Biomedical subjects

Bert Poolman

Publications and source records attributed to Bert Poolman.

At least 37 records · Page 2Linked to original sources

PubMed2005

Specificity and selectivity determinants of peptide transport in Lactococcus lactis and other microorganisms.

Peptide transport in microorganisms is important for nutrition of the cell and various signalling processes including regulation of gene expression, sporulation, chemotaxis, competence and virulence development. Peptide transport is mediated via different combinations of ion-linked and ATP-binding cassette (ABC) transporters, the latter utilizing single or multiple peptide-binding proteins with overlapping specificities. The paradigm for research on peptide transport is Lactococcus lactis, in which the uptake of peptides containing essential amino acids is vital for growth on milk proteins. Differential expression and characteristics of peptide-binding proteins in several Lactococcus lactis strains resulted in apparent conflicts with older literature. Recent developments and new data now make the pieces of the puzzle fall back into place again and confirm the view that the oligopeptide-binding proteins determine the uptake selectivity of their cognate ABC transporters. Besides reviewing the current data on binding specificity and transport selectivity of peptide transporters in L. lactis, the possible implications for peptide utilization by other bacterial species are discussed.

ATP-Binding Cassette Transporters↗

PubMed2004

Lactococcus lactis uses MscL as its principal mechanosensitive channel.

The functions of the mechanosensitive channels from Lactococcus lactis were determined by biochemical, physiological, and electrophysiological methods. Patch-clamp studies showed that the genes yncB and mscL encode MscS and MscL-like channels, respectively, when expressed in Escherichia coli or if the gene products were purified and reconstituted in proteoliposomes. However, unless yncB was expressed in trans, wild type membranes of L. lactis displayed only MscL activity. Membranes prepared from an mscL disruption mutant did not show any mechanosensitive channel activity, irrespective of whether the cells had been grown on low or high osmolarity medium. In osmotic downshift assays, wild type cells survived and retained 20% of the glycine betaine internalized under external high salt conditions. On the other hand, the mscL disruption mutant retained 40% of internalized glycine betaine and was significantly compromised in its survival upon osmotic downshifts. The data strongly suggest that L. lactis uses MscL as the main mechanosensitive solute release system to protect the cells under conditions of osmotic downshift.

Amino Acid Sequence↗

PubMed2004

Two homologous oligopeptide binding protein genes (oppA) in Lactococcus lactis opp2 [corrected].

In previous studies, it has been shown that inactivation of opp or even oppA abolishes the capacity of Lactococcus lactis to utilize oligopeptides. We now show that the opp operon has been duplicated in L. lactis MG1363. The nucleotide sequence of the oppA and oppC homologues (appA and appC) and most of the oppB homologue (appB) indicate that the corresponding protein sequences are 83%, 92% and 91% identical, respectively. Inactivation of appA, via homologous recombination, as well as complementation studies were carried out to determine the possible function of appA in peptide utilization. As anticipated from studies with an oppA knock-out, peptide utilization was not impaired in an appA disruption mutant. Importantly, AppA expressed from a plasmid could restore the ability of oppA deletion mutants to utilize Leu-enkephalin, albeit with a lower efficiency than OppA. The differences in the ability to utilize this pentapeptide were not due to differences in expression levels but most likely reflect a different catalytic efficiency in oligopeptide utilization when AppA is used as ligand receptor.

Amino Acid Sequence↗

PubMed2004

Distribution, lateral mobility and function of membrane proteins incorporated into giant unilamellar vesicles.

GUVs have been widely used for studies on lipid mobility, membrane dynamics and lipid domain (raft) formation, using single molecule techniques like fluorescence correlation spectroscopy. Reports on membrane protein dynamics in these types of model membranes are by far less advanced due to the difficulty of incorporating proteins into GUVs in a functional state. We have used sucrose to prevent four distinct membrane protein(s) (complexes) from inactivating during the dehydration step of the GUV-formation process. The amount of sucrose was optimized such that the proteins retained 100% biological activity, and many proteo-GUVs were obtained. Although GUVs could be formed by hydration of lipid mixtures composed of neutral and anionic lipids, an alternate current electric field was required for GUV formation from neutral lipids. Distribution, lateral mobility, and function of an ATP-binding cassette transport system, an ion-linked transporter, and a mechanosensitive channel in GUVs were determined by confocal imaging, fluorescence correlation spectroscopy, patch-clamp measurements, and biochemical techniques. In addition, we show that sucrose slows down the lateral mobility of fluorescent lipid analogs, possibly due to hydrogen-bonding with the lipid headgroups, leading to larger complexes with reduced mobility.

