Biomedical subjects

B De Kruijff

Publications and source records attributed to B De Kruijff.

At least 19 recordsLinked to original sources

PubMed2001

Fructans insert between the headgroups of phospholipids.

Fructans are polysaccharides consisting of one glucose unit and two or more fructose units. It was hypothesized that fructans play a role in drought tolerance in plants by interacting directly with the membrane. In this paper we investigated this hypothesis by studying fructan-membrane interactions in hydrated mono- and bilayer systems. It was found that fructans inserted between the headgroups of different kinds of phospholipids with some preference for phosphatidylethanolamine. Insertion occurred even under conditions of very tight lipid packing. The presence of a surface associated layer of fructan was observed in both model systems. This layer was able to reduce the ability of a surface-active protein to interact with the lipids. Fructans showed a much stronger effect on the different lipid systems than other (poly)saccharides, which appears to be related to their hydrophobic properties. Fructans were able to stabilize the liquid-crystalline lamellar phase, which is consistent with a drought protecting role in plants.

Calorimetry, Differential Scanning↗

PubMed2000

Role of lipids in the translocation of proteins across membranes.

The architecture of cells, with various membrane-bound compartments and with the protein synthesizing machinery confined to one location, dictates that many proteins have to be transported through one or more membranes during their biogenesis. A lot of progress has been made on the identification of protein translocation machineries and their sorting signals in various organelles and organisms. Biochemical characterization has revealed the functions of several individual protein components. Interestingly, lipid components were also found to be essential for the correct functioning of these translocases. This led to the idea that there is a very intimate relationship between the lipid and protein components that enables them to fulfil their intriguing task of transporting large biopolymers through a lipid bilayer without leaking their contents. In this review we focus on the Sec translocases in the endoplasmic reticulum and the bacterial inner membrane. We also highlight the interactions of lipids and proteins during the process of translocation and integrate this into a model that enables us to understand the role of membrane lipid composition in translocase function.

Adenosine Triphosphatases↗

PubMed1999

Interaction of the anti-cancer drug cisplatin with phosphatidylserine in intact and semi-intact cells.

The anti-cancer drug cisplatin (cis-diamminedichloroplatinum(II)) forms a stable coordination complex with phosphatidylserine (PS) in model membrane systems (Speelmans et al., Biochemistry 36 (1997) 10545-10550). Because a similar interaction in vivo would be expected to have important physiological implications we studied cisplatin-PS interaction in human erythrocytes and tumor cell lines. Although cisplatin was efficiently taken up by intact erythrocytes, a cisplatin-PS complex was only detected in cells which had lysed as a result of prolonged storage or hypotonic shock. Despite the use of highly sensitive detection methods, and despite efficient cellular uptake of cisplatin, a complex could also not be detected in four human tumor cell lines, unless cells were permeabilized. In experiments in which cisplatin was incubated with PS-containing liposomes in the presence of an alternative cellular substrate, such as reduced glutathione, the relative affinity of cisplatin for PS was found to be low. Moreover, loading erythrocyte ghosts with physiological concentrations of glutathione strongly reduced cisplatin-PS complexation. Thus, in intact (tumor) cells a complex is not detected, most likely, because of the presence of higher affinity substrates. Though a transient complexation of cisplatin to PS cannot be excluded, our data suggest that cisplatin-PS does not play a direct role in the cellular (cyto)toxicity of cisplatin.

Antineoplastic Agents↗

PubMed1997

Influence of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling capacity.

Three mutants of the antibiotic nisin Z, in which the Val32 residue was replaced by a Glu, Lys or Trp residue, were produced and characterized for the purpose of establishing the role of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling properties. 1H-NMR analyses showed that all three mutants harbor an unmodified serine residue at position 33, instead of the usual dehydroalanine. Apparently, the nature of the residue preceding the serine to be dehydrated, strongly affects the efficiency of modification. Cleavage of [Glu32,Ser33]nisin Z by endoproteinase Glu-C yielded [Glu32]nisin Z(1-32)-peptide, which has a net charge difference of -2 relative to wild-type nisin Z. The activity of [Lys32,Ser33]nisin Z against Micrococcus flavus was similar to that of wild-type nisin, while [Trp32,Ser33]nisin Z, [Glu32,Ser33]nisin Z and [Glu32]nisin Z(1-32)-peptide exhibited 3-5-fold reduced activity, indicating that negative charges in the C-terminal part of nisin Z are detrimental for activity. All variants showed significant loss of activity against Streptococcus thermophilus. The potency of the nisin variants to act as signaling molecules for auto-induction of biosynthesis was significantly reduced. To obtain mutant production, extracellular addition of (mutant) nisin Z to the lactococcal expression strains was essential.

