Biomedical subjects

Patrick Van Gelder

Publications and source records attributed to Patrick Van Gelder.

10 recordsLinked to original sources

PubMed2006

Structure of a membrane-based steric chaperone in complex with its lipase substrate.

Secretion via the type II secretion pathway in Gram-negative bacteria often relies crucially on steric chaperones in the periplasm. Here, we report the crystal structure of the soluble form of a lipase-specific foldase (Lif) from Burkholderia glumae in complex with its cognate lipase. The structure reveals how Lif uses a novel alpha-helical scaffold to embrace lipase, thereby creating an unusually extensive folding platform.

Bacterial Proteins↗

PubMed2006

Assembly factor Omp85 recognizes its outer membrane protein substrates by a species-specific C-terminal motif.

Integral beta-barrel proteins are found in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. The assembly of these proteins requires a proteinaceous apparatus of which Omp85 is an evolutionary conserved central component. To study its molecular mechanism, we have produced Omp85 from Escherichia coli in inclusion bodies and refolded it in vitro. The interaction of Omp85 with its substrate proteins was studied in lipid-bilayer experiments, where it formed channels. The properties of these channels were affected upon addition of unfolded outer-membrane proteins (OMPs) or synthetic peptides corresponding to their C-terminal signature sequences. The interaction exhibited species specificity, explaining the inefficient assembly of OMPs from Neisseria in E. coli. Accordingly, the in vivo assembly of the neisserial porin PorA into the E. coli outer membrane was accomplished after adapting its signature sequence. These results demonstrate that the Omp85 assembly machinery recognizes OMPs by virtue of their C-terminal signature sequence.

Amino Acid Motifs↗

PubMed2005

Crystallization and crystal manipulation of a steric chaperone in complex with its lipase substrate.

Bacterial lipases that are secreted via the type II secretion pathway require a lipase-specific foldase in order to obtain their native and biologically active conformation in the periplasmic space. The lipase-foldase complex from Burkholderia glumae (319 and 333 residues, respectively) was crystallized in two crystal forms. One crystal form belongs to space group P3(1)21 (P3(2)21), with unit-cell parameters a = b = 122.3, c = 98.2 A. A procedure is presented which improved the diffraction of these crystals from approximately 5 to 2.95 A. For the second crystal form, which belonged to space group C2 with unit-cell parameters a = 183.0, b = 75.7, c = 116.6 A, X-ray data were collected to 1.85 A.

Bacterial Proteins↗

PubMed2005

Encapsulation of therapeutic nucleoside hydrolase in functionalised nanocapsules.

Liposomes are introduced as encapsulating carrier for prodrug activating enzymes. Inosineã-adenosineã-guanosine preferring nucleoside hydrolase of Trypanosoma vivax, a potential prodrug activating enzyme, was encapsulated in porin functionalized dioleyl-phosphatidylglycerol/egg-phosphatidylglycerol (DOPC/EPG) liposomes. Reactors had radiuses in the nanometer scale. First, transport of nucleosides through general diffusion porins OmpF and PhoE was measured in swelling assays, after which fully functional nanoreactors were developed. Enzyme catalysis of p-nitrophenylriboside, a substrate analogue for nucleoside hydrolases, was significantly higher in permeabilized vesicles than in control vesicles without porins. Residual activity of control vesicles possibly resides in an interaction between the enzyme and the liposomes. This interaction was not of electrostatic nature, since it remained unaffected after the addition of high salt or after perturbation of liposome surface charge and charge density. With these vesicles, we have introduced a new strategy for prodrug therapy, combining the benefits of ADEPT and liposome targeting strategies.

Drug Compounding↗

PubMed2005

Therapeutic nanoreactors: combining chemistry and biology in a novel triblock copolymer drug delivery system.

Triblock copolymeric nanoreactors are introduced as an alternative for liposomes as encapsulating carrier for prodrug activating enzymes. Inosine-adenosine-guanosine preferring nucleoside hydrolase of Trypanosoma vivax, a potential prodrug activating enzyme, was encapsulated in nanometer-sized vesicles constructed of poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-(2-methyloxazoline) triblock copolymers. The nanoreactor is functionalized by incorporation of bacterial porins, OmpF or Tsx, in the reactor wall. Efficient cleavage of three natural substrates and one prodrug, 2-fluoroadenosine, by the nanoreactors was demonstrated.

Animals↗

PubMed2005

The VirE1VirE2 complex of Agrobacterium tumefaciens interacts with single-stranded DNA and forms channels.

