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B de Kruijff

Publications and source records attributed to B de Kruijff.

At least 55 records · Page 3Linked to original sources
PubMed1999

Different membrane anchoring positions of tryptophan and lysine in synthetic transmembrane alpha-helical peptides.

Specific interactions of membrane proteins with the membrane interfacial region potentially define protein position with respect to the lipid environment. We investigated the proposed roles of tryptophan and lysine side chains as "anchoring" residues of transmembrane proteins. Model systems were employed, consisting of phosphatidylcholine lipids and hydrophobic alpha-helical peptides, flanked either by tryptophans or lysines. Peptides were incorporated in bilayers of different thickness, and effects on lipid structure were analyzed. Induction of nonbilayer phases and also increases in bilayer thickness were observed that could be explained by a tendency of Trp as well as Lys residues to maintain interactions with the interfacial region. However, effects of the two peptides were remarkably different, indicating affinities of Trp and Lys for different sites at the interface. Our data support a model in which the Trp side chain has a specific affinity for a well defined site near the lipid carbonyl region, while the lysine side chain prefers to be located closer to the aqueous phase, near the lipid phosphate group. The information obtained in this study may further our understanding of the architecture of transmembrane proteins and may prove useful for refining prediction methods for transmembrane segments.

Amino Acid Sequence↗
PubMed1999

Delta psi stimulates membrane translocation of the C-terminal part of a signal sequence.

For several proteins in Escherichia coli it has been shown that the protonmotive force (pmf) dependence of translocation can be varied with the signal sequence composition, suggesting an effect of the pmf on the signal sequence. To test this possibility, we analyzed the effect of the membrane potential on translocation of the signal sequence. For this purpose, a precursor peptide was used (SP+7), corresponding to the signal sequence of PhoE with the first seven amino acids of the mature part that can be processed by purified leader peptidase. Translocation was studied in pure lipid vesicles containing leader peptidase, with its active site inside the vesicles. In the presence of a positive inside Delta psi, the amount of processing of SP+7 was significantly higher than without a Delta psi, indicating that the translocation of the cleavage region is stimulated by Delta psi. Replacement of the helix-breaking glycine residue at position -10 in the signal sequence for a leucine abolished the effect of Delta psi on the translocation of the cleavage region. It is concluded that Delta psi directly acts on the wild type signal sequence by stimulating the translocation of its C terminus. We propose that Delta psi acts on the signal sequence by stretching it into a transmembrane orientation.

Amino Acid Sequence↗
PubMed1999

The structural flexibility of the preferredoxin transit peptide.

In order to obtain insight into the structural flexibility of chloroplast targeting sequences, the Silene pratensis preferredoxin transit peptide was studied by circular dichroism and nuclear magnetic resonance spectroscopy. In water, the peptide is unstructured, with a minor propensity towards helix formation from Val-9 to Ser-12 and from Gly-30 to Ser-40. In 50% (v/v) trifluoroethanol, structurally independent N- and C-terminal helices are stabilized. The N-terminal helix appears to be amphipathic, with hydrophobic and hydroxylated amino acids on opposite sides. The C-terminal helix comprises amino acids Met-29-Gly-50 and is destabilized at Gly-39. No ordered tertiary structure was observed. The results are discussed in terms of protein import into chloroplasts, in which the possible interactions between the transit peptide and lipids are emphasized.

Amino Acid Sequence↗
PubMed1999

Expression, isolation, and characterization of a chloroplast targeting peptide.

For the first time a method is described in which an N-terminal targeting peptide is isolated from Escherichia coli. After overexpression, purification, and cleavage of a fusion protein the protease-sensitive transit peptide from the chloroplast precursor protein preferredoxin could be isolated by HPLC. It was characterized by N-terminal amino acid sequencing and electrospray mass spectrometry. Its functionality was suggested by in vitro import competition experiments with isolated pea chloroplasts, in which the isolated peptide inhibited the import of radioactively labeled preferredoxin. Results from import competition experiments performed with a transit peptide deletion mutant suggested that the four extreme C-terminal amino acids lack information to interact with the chloroplast import machinery.

