Biomedical subjects

P Van Gelder

Publications and source records attributed to P Van Gelder.

At least 19 recordsLinked to original sources

PubMed2001

An Agrobacterium VirE2 channel for transferred-DNA transport into plant cells.

Transferred DNA (T-DNA) transfer from Agrobacterium tumefaciens into eukaryotic cells is the only known example of interkingdom DNA transfer. T-DNA is a single-stranded segment of Agrobacterium's tumor-inducing plasmid that enters the plant cell as a complex with the bacterial virulence proteins VirD2 and VirE2. The VirE2 protein is highly induced on contact of A. tumefaciens with a plant host and has been reported to act in late steps of transfer. One of its previously demonstrated functions is binding to the single-stranded (ss) T-DNA and protecting it from degradation. Recent experiments suggest other functions of the protein. A combination of planar lipid bilayer experiments, vesicle swelling assays, and DNA transport experiments demonstrated that VirE2 can insert itself into artificial membranes and form channels. These channels are voltage gated, anion selective, and single-stranded DNA-specific and can facilitate the efficient transport of single-stranded DNA through membranes. These experiments demonstrate a VirE2 function as a transmembrane DNA transporter, which could have applications in gene delivery systems.

Agrobacterium tumefaciens↗

PubMed2001

Sucrose transport through maltoporin mutants of Escherichia coli.

Maltoporin (LamB) and sucrose porin (ScrY) reside in the bacterial outer membrane and facilitate the passive diffusion of maltodextrins and sucrose, respectively. To gain further insight into the determinants of solute specificity, LamB mutants were designed to allow translocation of sucrose, which hardly translocates through wild-type LamB. Three LamB mutants were studied. (a) Based on sequence and structure alignment of LamB with ScrY, two LamB triple mutants were generated (R109D, Y118D,D121F; R109N,Y118D,D121F) to mimic the ScrY constriction. The crystal structure of the first of these mutants was determined to be 3.2 A and showed an increased ScrY-like cross-section except for D109 that protrudes into the channel. (b) Based on this crystal structure a double mutant was generated by truncation of the two residues that obstruct the channel most in LamB (R109A,Y118A). Analysis of liposome swelling and in vivo sugar uptake demonstrated substantial sucrose permeation through all mutants with the double alanine mutant performing best. The triple mutants did not show a well-defined binding site as indicated by sugar-induced ion current noise analysis, which can be explained by remaining steric interference as deduced from the crystal structure. Binding, however, was observed for the double mutant that had the obstructing residues truncated to alanines.

Alanine↗

PubMed2000

Role of the lipase-specific foldase of Burkholderia glumae as a steric chaperone.

Most lipases of Gram-negative bacteria require a lipase-specific foldase (Lif) in order to fold in the periplasm into their active, protease-resistant conformation prior to their secretion. The periplasmic domain of the Lif (amino acids 44-353) of Burkholderia glumae was purified as a His-tagged protein, and its function in the folding of lipase was studied in vitro. Refolding of the denatured lipase into its active conformation was dependent on the presence of the Lif. Circular dichroism revealed that the lipase refolded in the absence of Lif into a form with a native-like conformation, which was more stable against heat-induced denaturation than the native form, but was enzymatically inactive. This form of the protein could be activated by adding Lif after several hours, which demonstrates that the function of this chaperone is to help lipase to overcome an energetic barrier in the productive folding pathway rather than to prevent it from entering a non-productive pathway. The Lif was shown to interact with the native lipase in protease-protection experiments as well as by affinity chromatography, consistent with a role of the Lif late in the folding process. These results demonstrate that the Lif functions in a way analogous to the propeptides of many bacterial proteases and indicate that the amino acid sequence of the lipase does not contain all the information required for the protein to adopt its three-dimensional structure.

Burkholderia↗

PubMed2000

Extended sugar slide function for the periplasmic coiled coil domain of ScrY.