The VirE2 protein is crucial for the transfer of single-stranded DNA (ssDNA) from Agrobacterium tumefaciens to the nucleus of the plant host cell because of its ssDNA binding activity, assistance in nuclear import and putative ssDNA channel activity. The native form of VirE2 in Agrobacterium's cytoplasm is in complex with its specific chaperone, VirE1. Here, we describe the ability of the VirE1VirE2 complex to both bind ssDNA and form channels. The affinity of the VirE1VirE2 complex for ssDNA is slightly reduced compared with VirE2, but the kinetics of binding to ssDNA are unaffected by the presence of VirE1. Upon binding of VirE1VirE2 to ssDNA, similar helical structures to those reported for the VirE2-ssDNA complex were observed by electron microscopy. The VirE1VirE2 complex can release VirE1 once the VirE2-ssDNA complexes assembled. VirE2 exhibits a low affinity for small unilamellar vesicles composed of bacterial lipids and a high affinity for lipid vesicles containing sterols and sphingolipids, typical components of animal and plant membranes. In contrast, the VirE1VirE2 complex associated similarly with all kind of lipids. Finally, black lipid membrane experiments revealed the ability of the VirE1VirE2 complex to form channels. However, the majority of the channels displayed a conductance that was a third of the conductance of VirE2 channels. Our results demonstrate that the binding of VirE1 to VirE2 does not inhibit VirE2 functions and that the effector-chaperone complex is multifunctional.

Agrobacterium tumefaciens↗

PubMed2004

Maltoporin: sugar for physics and biology.

Maltoporin has been studied for over 50 years. This trimeric bacterial outer membrane channel allows permeation of sugars such as maltodextrins. Its structure is described and functional studies resulting in a mechanistic transport model are critically discussed.

Bacterial Outer Membrane Proteins↗

PubMed2004

Structure and electrophysiological properties of the YscC secretin from the type III secretion system of Yersinia enterocolitica.

YscC is the integral outer membrane component of the type III protein secretion machinery of Yersinia enterocolitica and belongs to the family of secretins. This group of proteins forms stable ring-like oligomers in the outer membrane, which are thought to function as transport channels for macromolecules. The YscC oligomer was purified after solubilization from the membrane with a nonionic detergent. Sodium dodecyl sulfate did not dissociate the oligomer, but it caused a change in electrophoretic mobility and an increase in protease susceptibility, indicating partial denaturation of the subunits within the oligomer. The mass of the homo-oligomer, as determined by scanning transmission electron microscopy, was approximately 1 MDa. Analysis of the angular power spectrum from averaged top views of negatively stained YscC oligomers revealed a 13-fold angular order, suggesting that the oligomer consists of 13 subunits. Reconstituted in planar lipid bilayers, the YscC oligomer displayed a constant voltage-independent conductance of approximately 3 nS, thus forming a stable pore. However, in vivo, the expression of YscC did not lead to an increased permeability of the outer membrane. Electron microscopy revealed that the YscC oligomer is composed of three domains, two stacked rings attached to a conical domain. This structure is consistent with the notion that the secretin forms the upper part of the basal body of the needle structure of the type III secreton.

Bacterial Outer Membrane Proteins↗

PubMed2002

Virus-assisted loading of polymer nanocontainer.

We present a DNA-containing polymeric nanocontainer using the self-assembled superstructure of amphiphilic block copolymers in aqueous solutions. To demonstrate that DNA translocation is possible across a completely synthetic block copolymer membrane, we have used a phage transfection strategy as a DNA-transfer model system. For this purpose the bacterial channel forming protein LamB was reconstituted in ABA-triblock copolymer vesicles. The outer membrane protein LamB is a specific transporter for maltodextrins but also serves as a receptor for lambda phage to trigger the ejection of lambda phage DNA. We demonstrate that the functionality of the LamB protein is fully preserved despite the artificial surrounding. This leads to a type of polymeric vehicle for DNA that could be useful for gene therapy.

Bacterial Outer Membrane Proteins↗

PubMed2002

Sugar transport through maltoporin of Escherichia coli: role of the greasy slide.

The lining of the maltodextrin-specific maltoporin (LamB) channel exhibits a string of aromatic residues, the greasy slide, part of which has been shown previously by crystallography to be involved in substrate binding. To probe the functional role of the greasy slide, alanine scanning mutagenesis has been performed on the six greasy slide residues and Y118 at the channel constriction. The mutants were characterized by an in vivo uptake assay and sugar-induced-current-noise analysis. Crystallographic analysis of the W74A mutant showed no perturbation of the structure. All mutants showed considerably decreased maltose uptake rates in vivo (<10% of the wild-type value), indicating the functional importance of the investigated residues. Substitutions at the channel center revealed appreciably increased (up to 100-fold) in vitro half-saturation concentrations for maltotriose and maltohexaose binding to the channel. Sugar association rates, however, were significantly affected also by the mutations at either end of the slide (W74A, W358A, and F227A), an effect which became most apparent upon nonsymmetrical sugar addition. The kinetic data are discussed on the basis of an asymmetric one-site two-barrier model, which suggests that, at low substrate concentrations, as are found under physiological conditions, only the heights of the extracellular and periplasmic barriers, which are reduced by the presence of the greasy slide, determine the efficiency of this facilitated diffusion channel.

Alanine↗