Amino Acid Sequence↗

PubMed1996

Influence of the signal sequence and chaperone SecB on the interaction between precursor protein prePhoE and phospholipids.

To investigate in a direct way the interaction between a precursor protein and phospholipids, monolayer studies were performed using the purified precursor of Escherichia coli outer-membrane protein PhoE. It was demonstrated that prePhoE can insert efficiently into monolayers of dioleoylglycerophosphoglycerol (Ole2GroPGro) and dioleoylglycerophosphoethanolamine (Ole2GroPEtn), this insertion was mainly driven by hydrophobic forces. Compared with previous results obtained with PhoE signal peptide, the full-length precursor protein does not show the specific interaction with acidic lipids. PrePhoE inserted into a Ole2GroPGro monolayer occupies an area of 28 +/- 3 [corrected] nm2/molecule, which is approximately 10-fold larger than the area occupied by the PhoE signal peptide. The purified mature PhoE protein has a lower capacity to insert into Ole2GroPGro and Ole2GroPEtn monolayers and is, in contrast to prePhoE, fully accessible to proteinase K after interacting with a Ole2GroPGro monolayer. The results demonstrate that in the context of the precursor protein both the signal sequence and mature domain of prePhoE insert into lipid monolayers. It was found that PhoE, like prePhoE, can form in vitro a complex with the cytosolic chaperone SecB. Complexation with SecB increases the insertion of (pre)PhoE into acidic lipid monolayers. The high lipid affinity of prePhoE was also demonstrated by vesicle-binding experiments which showed that SecB dissociates from the SecB-prePhoE complex upon binding of the precursor to the bilayer. The implications of these findings for preprotein translocation are discussed and in addition some extrapolations to the insertion of PhoE into the outer membrane are made.

Bacterial Proteins↗

PubMed1996

Nisin Z, mutant nisin Z and lacticin 481 interactions with anionic lipids correlate with antimicrobial activity. A monolayer study.

Monomolecular layers of lipids at the air/water interface have been used as a model membrane to study membrane interactions of the lantibiotic nisin. The natural lantibiotics nisin A and nisin Z proved to have a high affinity for the anionic lipids phosphatidylglycerol and bis(phosphatidyl)glycerol (cardiolipin). The interaction with zwitterionic phopholipids or neutral lipids is very low at surface pressures higher than 32 mN/m. Nisin, nisin mutants and lacticin 481 show a remarkable correlation between anti-microbial activity and anionic lipid interaction. The results indicate that primarily the N-terminal part (residues 1-22) penetrates into the lipid phase. Reduction of the flexibility at positions 20-21 has a negative effect on monolayer interaction and activity. The C-terminal part is probably responsible for ionic interactions of nisin in monomeric or oligomeric form with anionic lipids. In mixtures of anionic and zwitterionic lipids maximal interactions are found at approximately 70 mol/100 mol anionic lipid. Gram-positive bacteria, which form the main target for nisin, are characterized by a high content of anionic lipids in the membrane. Monolayers formed of lipid extracts of bacteria sensitive to nisin were more strongly penetrated than those of bacteria relatively insensitive to nisin.

Amino Acid Sequence↗

PubMed1995

Verapamil competes with doxorubicin for binding to anionic phospholipids resulting in increased internal concentrations and rates of passive transport of doxorubicin.

It is well documented that the Ca2+ channel antagonist verapamil can reverse multidrug resistance in cancer cells by decreasing P-glycoprotein mediated drug efflux. However, less information is available about effects of verapamil on drug-phospholipid interactions and on passive diffusion of drugs across the membrane, which both may play an important role in resensitizing cells to anti-cancer drugs. Therefore we studied the binding of verapamil to model membranes (large unilamellar vesicles) composed of various phospholipids and biological membranes. An increase of the amount of anionic phospholipids resulted in an enhanced binding of verapamil. Competition between verapamil and the anti-cancer drug and P-glycoprotein substrate doxorubicin for binding to anionic phospholipids was observed in model membranes composed of synthetic lipids, or composed of native Escherichia coli phospholipid mixtures, and in cytoplasmic membrane vesicles of this organism. Furthermore, verapamil specifically increased the rate of passive diffusion of doxorubicin across model membranes containing anionic phospholipids. It can be concluded that besides the decrease of P-glycoprotein mediated efflux at least two other effects may account for an increase of the internal (free and DNA-bound) doxorubicin concentration in the presence of verapamil; (i) a decrease of binding to anionic phospholipids in plasma-and intracellular membranes and (ii) an increase of the rate of passive import of doxorubicin across the plasma membrane.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Membrane potential-driven translocation of a lipid-conjugated rhodamine.