ATP-Binding Cassette Transporters↗

PubMed2004

Bacterial osmosensing: roles of membrane structure and electrostatics in lipid-protein and protein-protein interactions.

Bacteria act to maintain their hydration when the osmotic pressure of their environment changes. When the external osmolality decreases (osmotic downshift), mechanosensitive channels are activated to release low molecular weight osmolytes (and hence water) from the cytoplasm. Upon osmotic upshift, osmoregulatory transporters are activated to import osmolytes (and hence water). Osmoregulatory channels and transporters sense and respond to osmotic stress via different mechanisms. Mechanosensitive channel MscL senses the increasing tension in the membrane and appears to gate when the lateral pressure in the acyl chain region of the lipids drops below a threshold value. Transporters OpuA, BetP and ProP are activated when increasing external osmolality causes threshold ionic concentrations in excess of about 0.05 M to be reached in the proteoliposome lumen. The threshold activation concentrations for the OpuA transporter are strongly dependent on the fraction of anionic lipids that surround the cytoplasmic face of the protein. The higher the fraction of anionic lipids, the higher the threshold ionic concentrations. A similar trend is observed for the BetP transporter. The lipid dependence of osmotic activation of OpuA and BetP suggests that osmotic signals are transmitted to the protein via interactions between charged osmosensor domains and the ionic headgroups of the lipids in the membrane. The charged, C-terminal domains of BetP and ProP are important for osmosensing. The C-terminal domain of ProP participates in homodimeric coiled-coil formation and it may interact with the membrane lipids and soluble protein ProQ. The activation of ProP by lumenal, macromolecular solutes at constant ionic strength indicates that its structure and activity may also respond to macromolecular crowding. This excluded volume effect may restrict the range over which the osmosensing domain can electrostatically interact. A simplified version of the dissociative double layer theory is used to explain the activation of the transporters by showing how changes in ion concentration could modulate interactions between charged osmosensor domains and charged lipid or protein surfaces. Importantly, the relatively high ionic concentrations at which osmosensors become activated at different surface charge densities compare well with the predicted dependence of 'critical' ion concentrations on surface charge density. The critical ion concentrations represent transitions in Maxwellian ionic distributions at which the surface potential reaches 25.7 mV for monovalent ions. The osmosensing mechanism is qualitatively described as an "ON/OFF switch" representing thermally relaxed and electrostatically locked protein conformations.

Bacteria↗

PubMed2004

Lipid-mediated light activation of a mechanosensitive channel of large conductance.

This paper describes the reversible activation of a mechanosensitive channel via a light-sensitive lipid mimic. For these experiments, the mechanosensitive channel of large conductance (MscL) protein from Lactococcus lactis and Escherichia coli was reconstituted in lipid bilayers composed of 80 mol % 1,2-dioleoyl-sn-glycero-3-phosphocholine and 20 mol % di-(5-[[4-(4-butylphenyl)azo]phenoxy]pentyl)phosphate (4-Azo-5P). Light-induced isomerization of the azobenzene moiety of 4-Azo-5P from trans to cis was used to activate MscL.

Azo Compounds↗

PubMed2004

The binding specificity of OppA determines the selectivity of the oligopeptide ATP-binding cassette transporter.

The purification and functional reconstitution of a five-component oligopeptide ATP-binding cassette transporter with a remarkably wide substrate specificity are described. High-affinity peptide uptake was dependent on liganded substrate-binding protein OppA, which interacts with the translocator OppBCDF with higher affinity than unliganded OppA. Transport screening with combinatorial peptide libraries revealed that (i) the Opp transporter is not selective with respect to amino acid side chains of the transported peptides; (ii) any peptide that can bind to OppA is transported via Opp, including very long peptides up to 35 residues long; and (iii) the binding specificity of OppA largely determines the overall transport selectivity.