Several bacterial outer membrane proteins have a periplasmic extension whose structure and function remain elusive. Here, the structure/function relationship of the N-terminal periplasmic domain of the sucrose-specific outer membrane channel ScrY was investigated. Circular dichroism and analytical centrifugation demonstrated that the N-terminal domain formed a parallel, three-stranded coiled coil. When this domain was fused to the maltose-specific channel LamB, permeation of maltooligosaccharides in liposomes increased with increasing sugar chain length whereas wild-type LamB showed the opposite effect. Current fluctuation analysis demonstrated increased off-rates for sugar transport through the fusion protein. Moreover, equilibrium dialysis showed an affinity of sucrose for the isolated N-terminal peptide. Together these results demonstrate a novel function for coiled coil domains, operating as an extended sugar slide.

Alkylation↗

PubMed2000

Sugar transport through maltoporin of Escherichia coli. Role of polar tracks.

The three-dimensional structure of the maltooligosaccharide specific outer membrane channel LamB of Escherichia coli complexed with sugar molecules revealed a hypothetical transport pathway. Sugars are supposed to slide over a stretch of aromatic residues facilitated by continuous making/breaking of hydrogen bonds between the hydroxyl groups of the sugars and charged amino acids, the "polar tracks." The effect of nine single and three multiple mutations in the polar track residues was investigated by current fluctuations, liposome swelling assays, and in vivo uptake of radiolabeled substrates. Additionally, sugar transport through wild-type LamB was investigated by current fluctuation analysis in water and deuterium. This way the effects on k(on) and k(off) could be investigated separately. Analyses of the various mutants revealed a strong effect on the k(on) values. Because steering to the binding site requires only a few interactions, consequently the loss of even one bond will have a strong effect. Deuterium experiments, which changed the characteristic of all hydrogen bonds, showed a strong effect on k(off) rates, because at this stage the sugar has numerous interactions with the channel. Furthermore, all the mutations induces a strong decrease of in vivo uptake of sugars. These results clearly demonstrate the importance of the polar track residues on both on and off rates in sugar transport and reveal a strong cooperative effect of hydrogen bond formation.

Bacterial Outer Membrane Proteins↗

PubMed2000

Structure and function of bacterial outer membrane proteins: barrels in a nutshell.

The outer membrane protects Gram-negative bacteria against a harsh environment. At the same time, the embedded proteins fulfil a number of tasks that are crucial to the bacterial cell, such as solute and protein translocation, as well as signal transduction. Unlike membrane proteins from all other sources, integral outer membrane proteins do not consist of transmembrane alpha-helices, but instead fold into antiparallel beta-barrels. Over recent years, the atomic structures of several outer membrane proteins, belonging to six families, have been determined. They include the OmpA membrane domain, the OmpX protein, phospholipase A, general porins (OmpF, PhoE), substrate-specific porins (LamB, ScrY) and the TonB-dependent iron siderophore transporters FhuA and FepA. These crystallographic studies have yielded invaluable insight into and decisively advanced the understanding of the functions of these intriguing proteins. Our review is aimed at discussing their common principles and peculiarities as well as open questions associated with them.

Bacterial Outer Membrane Proteins↗

PubMed2000

Oriented channels reveal asymmetric energy barriers for sugar translocation through maltoporin of Escherichia coli.

Sugar transport through maltoporin of Escherichia coli was investigated. This protein facilitates maltooligosaccharide translocation via a binding site in the channel. Because incorporation of the protein into the bilayer results in randomly orientated channels, we re-examined the postulated symmetric translocation model by reconstitution of maltoporin under an externally applied field. Upon binding of bacteriophage lambda, which exploit surface-exposed loops of maltoporin as the receptor, sugar permeation, but not the ion current, was blocked. Thus using the phage-to-probe orientation we were able to show that the channels were approximately 80% directionally inserted into the bilayer. Moreover, asymmetry of the channel was revealed because sugar entrance through the 'open' periplasmic side of maltoporin was similarly reduced. Here a new asymmetrical two-barrier model is presented. Based on liposome-swelling assays and current-fluctuation analysis we conclude that the periplasmic side of the porin shows a two- to threefold higher energy barrier than the extracellular loop-side of the channels.