The present study demonstrates that the permanently positively charged, lipid-conjugated rhodamine, R18, can be transported from the outer to the inner leaflet of lipid bilayers in response of a transmembrane potential (negative inside). This conclusion was based on the following observations. (i) A fast decrease of the R18 fluorescence, when present at self-quenching concentrations in DOPC large unilamellar vesicles, was revealed upon induction of a valinomycin-induced K(+)-diffusion potential. (ii) Iodide quenching experiments demonstrated that R18 was no longer accessible to externally added aqueous quencher after application of a transmembrane potential. (iii) 2H-NMR measurements, using DOPC, specifically deuterated at the alpha-position of the phosphocholine head group, revealed a massive transbilayer movement of R18 upon induction of a membrane potential. The extent of the fluorescence changes were found to be dependent on the magnitude of the applied transmembrane potential, which opens possibilities for novel applications of R18 as an internal lipid-conjugated membrane potential probe.

Biological Transport↗

PubMed1995

Characterization of the resonance energy transfer couple coumarin-BODIPY and its possible applications in protein-lipid research.

In our search for suitable resonance energy transfer (RET) couples for studying protein-lipid interactions, the promising couple coumarin-BODIPY was found and characterized. We characterized the RET from the donor coumarin to two different dipyrrometheneboron difluoride (BODIPY)-labeled phospholipid analogs both experimentally and theoretically. Calculations using the spectral overlap revealed a Förster energy transfer radius (RO) of 50 +/- 2 A and 40 +/- 2 A for the coumarin-(beta-BODIPY FL C12-HPC) and the coumarin-(beta-BODIPY 530/550 C12-HPC) couple respectively. Experimentally this was estimated to be 49.0-51.5 A and 38.5-42.5 A respectively. The use of this couple for studying protein-lipid interactions is exemplified by measurements on a bacterial precursor protein.

Bacterial Proteins↗

PubMed1994

The influence of acylation on the lipid structure modulating properties of the transmembrane polypeptide gramicidin.

In order to get insight into the effect of acylation of a transmembrane polypeptide on the interaction of the polypeptide with the membrane lipids we used 31P-NMR to investigate the influence of acylated gramicidins on the polymorphic phase behavior of hydrated dispersions of 1-palmitoyllysophosphatidylcholine (lyso-PC), 1,2-dioleoylphosphatidylcholine (DOPC) and 1,2-dielaidoylphosphatidylethanolamine (DEPE). Palmitoylgramicidin induces a micelle to extended bilayer organization in lyso-PC with a slightly lower efficiency than the parent gramicidin molecule. In DOPC and DEPE acylgramicidins induce the formation of HII phase at the expense of a bilayer organization with a similar high efficiency as gramicidin. The ability of acylgramicidin to induce lipid mixing between vesicles prepared of DOPC was decreased relative to gramicidin. The results are discussed in the light of the proposed models for gramicidin-induced HII phase formation and emphasize that gramicidin itself has a very strong lipid structure modulating activity.

Acylation↗

PubMed1994

Structure and dynamics of the acyl chain of a transmembrane polypeptide.

We have used acylated analogs of gramicidin as a model to study the interaction between a covalently coupled fatty acid and the hydrophobic part of a membrane-spanning protein in a bilayer environment. The acyl chain was covalently coupled to the C-terminal ethanolamine group of gramicidin which is located near the membrane interface, mimicking a situation found in acylated proteins. Either perdeuterated palmitic acid or palmitic acid deuterated at only C2, C3, C5-6, C7-8, C9, or C13 was coupled to gramicidin and examined by 2H-NMR in oriented bilayers of dimyristoylphosphatidylcholine. In this way, quadrupolar splittings of deuterons at specific carbons were assigned. The quadrupolar splittings and T1 values were compared to those of free palmitic acid in oriented bilayers, with and without gramicidin. The results indicate that the covalently coupled fatty acid is highly immobilized near the carboxyl terminus because double quadrupolar splittings and very low T1 values (4 ms) were found for the -CD2- deuterons at carbon atoms C2 and C3. Control experiments with free fatty acid showed single quadrupolar splittings and higher T1 values for this segment of the fatty acid. Molecular modeling of the carboxy-terminal segment of the covalently coupled acyl chain suggested that it has a defined structure with a bend near its attachment site. In contrast, the methyl end (C10-C16) of the covalently coupled fatty acid had quadrupolar splittings and T1 values very similar to those found for free fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Gramicidin↗

PubMed1992

In-vitro studies on the folding characteristics of the Escherichia coli precursor protein prePhoE. Evidence that SecB prevents the precursor from aggregating by forming a functional complex.