Chloroplasts↗
PubMed1999

Blistering of langmuir-blodgett bilayers containing anionic phospholipids as observed by atomic force microscopy.

Asymmetric bilayers of different phospholipid compositions have been prepared by the Langmuir-Blodgett (L-B) method, and imaged by atomic force microscopy (AFM). Such bilayers can function as a model for biological membranes. The first leaflet consisted of zwitterionic phospholipids phosphatidylcholine (PC) or phosphatidylethanolamine (PE). The second leaflet consisted of the anionic phospholipid phosphatidylglycerol (PG), in either the condensed or liquid phase or, for comparison, of PC. Different bilayers showed different morphology. In all bilayers defects in the form of holes were present. In some bilayers with a first leaflet consisting of PC, polygonal line-shaped defects were observed, whereas when the first leaflet consisted of PE, mainly round defects were seen. Not only the shape, but also the amount of defects varied, depending on the condition and the composition of the second leaflet. In most of the PG-containing systems the defects were surrounded by elevations, which reversibly disappeared in the presence of divalent cations. This is the first time that such elevations have been observed on phospholipid bilayers. We propose that they are induced by phospholipid exchange between the two leaflets around the defects, leading to the presence of negatively charged phospholipids in the first leaflet. Because the substrate is also negatively charged, the bilayer around the edges is repelled and lifted up. Since it was found that the elevations are indeed detached from the substrate, we refer to this effect as bilayer blistering.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1998

An investigation into the lipid interactions of peptides corresponding to the C-terminal anchoring domains of Escherichia coli penicillin-binding proteins 4, 5 and 6.

The Escherichia coli low molecular mass penicillin-binding proteins PBP4, PBP5 and PBP6 are DD-peptidases involved in murein biosynthesis. It has been suggested that these proteins may be anchored to the periplasmic face of the inner membrane via their C termini. Here, peptide homologues (P4, P5 and P6) of the PBP4, PBP5 and PBP5 C-terminal regions have been used to investigate potential protein-lipid interactions involved in this anchoring mechanism. Surface pressure changes observed for the interactions of P5 and P6 with a range of monolayers indicated that the peptides are membrane interactive and that the interactions proceeded via predominantly hydrophobic forces with only minor requirements for anionic lipid. In contrast, P4 interactions with monolayers appeared to proceed via predominantly electrostatic forces with a major requirement for anionic lipid. The lipid interactions of all three peptides were generally enhanced by low pH and for P5 and P6 were in the range of 10-15 mN m-1 whereas for P4 interactions they were in the range of 3-7 mN m-1. CD analysis implied the presence of alpha-helical structure in P5 and P6 and molecular area determinations implied that P4 may also possess helical architecture in the presence of dioleoylphosphatidylglycerol monolayers. Overall, our results support the view that C-terminal amphiphilic alpha-helices are involved in the membrane anchoring of PBP5 and PBP6 and suggest that a similar mechanism could contribute to PBP4-membrane anchoring. Furthermore, we have speculated that the presence of cationic residues in the hydrophilic face of these alpha-helices may help facilitate membrane interaction.

Amino Acid Sequence↗
PubMed1998

Pore formation by nisin involves translocation of its C-terminal part across the membrane.