Bacterial Outer Membrane Proteins↗

PubMed1999

The C-terminal domain of the Pseudomonas secretin XcpQ forms oligomeric rings with pore activity.

The Pseudomonas secretin XcpQ forms an oligomeric complex, which is involved in the translocation of proteins across the outer membrane via the type II secretion pathway. Pseudomonas aeruginosa produces only small amounts of this complex, 50 to 100 copies per bacterium, and overexpression is lethal to these cells. However, overexpression of Pseudomonas alcaligenes XcpQ could be achieved in the P. alcaligenes mutant strain 537. Protease protection experiments with P. alcaligenes XcpQ showed that the C-terminal domain of XcpQ, which is conserved in all the different members of the secretin family, is largely resistant to proteinase K. This protease-resistant fragment is embedded in the membrane and remains a stable complex, indicating that this domain is involved in complex formation. Both the intact and the protease-protected XcpQ complex showed a tendency to form two-dimensional crystal-like structures. Electron microscopic analysis of these structures showed that the overall oligomeric rings of the intact and of the protease-resistant complex are highly similar. The central cavity of the intact XcpQ complex contains structured mass. Both the intact and the protease-protected XcpQ complex showed pore-forming activity in planar lipid bilayers, consistent with their role as a translocation channel. However, the single-channel conductances observed were not uniform. Together, these results demonstrate that the C-terminal secretin homology domain of XcpQ is the structural domain that forms the channel through which macromolecules are being transported.

Bacterial Proteins↗

PubMed1998

Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli.

Combination of an origin repair mutagenesis system with a new mutS host strain increased the efficiency of mutagenesis from 46% to 75% mutant clones. Overexpression with the T7 expression system afforded large quantities of proteins from mutant strains. A series of E. coli BE host strains devoid of major outer membrane proteins was constructed, facilitating the purification of mutant porins to homogeneity. This allowed preparation of 149 porin mutants in E. coli used in detailed explorations of the structure and function of this membrane protein to high resolution.

Adenosine Triphosphatases↗

PubMed1997

Role of the constriction loop in the gating of outer membrane porin PhoE of Escherichia coli.

Porins form voltage-gated channels in the bacterial outer membrane. These proteins are composed of three identical subunits, each forming a 16-stranded beta-barrel. In this study, the role in voltage gating of a loop that forms a constriction within the pore was studied. The channel characteristics of mutant PhoE porins, in which the tip of the constriction loop was connected to the barrel wall, were determined. Whereas the properties of several mutant channels were changed, all of these channels could still be closed at high potential, showing that a gross movement of the constriction loop within the channel is not implicated in voltage gating.

Anti-Bacterial Agents↗

PubMed1997

Voltage sensing in the PhoE and OmpF outer membrane porins of Escherichia coli: role of charged residues.

The porins PhoE and OmpF form anion and cation-selective pores, respectively, in the outer membrane of Escherichia coli. Each monomer of these trimeric proteins consists of a 16-stranded beta-barrel, which contains a constriction at half the height of the channel. The functional significance of a transverse electrical field that is formed by charged amino acid residues within the constriction zone was investigated. For this purpose, the PhoE residues R37, R75, K18 and E110 were substituted by neutral amino acids. The mutant pores allowed an increased permeation of beta-lactam antibiotics across the outer membrane in vivo, although the single channel conductance, measured in planar lipid bilayers, was not increased or even slightly decreased. Replacement of the positively charged residues resulted in a decreased voltage sensitivity, whereas the substitution of a negatively charged residue resulted in an increased voltage sensitivity. Similar substitutions in OmpF caused the opposite effects, i.e. the substitution of positive and negative charges resulted in increased and decreased voltage sensitivity, respectively. Together, the results suggest that opposite charges, i.e. positive charges in anion-selective and negative charges in cation-selective porins, act as sensors for voltage gating.

Bacterial Outer Membrane Proteins↗

PubMed1997

Peak velocities of visually and nonvisually guided saccades in smooth-pursuit and saccadic tasks.