We characterised the behaviour of the purified precursor protein prePhoE upon dilution from 8 M urea by CD, fluorescence spectroscopy and gel-filtration techniques. It is demonstrated that prePhoE rapidly adopts beta structure, folds and aggregates upon dilution to urea concentrations below 3 M. These processes are paralleled by a loss of translocation competence. Furthermore the interaction of prePhoE with SecB was investigated. SecB is shown to have a very high content of beta structure, therefore we propose that precursor recognition by SecB is mediated through beta-beta interaction. It is shown that SecB has little effect on the adoption of secondary structure and tertiary folding upon dilution of the precursor from urea. However, SecB prevents the precursor from aggregating by forming a functional and stable complex.

Bacterial Outer Membrane Proteins↗

PubMed1992

Influence of acylation on the channel characteristics of gramicidin A.

The influence of acylation on the conductance, average duration, and channel-forming potency of channels formed by gramicidin A analogues was investigated using single-channel and multichannel techniques. Lauroyl-, myristoyl-, palmitoyl-, stearoyl-, and oleoylgramicidin A were prepared by covalent coupling of that fatty acid to the C-terminal ethanolamine group. Acylation of gramicidin A does not affect the single-channel conductance or the minichannel frequency in diphytanoylphosphatidylcholine/n-decane black lipid membranes. However, the average duration of all acylgramicidin channels was increased approximately 5-fold as compared to unmodified gramicidin A, which has a duration of 0.9 s at 200-mV applied potential. Somewhat surprisingly the rate of channel formation of the acylgramicidins is decreased relative to gramicidin A: lauroyl- and stearoylgramicidin are approximately 200 times less effective in channel formation as compared to gramicidin A. We conclude that channels formed by the acylgramicidins and by gramicidin A are structurally and conformationally equivalent.

Acylation↗

PubMed1991

Synthesis of acylated gramicidins and the influence of acylation on the interfacial properties and conformational behavior of gramicidin A.

Five gramicidin A analogs were synthesized in which various acyl chains, differing in length and unsaturation, were covalently coupled to the C-terminal ethanolamine group. The analogs were characterized by various spectroscopic techniques and their molecular properties were investigated using monolayer techniques and circular dichroism. It is demonstrated that neither the interfacial properties nor the conformational behavior of gramicidin A at the air/water interface are seriously affected upon acylation. It is proposed that at the limiting area the gramicidin molecule is oriented with its C-terminus towards the subphase with the covalently coupled acylchain located parallel to the helical axis in between the protruding tryptophans. Circular dichroism experiments, in which gramicidin-containing vesicles were prepared from different organic solvents, indicate that the presence of a covalently coupled fatty acylchain tends to stabilize the beta 6.3 helical conformation. It is demonstrated that, like for gramicidin A, also for the acylgramicidins the single-stranded beta 6.3 helical conformation, or channel conformation, is the preferred conformation upon incorporation in bilayers.

Acylation↗

PubMed1990

The role of charge and hydrophobicity in peptide-lipid interaction: a comparative study based on tryptophan fluorescence measurements combined with the use of aqueous and hydrophobic quenchers.

