMed2002

Aerobic sn-glycerol-3-phosphate dehydrogenase from Escherichia coli binds to the cytoplasmic membrane through an amphipathic alpha-helix.

sn-Glycerol-3-phosphate dehydrogenase (GlpD) from Escherichia coli is a peripheral membrane enzyme involved in respiratory electron transfer. For it to display its enzymic activity, binding to the inner membrane is required. The way the enzyme interacts with the membrane and how this controls activity has not been elucidated. In the present study we provide evidence for direct protein-lipid interaction. Using the monolayer technique, we observed insertion of GlpD into lipid monolayers with a clear preference for anionic phospholipids. GlpD variants with point mutations in their predicted amphipathic helices showed a decreased ability to penetrate anionic phospholipid monolayers. From these data we propose that membrane binding of GlpD occurs by insertion of an amphipathic helix into the acyl-chain region of lipids mediated by negatively charged phospholipids.

Base Sequence↗

PubMed2002

Clavanin permeabilizes target membranes via two distinctly different pH-dependent mechanisms.

The pH dependence of the antimicrobial and membrane activity of clavanin A, a peptide antibiotic that is rich in histidines and glycines, was analyzed in growth and membrane leakage experiments. Clavanin A more effectively inhibited the growth of the test organism Lactobacillus sake when the pH of the medium was lowered. Whereas the wild-type peptide efficiently released fluorophores from unilamellar vesicles at neutral pH according to a nonspecific permeabilization mechanism, it did not permeabilize model bilayers at low pH. It was therefore suggested that this peptide uses a distinct mode of action under acidic conditions different than that used around neutral pH. However, at low pH, the membrane is still the target for clavanin A, as the peptide collapsed both vital transmembrane proton gradients and ion gradients under these conditions. Clavanin A did not act as a ionophore across phospholipid bilayers, indicating that membrane constituents other than membrane phospholipids are involved in the dissipation of transmembrane ion gradients. Membrane proteins that generate transmembrane ion gradients are suggested to be the targets for clavanin A at low pH. In addition to the histidines, the three glycine residues of clavanin A are shown to play an important role in the specific mode of interaction with these membrane targets. These residues may induce a flexible hydrophobic conformation that allows the peptide to exert different membrane activities. This study demonstrates that clavanin A is a special membrane-active peptide that has access to two markedly distinct pH-dependent modes of actions.

Amino Acid Sequence↗

PubMed2002

Mapping the targeted membrane pore formation mechanism by solution NMR: the nisin Z and lipid II interaction in SDS micelles.

Nisin is an example of type-A lantibiotics that contain cyclic lanthionine rings and unusual dehydrated amino acids. Among the numerous pore-forming antimicrobial peptides, type-A lantibiotics form an unique family of post-translationally modified peptides. Via the recognition of cell wall precursor lipid II, nisin has the capacity to form pores against Gram-positive bacteria with an extremely high activity in the nanomolar (nM) range. Here we report a high-resolution NMR spectroscopy study of nisin/lipid II interactions in SDS micelles as a model membrane system in order to elucidate the mechanism of molecular recognition at residue level. The binding to lipid II was studied through (15)N-(1)H HSQC titration, backbone amide proton temperature coefficient analysis, and heteronuclear (15)N[(1)H]-NOE relaxation dynamics experiments. Upon the addition of lipid II, significant changes were monitored in the N-terminal part of nisin. An extremely low amide proton temperature coefficient (Delta delta/Delta T) was found for the amide proton of Ala3 (> -0.1 ppb/K) in the complex form. This suggests tight hydrogen bonding and/or isolation from the bulk solvent for this residue. Large chemical shift perturbations were also observed in the first two rings. In contrast, the C-terminal part of nisin was almost unaffected. This part of the molecule remains flexible and solvent-exposed. On the basis of our results, a multistep pore-forming mechanism is proposed. The N-terminal part of nisin first binds to lipid II, and a subsequent structural rearrangement takes place. The C-terminal part of nisin is possibly responsible for the activation of the pore formation. In light of the emerging antibiotic resistance problems, an understanding of the specific recognition mechanism of nisin with lipid II at the residue specific level may therefore aid in the development of novel antibiotics.

Amides↗

PubMed2002

Photolabeling identifies an interaction between phosphatidylcholine and glycerol-3-phosphate dehydrogenase (Gut2p) in yeast mitochondria.

In search of mitochondrial proteins interacting with phosphatidylcholine (PC), a photolabeling approach was applied, in which photoactivatable probes were incorporated into isolated yeast mitochondria. Only a limited number of proteins were labeled upon photoactivation, using either the PC analogue [125I]TID-PC or the small hydrophobic probe [125I]TID-BE. The most prominent difference was the very specific labeling of a 70 kDa protein by [125I]TID-PC. Mass spectrometric analysis of a tryptic digest of the corresponding 2D-gel spot identified the protein as the GUT2 gene product, the FAD-dependent mitochondrial glycerol-3-phosphate dehydrogenase. This was confirmed by the lack of specific labeling in mitochondria from a gut2 deletion strain. Only under conditions where the inner membrane was accessible to the probe, Gut2p was labeled by [125I]TID-PC, in parallel with increased labeling of the phosphate carrier (P(i)C) in the inner membrane. A hemagglutinin-tagged version of Gut2p was shown to be membrane-bound. Carbonate extraction released the protein from the membrane, whereas a high concentration of NaCl did not, demonstrating that Gut2p is a peripheral membrane protein bound to the inner membrane via hydrophobic interactions. The significance of the observed interactions between Gut2p and PC is discussed.

Amino Acid Sequence↗