Smooth pursuit typically includes corrective catch-up saccades, but may also include such intrusive saccades away from the target as anticipatory or large overshooting saccades. We sought to differentiate catch-up from anticipatory and overshooting saccades by their peak velocities, to see whether the higher velocities of visually rather than nonvisually guided saccades in saccadic tasks may be found also in saccades in pursuit. In experiment 1, 12 subjects showed catch-up, anticipatory, and overshooting saccades to comprise 70.4% of all saccades in pursuit of periodic, 30 degrees/s constant-velocity targets. Catch-up saccades were faster than the others. Saccadic tasks were run as well, on 19 subjects, including the 12 whose pursuit data were analyzed, with target-onset, target-remaining (saccade to the remaining target when the other three extinguish), and antisaccade tasks. For 17 of the 19 subjects, antisaccade velocities were lower than for either target-onset or target-remaining tasks. Velocities for the target-remaining task were near those for target onset, indicating that target presence, not its onset, defines visually guided saccades. Error and reaction-time data suggest greater cognitive difficulty for target remaining than for target onset, so that the cognitive difficulty of typical nonvisually guided saccade tasks is not sufficient to produce their lowered velocity. To produce reliably, in each subject, catch-up and anticipatory saccades with comparable amplitude distributions, nine new subjects were asked in experiment 2 to make intentional catch-up and anticipatory saccades in pursuit, and were presented with embedded target jumps to elicit catch-up saccades, all with periodic target trajectories of 15 degrees/s and 30 degrees/s. Velocities of intentional anticipatory saccades were lower than velocities of intentional catch-up saccades, while velocities of intentional and embedded catch-up saccades were similar. Target-onset and remembered-target saccadic tasks were run, showing the expected higher velocity for the target-onset task in each subject. Both experiments demonstrate higher peak velocities for catch-up saccades than for anticipatory saccades, suggesting that cortical structures preferentially involved in nonvisually guided saccades may initiate the anticipatory and overshooting saccades in pursuit.

Calibration↗

PubMed1997

Pore functioning of outer membrane protein PhoE of Escherichia coli: mutagenesis of the constriction loop L3.

Each monomer of the trimeric outer membrane porin PhoE of Escherichia coli consists of a 16-stranded beta-barrel with short turns at the periplasmic side and large loops at the cell surface. One of these loops, L3, is folded inside the beta-barrel and forms a constriction within the channel. Therefore, it is assumed to play an important role in the permeability properties of this general diffusion pore. Several site-directed mutations were introduced in loop L3 to investigate its function. The loop L3 contains a short alpha-helix and, at the tip of the loop, a highly conserved PEFGG sequence. The alpha-helix was deleted and the two glycines in the PEFGG sequence were either replaced by alanines or deleted. A serine residue, supposed to play an indirect role in the anion selectivity of the pore, was removed. The mutant porins were analysed both in vitro and in vivo. The results suggest that flexibility of the third loop is important for solute passage and that this flexibility is determined by the two glycine residues in the PEFGG sequence. Furthermore, the alpha-helix is probably important for the folding of the protein. The supposed involvement of Ser115 (Ser121A in OmpF nomenclature) in anion selectivity was confirmed.

Anti-Bacterial Agents↗

PubMed1996

Structural and functional characterization of a His-tagged PhoE pore protein of Escherichia coli.

The recent elucidation of the 3-D structure of the outer membrane protein PhoE of Escherichia coli provides an excellent tool for a detailed analysis of the structure-function relationship of this pore-forming protein. For this purpose, a fast and efficient method for the purification of mutant porins is needed. A histidine-tag was engineered between the signal sequence and the N terminus of mature PhoE. The recombinant PhoE protein was normally assembled into the outer membrane and could be purified by immobilized metal affinity chromatography. Part of the total amount of the trimers dissociated into folded monomers during purification. The histidine-tag did not change the electrophysical characteristics of the protein in lipid bilayers. Hence, the method is useful for the fast purification of mutant porins for functional and structural characterization.