The interaction of interrelated model peptides with model membranes has been studied by techniques based on tryptophan fluorescence. The peptides used are derivatives of the sequence H-Ala-Met-Leu-Trp-Ala-OH, which was designed for this purpose. Several modifications yielded a set of 13 penta- and hexapeptides varying in net charge, hydrophobicity, charge distribution, and the intramolecular position of the tryptophan residue with respect to the charge(s). The affinity of these peptides for small unilamellar vesicles (SUV) consisting of zwitterionic egg phosphatidylcholine (eggPC) and negatively charged beef heart cardiolipin (bhCL) has been investigated in a comparative way. The criteria for affinity comprise (1) intrinsic fluorescence changes upon titration of the peptides with the lipid vesicles, (2) reduced accessibility of the peptides to aqueous quenchers of tryptophan fluorescence (I- and acrylamide) in the presence of lipid, and (3) exposure to membrane-incorporated fluorescence quenchers, brominated phosphatidylcholines (BrPC). Application of BrPC brominated at different positions along the acyl chains provided information on the membrane topology of the peptides. With respect to the extent of affinity for zwitterionic membranes, the overall hydrophobicity of the peptides is the main determinant. A comparison of the affinity for PC of equally hydrophobic peptides carrying either a single positive or negative charge reveals preferential interaction of the cationic peptide. Both hydrophobic and electrostatic interactions determine the affinity of positively charged mono- and divalent peptides for CL vesicles. The distribution of the charged moieties in divalent positively charged peptides, either both at one end of the molecule or one at each end, has little influence on the affinity of these peptides for CL but does affect the extent of exposure to BrPC. Upon decreasing the surface charge density of the vesicles by diluting CL with increasing amounts of PC, both types of peptides show different behavior. The position of the tryptophan relative to the charged moiety in the peptide molecule is shown to affect the fluorescent properties upon interaction with vesicles. Concerning the membrane topology, all peptides adopt a localization near the membrane surface, with the neutral peptides inserting slightly deeper into the bilayer than the charged peptides. The results allow a comparative analysis of the factors determining the extents and modes of lipid-model peptide interaction; in addition, the validity of the methods applied is discussed.

Amino Acid Sequence↗

PubMed1989

Inhibition of PhoE translocation across Escherichia coli inner-membrane vesicles by synthetic signal peptides suggests an important role of acidic phospholipids in protein translocation.

To obtain insight into the mechanism of precursor protein translocation across membranes, the effect of synthetic signal peptides and other relevant (poly)peptides on in vitro PhoE translocation was studied. The PhoE signal peptide, associated with inner membrane vesicles, caused a concentration-dependent inhibition of PhoE translocation, as a result of a specific interaction with the membrane. Using a PhoE signal peptide analog and PhoE signal peptide fragments, it was demonstrated that the hydrophobic part of the peptide caused the inhibitory effect, while the basic amino terminus is most likely important for an optimal interaction with the membrane. A quantitative analysis of our data and the known preferential interaction of synthetic signal peptides with acidic phospholipids in model membranes strongly suggest the involvement of negatively charged phospholipids in the inhibitory interaction of the synthetic PhoE signal peptide with the inner membrane. The important role of acidic phospholipids in protein translocation was further confirmed by the observation that other (poly)peptides, known to have both a high affinity for acidic lipids and hydrophobic interactions with model membranes, also caused strong inhibition of PhoE translocation. The implication of these results with respect to the role of signal peptides in protein translocation is indicated.

Amino Acid Sequence↗

PubMed1988

Relationship between gramicidin conformation dependent induction of phospholipid transbilayer movement and hexagonal HII phase formation in erythrocyte membranes.

Addition of gramicidin in sufficient concentration from dimethylsulfoxide or trifluoroethanol to isolated erythrocyte membranes induces hexagonal HII phase formation for the phospholipids. In contrast, addition from ethanol does not change the overall bilayer organization despite a similar extent of peptide incorporation. The same solvent dependence is observed for the enhancement of transbilayer reorientation of lysophospholipids and unspecific leak formation in intact erythrocytes at lower gramicidin concentrations. These results indicate that the (beta 6.3) conformation of the peptide is essential for all three membrane perturbing effects.

Chemical Phenomena↗

PubMed1988

Influence of heme and importance of the N-terminal part of the protein and physical state of model membranes for the apocytochrome c-lipid interaction.

The interaction between cytochrome c and its heme-free precursor apocytochrome c and chemically prepared fragments of these basic proteins with phosphatidylserine containing model membrane systems was studied by differential scanning calorimetry and carboxyfluorescein release experiments. Addition of apocytochrome c and fragments derived from the N-terminus cause a pronounced and linear decrease of the enthalpy (delta H) of the gel to liquid-crystalline phase transition of dielaidoylphosphatidylserine. In contrast, fragments derived from the C-terminus cause a smaller reduction in delta H; a similar trend was observed for the ability of the fragments to cause an increased carboxyfluorescein release from unilamellar vesicles. In addition, the covalent attachment of the heme at cysteine residues 14 and 17 greatly reduced the ability of both the intact protein and the N-terminal fragments to decrease delta H. Using a protein translocation assay based on large unilamellar vesicles containing enclosed trypsin it was found that at gel state temperatures the ability of apocytochrome c to partially translocate the bilayer (reach the opposite membrane/water interface) was greatly reduced. The implications of these findings for the import mechanism of apocytochrome c in mitochondria are shortly indicated.

Animals↗