Nisin is an amphiphilic peptide with a strong antimicrobial activity against various Gram-positive bacteria. Its activity results from permeabilization of bacterial membranes, causing efflux of cytoplasmic compounds. To get information on the molecular mechanism of membrane permeabilization, a mutant of nisin Z containing the C-terminal extension Asp-(His)6 was produced. The biological and anionic lipid-dependent membrane activity of this peptide was very similar to that of nisin Z. Analysis of the pH dependence of model membrane interactions with the elongated peptide indicated the importance of electrostatic interactions of the C-terminus with the target membrane for membrane permeabilization. Most importantly, the membrane topology of the C-terminus of the molecule could be determined by trypsin digestion experiments, in which trypsin was encapsulated in the lumen of large unilamellar vesicles. The results show that the C-terminal part of the peptide translocates across model membranes. The pH and anionic lipid dependence of translocation closely paralleled the results of membrane permeabilization studies. Binding of nickel ions to the histidines blocked translocation of the C-terminus and concomitantly resulted in a 4-fold reduced capacity to induce K+ leakage. The results demonstrate for the first time that pore formation of nisin involves translocation of the C-terminal region of the molecule across the membrane.

Amino Acid Sequence↗
PubMed1998

Fructans interact strongly with model membranes.

Bacterial fructans with a high degree of polymerisation cause a very large increase in surface pressure of lipid monolayers at the air-water interface with a broad range of lipids, including phosphatidylethanolamine and several types of phosphatidylcholines. The surface active effect of fructans contrasts strongly with the maximal effects observed for trehalose, sucrose and glucose under comparable conditions (20 and 0.6 mN/m for fructans and the other sugars, respectively). The results demonstrate a profound and specific membrane interaction of the fructans which is probably very different from the effect of the smaller carbohydrates. The fructan concentrations used in this study are within the physiological range observed in fructan-accumulating plants. The suggested water-stress protective effect of fructans may be induced by membrane-fructan interaction which prevent lipid condensation and phase transitions to take place.

Bacillus subtilis↗
PubMed1998

Translocase-bound SecA is largely shielded from the phospholipid acyl chains.

Protein translocation in Escherichia coli is mediated by the SecA ATPase bound to the SecYEG membrane protein complex. SecA translocation ATPase activity as well as protein translocation is dependent on the presence of negatively charged lipids. By using a phospholipid with an acyl chain linked photoactivatable group, the lipid accessibility of SecA bound at the translocase was explored. SecA bound to lipid vesicles containing negatively charged lipids was found to be readily accessible for labeling by the photoactivatable phospholipid. The presence of an excess amount of SecYEG complex resulted in a remarkable reduction in the amount of lipid-accessible SecA irrespective of the nucleotide-bound form of SecA. These data demonstrate that the SecYEG-bound SecA is largely shielded from the phospholipid acyl chains and suggest the presence of two distinct pools of membrane-bound SecA that differ in the degree of lipid association.

Adenosine Triphosphatases↗
PubMed1998

Rapid transmembrane movement of newly synthesized phosphatidylethanolamine across the inner membrane of Escherichia coli.

For the first time the transmembrane movement of an endogenously synthesized phospholipid across the inner membrane of E. coli is reported. [14C]phosphatidylethanolamine (PE) was biosynthetically introduced into inner membrane vesicles from the PE-deficient strain AD93, by reconstitution with the enzyme phosphatidylserine (PS) synthetase. Upon addition of wild type cell lysate containing PS synthetase, and the metabolic substrates CTP and [14C]serine to inside-out vesicles from AD93, [14C]PS was synthesized, which was for the most part converted into [14C]PE. [14C]PE was introduced in right-side out vesicles by enclosing PS synthetase and CTP in the vesicle lumen and adding [14C]serine. The newly synthesized [14C]PE immediately equilibrated over both membrane leaflets (t1/2 less than one min), as determined by its accessibility toward the amino-reactive chemical fluorescamine. In both inside- out and right-side out vesicles, a 35-65% distribution was found of the newly synthesized PE over the cytoplasmic and periplasmic leaflet, respectively. The transport process of PE was not influenced by the presence of ATP or the proton motive force in inside out vesicles. Pretreatment of both types of vesicles with sulfhydryl reagents, or of right-side out vesicles with proteinase K, did not affect the rate and extent of the transmembrane distribution of the newly synthesized PE.

Biological Transport↗
PubMed1998

Phosphatidylethanolamine mediates insertion of the catalytic domain of leader peptidase in membranes.