Adenosine Triphosphate↗

PubMed2004

Evaluation of the flow-dialysis technique for analysis of protein-ligand interactions: an experimental and a monte carlo study.

Flow dialysis has found widespread use in determining the dissociation constant (KD) of a protein-ligand interaction or the amount of available binding sites (E0). This method has the potency to measure both these parameters in a single experiment and in this article a method to measure simultaneously the KD and E0 is presented, together with an extensive error analysis of the method. The flow-dialysis technique is experimentally simple to perform. However, a number of practical aspects of this method can have a large impact on the outcome of KD and E0. We have investigated all sources of significant systematic and random errors, using the interaction between mannitol and its transporter from Escherichia coli as a model. Monte Carlo simulations were found to be an excellent tool to assess the impact of these errors on the binding parameters and to define the experimental conditions that allow their most accurate estimation.

Computer Simulation↗

PubMed2003

On the role of the two extracytoplasmic substrate-binding domains in the ABC transporter OpuA.

Members of two transporter families of the ATP-binding cassette (ABC) superfamily use two or even four extracytoplasmic substrate-binding domains (SBDs) for transport. We report on the role of the two SBDs in the translocation cycle of the ABC transporter OpuA from Lactococcus lactis. Heterooligomeric OpuA complexes with only one SBD or one functional and one non-functional SBD (inactivated by covalent linkage of a substrate mimic) have been constructed, and the substrate binding and transport kinetics of the purified transporters, reconstituted in liposomes, have been determined. The data indicate that the two SBDs of OpuA interact in a cooperative manner in the translocation process by stimulating either the docking of the SBDs onto the translocator or the delivery of glycine betaine to the translocator. It appears that one of these initial steps, but not the later steps in translocation or resetting of the system to the initial state, is rate determining for transport. These new insights on the functional role of the extracytoplasmic SBDs are discussed in the light of the current knowledge of substrate-binding-protein-dependent ABC transporters.

ATP-Binding Cassette Transporters↗

PubMed2003

Specificity of the second binding protein of the peptide ABC-transporter (Dpp) of Lactococcus lactis IL1403.

The genome sequence of Lactococcus lactis IL1403 revealed the presence of a putative peptide-binding protein-dependent ABC-transporter (Dpp). The genes for two peptide-binding proteins (dppA and dppP) precede the membrane components, which include two transmembrane protein genes (dppB and dppC) and two ATP-binding protein genes (dppD and dppF). In this work, the gene specifying the second peptide-binding protein (DppP) was cloned under the control of the nisin promoter. The protein fused to a carboxyl-terminal histidine tag (DppP-His(6)) was purified and its binding properties were determined by monitoring the intrinsic fluorescence changes observed upon ligand binding. The major features of peptide binding to DppP-His(6) include: (i) a requirement for a free N-terminal alpha-amino group in the ligand; (ii) a high affinity for di-, tri- and tetra-peptides; (iii) affinity constants for peptide binding independent of pH; and (iv) a high affinity for D-isomer-containing peptides. Remarkably, the features (ii), (iii) and (iv) differ from those previously reported for DppA-His(6), suggesting that DppP-His(6) is a more versatile peptide-binding protein that could have additional functions.

ATP-Binding Cassette Transporters↗

PubMed2003

Identification of the dimer interface of the lactose transport protein from Streptococcus thermophilus.

The lactose transporter from Streptococcus thermophilus catalyses the symport of galactosides and protons. The carrier domain of the protein harbours the contact sites for dimerization, and the individual subunits in the dimer interact functionally during the transport reaction. As a first step towards the elucidation of the mechanism behind the cooperation between the subunits, regions involved in the dimer interface were determined by oxidative and chemical cross-linking of 12 cysteine substitution mutants. Four positions in the protein were found to be susceptible to intermolecular cross-linking. To ensure that the observed cross-links were not the result of randomly colliding particles, the cross-linking was studied in samples in which either the concentration of LacS in the membrane was varied or the oligomeric state was manipulated. These experiments showed that the cross-links were formed specifically within the dimer. The four regions of the protein located at the dimer interface are close to the extracellular ends of transmembrane segments V and VIII and the intracellular ends of transmembrane segments VI and VII.