Escherichia coli↗

PubMed1996

Demonstration of a folded monomeric form of porin PhoE of Escherichia coli in vivo.

The porins in the outer membranes of gram-negative bacteria are trimeric proteins. A folded monomeric form of the Escherichia coli porin PhoE, with a higher electrophoretic mobility than that of the denatured protein, has recently been detected in in vitro folding studies. To investigate the possible biological significance of the folded monomer, we attempted to detect this form in vivo. After pulse-labeling, folded monomers could be detected by immunoprecipitation. Furthermore, folded monomers were detected in a preparation of mutant PhoE porins, in which the subunit interactions were weakened by a E-66-->R substitution. Together, these results show that the folded monomer is not an in vitro folding artifact but an integral part of the native trimer.

Electrophoresis, Gel, Pulsed-Field↗

PubMed1996

Square-root relations between main saccadic parameters.

PURPOSE: To derive and evaluate two equations in which saccade duration and peak velocity are proportional to the square root of saccade amplitude. METHODS: A population of horizontal visually guided saccades in a range of amplitudes from 1.5 degrees to 30 degrees was recorded by means of electro-oculography in eight normal adult subjects. The peak velocity-amplitude data of this population were fitted to four models: inverse linear, exponential, power law, and square root. To approximate the duration-amplitude relation, the square root was tested against the linear and power law models. For each model, the best-fit values of its parameters were estimated by the method of least squares. RESULTS: When the entire population was used, all tested models displayed comparable goodness of fit, but when different subranges of this population were used, only the square root equations appeared to be robust and acceptably accurate. CONCLUSIONS: In a restricted range of saccade amplitudes from 1.5 degrees to 30 degrees, the square root model has some advantages over the others commonly used: to express peak velocity and duration as functions of amplitude, it requires the estimation of only two parameters, whereas the others require four. Because of its robustness, this model can be used to evaluate populations of saccadic eye movements with different ranges of amplitudes. The two parameters of the model equations allow a simple and clear physical interpretation.

Adult↗

PubMed1995

Anticipatory saccades in smooth pursuit: task effects and pursuit vector after saccades.

The dramatic improvement in smooth pursuit performance seen while analyzing the pursuit target has been ascribed to attention enhancement. With a periodic constant velocity target trajectory we ran a concurrent listening condition instead, to see if this mild distraction would degrade performance. Performance improved somewhat with the listening task, suggesting that displacing attentional effort from pursuit accuracy, rather than increasing it, brings better pursuit performance. Catch-up saccades were evenly distributed across tracking, listening, and target analysis conditions, but anticipatory and overshooting saccades were almost eliminated with target analysis. Thus the poor pursuit seems to have been caused by anticipatory and overshooting saccades, produced erroneously in the attempt to perform purposive smooth pursuit. Pursuit velocity immediately following anticipatory saccades was reduced such that the target would catch up with the point of gaze when it reached the endpoint of its trajectory, indicating a predictive goal other than instantaneous target foveation and velocity match.

Adult↗

PubMed1994

Detergent-induced folding of the outer-membrane protein PhoE, a pore protein induced by phosphate limitation.

The folding of the in vitro synthesized outer-membrane protein PhoE, a protein induced by phosphate limitation, has previously been studied using immunoprecipitation experiments with monoclonal antibodies that recognize conformational epitopes [De Cock, H., Hendricks, R., de Vrije, T. & Tommassen, J. (1990) J. Biol. Chem. 265, 4646-4651]. A folded monomer of the protein was detected in this way, while the addition of outer membranes was required to induce trimerization. In this study, we demonstrate that the folding of the in vitro synthesized PhoE protein did not occur spontaneously, but was dependent on the detergent that was present in the immunoprecipitation buffer. A remarkable specificity of phenyl-containing detergents on the efficient in vitro folding of PhoE molecules was observed. Furthermore, trimerization was detected in the absence of outer membranes when such detergents were present. However, the rate of trimerization was increased by the addition of crude cell envelopes containing outer membranes. The outer membranes probably enhanced trimerization by concentrating the folded PhoE molecules.

Cell Membrane↗