Leader peptidase is an integral membrane protein of E. coli and it catalyses the removal of most signal peptides from translocated precursor proteins. In this study it is shown that when the transmembrane anchors are removed in vivo, the remaining catalytic domain can bind to inner and outer membranes of E. coli. Furthermore, the purified catalytic domain binds to inner membrane vesicles and vesicles composed of purified inner membrane lipids with comparable efficiency. It is shown that the interaction is caused by penetration of a part of the catalytic domain between the lipids. Penetration is mediated by phosphatidylethanolamine, the most abundant lipid in E. coli, and does not seem to depend on electrostatic interactions. A hydrophobic segment around the catalytically important residue serine 90 is required for the interaction with membranes.

Amino Acid Sequence↗
PubMed1998

The orientation of nisin in membranes.

Nisin is a 34 residue long peptide belonging to the group A lantibiotics with antimicrobial activity against Gram-positive bacteria. The antimicrobial activity is based on pore formation in the cytoplasmic membrane of target organisms. The mechanism which leads to pore formation remains to be clarified. We studied the orientation of nisin via site-directed tryptophan fluorescence spectroscopy. Therefore, we engineered three nisin Z variants with unique tryptophan residues at positions 1, 17, and 32, respectively. The activity of the tryptophan mutants against Gram-positive bacteria and in model membrane systems composed of DOPC or DOPG was established to be similar to that of wild type nisin Z. The tryptophan fluorescence emission maximum showed an increasing blue-shift upon interaction with vesicles containing increased amounts of DOPG, with the largest effect for the 1W peptide. Studies with the aqueous quencher acrylamide showed that all tryptophans became inaccessible from the aqueous phase in the presence of negatively charged lipids in the vesicles. From these results it is concluded that anionic lipids mediate insertion of the tryptophan residues in at least three positions of the molecule into the lipid bilayer. The depth of insertion of the tryptophan residues was determined via quenching of the tryptophan fluorescence by spin-labeled lipids. The results showed that the depth of insertion was dependent on the amount of negatively charged lipids. In membranes containing 50% DOPG, the distances from the bilayer center were determined to be 15.7, 15.0, and 18.4 A for the tryptophan at position 1, 17, and 32, respectively. In membranes containing 90% DOPG, these distances were calculated to be 10.8, 11.5, and 13.1 A, respectively. These results suggest an overall parallel average orientation of nisin in the membrane, with respect to the membrane surface, with the N-terminus more deeply inserted than the C-terminus. These data were used to model the orientation of nisin in the membrane.

Acrylamide↗
PubMed1997

Charge-dependent insertion of beta-lactoglobulin into monoglyceride monolayers.

The interactions between beta-lactoglobulin and 1-monostearoyl-glycerol were studied in order to gain insight into protein-gel-phase monoglyceride interactions. Using a monomolecular layer at the air-water interface, we determined the insertion of beta-lactoglobulin into the monoglycerides under different conditions of protein and surface charge by varying the pH and/or incorporating charged amphiphiles into the monolayer, respectively, and using subphases with either a low or high ionic strength. The interactions were quantified by determining the binding of 14C-labeled beta-lactoglobulin to the monolayer. Our results show the importance of electrostatics for binding of beta-lactoglobulin to condensed monoglycerides. Moreover, electrostatic interactions were found to be important for specific insertion of beta-lactoglobulin into the monolayer. A negatively charged surface in particular allowed positively charged beta-lactoglobulin to insert in a surface charge density-dependent manner, even at surface pressures as high as 36 mN/m, whereas under other conditions, the limiting insertion pressure was 32 mN/m. The rheological properties of the monolayer were not affected by the interactions with beta-lactoglobulin.

Animals↗
PubMed1997

Cisplatin complexes with phosphatidylserine in membranes.