Amino Acid Sequence↗

PubMed2003

The ATP/substrate stoichiometry of the ATP-binding cassette (ABC) transporter OpuA.

ATP-binding cassette (ABC) transport proteins catalyze the translocation of substrates at the expense of hydrolysis of ATP, but the actual ATP/substrate stoichiometry is still controversial. In the osmoregulated ABC transporter (OpuA) from Lactococcus lactis, ATP hydrolysis and substrate translocation are tightly coupled, and the activity of right-side-in and inside-out reconstituted OpuA can be determined accurately. Although the ATP/substrate stoichiometry determined from the uptake of glycine betaine and intravesicular ATP hydrolysis tends to increase with decreasing average size of the liposomes, the data from inside-out reconstituted OpuA indicate that the mechanistic stoichiometry is 2. Moreover, the two orientations of OpuA in proteoliposomes allowed possible contributions from substrate (glycine betaine) inhibition on the trans-side of the membrane and inhibition by ADP to be determined. Here we show that OpuA is not inhibited by up to 400 mm glycine betaine on the trans-side of the membrane. ADP is an inhibitor, but accumulation of ADP was negligible in the assays with inside-out-oriented OpuA, and potential effects of the ATP/ADP ratio on the ATP/substrate stoichiometry determinations could be eliminated.

ATP-Binding Cassette Transporters↗

PubMed2003

Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy.

Confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS) have been employed to investigate the lipid spatial and dynamic organization in giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine/sphingomyelin/cholesterol. For a certain range of cholesterol concentration, formation of domains with raft-like properties was observed. Strikingly, the lipophilic probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C18) was excluded from sphingomyelin-enriched regions, where the raft marker ganglioside GM1 was localized. Cholesterol was shown to promote lipid segregation in dioleoyl-phosphatidylcholine-enriched, liquid-disordered, and sphingomyelin-enriched, liquid-ordered phases. Most importantly, the lipid mobility in sphingomyelin-enriched regions significantly increased by increasing the cholesterol concentration. These results pinpoint the key role, played by cholesterol in tuning lipid dynamics in membranes. At cholesterol concentrations >50 mol%, domains vanished and the lipid diffusion slowed down upon further addition of cholesterol. By taking the molecular diffusion coefficients as a fingerprint of membrane phase compositions, FCS is proven to evaluate domain lipid compositions. Moreover, FCS data from ternary and binary mixtures have been used to build a ternary phase diagram, which shows areas of phase coexistence, transition points, and, importantly, how lipid dynamics varies between and within phase regions.

Carbocyanines↗

PubMed2003

Lactococcus lactis as host for overproduction of functional membrane proteins.

Lactococcus lactis has many properties that are ideal for enhanced expression of membrane proteins. The organism is easy and inexpensive to culture, has a single membrane and relatively mild proteolytic activity. Methods for genetic manipulation are fully established and a tightly controlled promoter system is available, with which the level of expression can be varied with the inducer concentration. Here we describe our experiences with lactococcal expression of the mechanosensitive channel, the human KDEL receptor and transporters belonging to the ABC transporter family, the major facilitator superfamily, the mitochondrial carrier family and the peptide transporter family. Previously published expression studies only deal with the overexpression of prokaryotic membrane proteins, but in this paper, experimental data are presented for the overproduction of mitochondrial and hydrogenosomal carriers and the human KDEL receptor. These eukaryotic membrane proteins were expressed in a functional form and at levels amenable to structural work.

Animals↗

PubMed2002

Combined in-gel tryptic digestion and CNBr cleavage for the generation of peptide maps of an integral membrane protein with MALDI-TOF mass spectrometry.

A limitation of the in-gel approaches for the generation of peptides of membrane proteins is the size and hydrophobicity of the fragments generated. For membrane proteins like the lactose transporter (LacS) of Streptococcus thermophilus, tryptic digestion or CNBr cleavage yields several hydrophobic fragments larger than 3.5 kDa. As a result, the sequence coverage of the membrane domain is low when the in-gel tryptic-digested or CNBr-cleaved fragments are analyzed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS). The combination of tryptic digestion and subsequent CNBr cleavage on the same gel pieces containing LacS approximately doubled the coverage of the hydrophobic membrane domain compared to the individual cleavage methods, while the coverage of the soluble domain remained complete. The fragments formed are predominantly below m/z 2500, which allows accurate mass measurement.

Amino Acid Sequence↗

PubMed2002

Spatial organization of bacteriorhodopsin in model membranes. Light-induced mobility changes.

Bacteriorhodopsin is a proton-transporting membrane protein in Halophilic archaea, and it is considered a prototype of membrane transporters and a model for G-protein-coupled receptors. Oligomerization of the protein has been reported, but it is unknown whether this feature is correlated with, for instance, light activation. Here, we have addressed this issue by reconstituting bacteriorhodopsin into giant unilamellar vesicles. The dynamics of the fully active protein was investigated using fluorescence correlation spectroscopy and freeze fracture electron microscopy. At low protein-to-lipid ratios (<1:10 w/w), a decrease in mobility was observed upon protein photoactivation. This process occurred on a second time scale and was fully reversible, i.e. when the dark-adapted state was reestablished the lateral diffusion rate of the protein was returned to that prior to activation. A similar decrease in lateral mobility as observed upon photoactivation was obtained when bacteriorhodopsin was reconstituted at high protein-to-lipid ratios (>1:10 w/w). We interpret the shifts in mobility during light adaptation as being caused by transient photoinduced oligomerization of bacteriorhodopsin. These observations are fully supported by freeze-fracture electron microscopy, and the size of the clusters during photoactivation was estimated to consist of two or three trimers.

Bacteriorhodopsins↗

PubMed2002

Oligomeric state of membrane transport proteins analyzed with blue native electrophoresis and analytical ultracentrifugation.

Blue native electrophoresis is used widely for the analysis of non-dissociated protein complexes with respect to composition, oligomeric state and molecular mass. However, the effects of detergent or dye binding on the mass and stability of the integral membrane proteins have not been studied. By comparison with analytical ultracentrifugation, we have evaluated whether the oligomeric state of membrane transport proteins is reflected reliably with blue native electrophoresis. For the analysis we have used two well-characterized transporters, that is, the major facilitator superfamily protein LacS and the phosphotransferase system EII(Mtl). For another member of the major facilitator superfamily, the xyloside transporter XylP from Lactobacillus pentosus, the complete analysis of the quaternary structure determined by analytical ultracentrifugation and freeze-fracture electron microscopy is presented. Our experiments show that during blue native electrophoresis the detergent bound to the proteins is replaced by the amphipathic Coomassie brilliant blue (CBB) dye. The mass of the bound CBB dye was quantified. Provided this additional mass of bound CBB dye is accounted for and care is taken in the choice and concentration of the detergent used, the mass of LacS, XylP and EII(Mtl) and four other membrane (transport) proteins could be deduced within 10 % error. Our data underscore the fact that the oligomeric state of many membrane transport proteins is dimeric.

Bacterial Proteins↗

PubMed2002

Mapping of the dimer interface of the Escherichia coli mannitol permease by cysteine cross-linking.

A cysteine cross-linking approach was used to identify residues at the dimer interface of the Escherichia coli mannitol permease. This transport protein comprises two cytoplasmic domains and one membrane-embedded C domain per monomer, of which the latter provides the dimer contacts. A series of single-cysteine His-tagged C domains present in the native membrane were subjected to Cu(II)-(1,10-phenanthroline)(3)-catalyzed disulfide formation or cysteine cross-linking with dimaleimides of different length. The engineered cysteines were at the borders of the predicted membrane-spanning alpha-helices. Two residues were found to be located in close proximity of each other and capable of forming a disulfide, while four other locations formed cross-links with the longer dimaleimides. Solubilization of the membranes did only influence the cross-linking behavior at one position (Cys(73)). Mannitol binding only effected the cross-linking of a cysteine at the border of the third transmembrane helix (Cys(134)), indicating that substrate binding does not lead to large rearrangements in the helix packing or to dissociation of the dimer. Upon mannitol binding, the Cys(134) becomes more exposed but the residue is no longer capable of forming a stable disulfide in the dimeric IIC domain. In combination with the recently obtained projection structure of the IIC domain in two-dimensional crystals, a first proposal is made for alpha-helix packing in the mannitol permease.

Amino Acid Sequence↗