Upon incubation of the anticancer drug cisplatin [cis-diamminedichloroplatinum(II)] with model membranes composed of phosphatidylserine (PS), a stable product is formed that has been isolated after chloroform/methanol extraction of the sample. The product formation is specific for PS and does not occur with other major membrane phospholipids. The rate and extent of product formation is dependent on the pH, chloride ion concentration, and temperature, with the highest rate at pH 6.0, in the absence of Cl- and at 37 degrees C, indicating that positively charged aquated cisplatin is the reactive species. Over 80% of PS is converted within 15 h under these conditions with a halftime of 5 h. PS can be regenerated by an excess of glutathione. Mass spectrometry experiments demonstrate that interaction of cisplatin with PS involves a loss of two chloride ions and coordination of platinum to the amine and carboxyl group of the serine moiety. Cisplatin forms complexes specifically with PS not only in model membranes but also in the plasma membrane of human erythrocytes. Since PS is essential in several cellular processes, its interaction with cisplatin may have important physiological implications.

Antineoplastic Agents↗
PubMed1997

In vitro membrane integration of leader peptidase depends on the Sec machinery and anionic phospholipids and can occur post-translationally.

A cell-free system based on a lysate and membrane vesicles from Escherichia coli is used to study characteristics of the membrane integration reaction of the polytopic membrane protein leader peptidase (Lep). Integration into inverted inner membrane vesicles was detected by partial protection against externally added protease. Integration is most efficient when coupled to translation but can also occur post-translationally and depends on the action of the proteinaceous Sec machinery and availability of anionic phospholipids. Lep is the first example of a membrane protein without cleavable signal sequence which requires anionic lipids for integration in vitro.

Adenosine Triphosphatases↗
PubMed1997

Anionic phospholipids are determinants of membrane protein topology.

The orientation of many membrane proteins is determined by the asymmetric distribution of positively charged amino acid residues in cytoplasmic and translocated loops. The positive-inside rule states that loops with large amounts of these residues tend to have cytoplasmic locations. Orientations of constructs derived from the inner membrane protein leader peptidase from Escherichia coli were found to depend on the anionic phospholipid content of the membrane. Lowering the contents of anionic phospholipids facilitated membrane passage of positively charged loops. On the other hand, elevated contents of acidic phospholipids in the membrane rendered translocation more sensitive to positively charged residues. The results demonstrate that anionic lipids are determinants of membrane protein topology and suggest that interactions between negatively charged phospholipids and positively charged amino acid residues contribute to the orientation of membrane proteins.

Alkaline Phosphatase↗
PubMed1997

The anionic phospholipid-mediated membrane interaction of the anti-cancer drug doxorubicin is enhanced by phosphatidylethanolamine compared to other zwitterionic phospholipids.

The interaction of doxorubicin and lipids has been studied using large unilamellar vesicles (LUVET) composed of mixtures of anionic phospholipids and various zwitterionic phospholipids. Dilution of anionic lipids with zwitterionic lipids leads to decreased membrane association of the drug because electrostatic forces are very important in doxorubicin-membrane interaction. However, binding of doxorubicin to LUVET composed of anionic phospholipids combined with phosphatidylethanolamine (PE) is much higher than binding to LUVET made of anionic lipids plus a range of other zwitterionic lipids such as phosphatidylcholine (PC) and the N-methylethanolamine and N, N-dimethylethanolamine derivatives of PE. This preferential interaction is observed with all negatively charged phospholipids tested and is, in the case of phosphatidylserine (PS), confirmed in monolayer experiments. The increase in surface area observed in a monolayer composed of PS and PE (1/3) was 3 times higher than in a monolayer of PS/PC (1/3). The preferential interaction appears not to be due to the ability of PE to adopt inverted nonbilayer structures, but probably involves a combination of the ability of PE to form additional hydrogen bonds and of the intrinsic curvature of a bilayer containing PE because of its small headgroup. Implications of our finding for the in vivo membrane interaction and transport of the drug will be discussed.

Anions↗