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V Chupin

Publications and source records attributed to V Chupin.

16 recordsLinked to original sources
PubMed2003

Effect of phospholipids and a transmembrane peptide on the stability of the cubic phase of monoolein: implication for protein crystallization from a cubic phase.

The cubic phase of monoolein has successfully been used for crystallization of a number of membrane proteins. However, the mechanism of protein crystallization in the cubic phase is still unknown. It was hypothesized, that crystallization occurs at locally formed patches of bilayers. To get insight into the stability of the cubic phase, we investigated the effect of different phospholipids and a model transmembrane peptide on the lipid organization in mixed monoolein systems. Deuterium-labeled 1-oleoyl-rac-[(2)H(5)]-glycerol was used as a selective probe for (2)H NMR. The phase behavior of the phospholipids was followed by (31)P NMR. Upon incorporation of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, or phosphatidic acid, the cubic phase of monoolein transformed into the L(alpha) or H(II) phase depending on the phase preference of the phospholipid and its concentration. The ability of phospholipids to destabilize the cubic phase was found to be dependent on the phospholipid packing properties. Electrostatic repulsion facilitated the cubic-to-L(alpha) transition. Incorporation of the transmembrane peptide KALP31 induced formation of the L(alpha) phase with tightly packed lipid molecules. In all cases when phase separation occurs, monoolein and phospholipid participate in both phases. The implications of these findings for protein crystallization are discussed.

Crystallization↗
PubMed2002

Thermotropic phase behavior of monoglyceride-dicetylphosphate dispersions and interactions with proteins: a (2)H and (31)P NMR study.

The phase behavior of a 1-[(2)H(35)]-stearoyl-rac-glycerol ([(2)H(35)]-MSG)/dicetylphosphate (DCP) mixture and its interaction with beta-lactoglobulin and lysozyme were studied by (2)H and (31)P nuclear magnetic resonance (NMR). The behavior of the lipids was monitored by using deuterium-labeled [(2)H(35)]-MSG as a selective probe for (2)H NMR and DCP for (31)P NMR. Both (2)H and (31)P NMR spectra exhibit characteristic features representative of different phases. In the lamellar phases, (31)P NMR spectra of DCP are different from the spectra of natural phospholipids, which is attributable to differences in the intramolecular motions and the orientation of the shielding tensor of DCP compared with phospholipids. The presence of the negatively charged amphiphile DCP has a large effect on the phase behavior of [(2)H(35)]-MSG. At low temperature, the presence of DCP inhibits crystallization of the gel phase into the coagel. Upon increasing the temperature, the gel phase of [(2)H(35)]-MSG transforms in the liquid-crystalline lamellar phase. In the presence of DCP, the gel phase directly transforms into an isotropic phase. The negatively charged beta-lactoglobulin and the positively charged lysozyme completely neutralize the destabilizing effect of DCP on the monoglyceride liquid-crystalline phase and they even stabilize this phase. Without DCP the proteins do not seem to interact with the monoglyceride. These results suggest that interaction is facilitated by electrostatic interactions between the negatively charged DCP and positively charged residues in the proteins. In addition, the nonbilayer-forming DCP creates insertion sites for proteins in the bilayer.

Animals↗
PubMed2002

The effect of protein stability on protein-monoglyceride interactions.

We have previously shown that proteins such as beta-lactoglobulin and lysozyme insert into monoglyceride monolayers and are able to induce an L(beta) to coagel phase transition in monoglyceride bilayers. These studies gave a first indication that protein stability could be an important factor for these interactions. This study therefore aims at further investigating the potential role of protein stability on protein-monoglyceride interactions. To this end we studied the interaction of stable and destabilized alpha-lactalbumin with monostearoylglycerol. Our results show that protein stability is important for the insertion of proteins into a monostearoylglycerol monolayer, such that the lower the stability of the protein the better the protein inserts. In marked contrast to beta-lactoglobulin and lysozyme we found that destabilized alpha-lactalbumin does not induce the L(beta) to coagel phase transition in monoglyceride bilayers. We propose that this is due to an increased surface coverage by the protein which could result from the unfolding of the protein upon binding to the interface.

Calorimetry, Differential Scanning↗
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↗
PubMed2001

The specificity of monoglyceride-protein interactions and mechanism of the protein induced L(beta) to coagel phase transition.

This study aims at gaining insight into the specificity and molecular mechanism of monoglyceride-protein interactions. We used beta-lactoglobulin (beta-LG) and lysozyme as model proteins and both monostearoylglycerol and monopalmitoylglycerol as defined gel phase monoglycerides. The monoglycerides were used in different combinations with the two negatively charged amphiphiles dicetylphosphate and distearylphosphate. The interactions were characterized using the monolayer technique, isothermal titration calorimetry, (2)H-nuclear magnetic resonance (NMR) using deuterium labelled monoglycerides and freeze fracture electron microscopy (EM). Our results show that lysozyme inserts efficiently into all monolayers tested, including pure monoglyceride layers. The insertion of beta-LG depends on the lipid composition of the monolayer and is promoted when the acylchains of the negatively charged amphiphile are shorter than that of the monoglyceride. The binding parameters found for the interaction of beta-LG and lysozyme with monoglyceride bilayers were generally similar. Moreover, in all cases a large exothermic binding enthalpy was observed which was found to depend on the nature of the monoglycerides but not of the proteins. (2)H-NMR and freeze fracture EM showed that this large enthalpy results from a protein mediated catalysis of the monoglyceride L(beta) to coagel phase transition. The mechanism of this phase transition consists of two steps, an initial protein mediated vesicle aggregation step which is followed by stacking and probably fusion of the bilayers.

Freeze Fracturing↗
PubMed2001

Lipid organization and dynamics of the monostearoylglycerol-water system. A 2H NMR study.

Deuterium labeled monostearoylglycerols with fully ([2H(35)]-MSG) and selectively ([11-(2)H(2)]-MSG) deuterated chains have been synthesized and used as a probe for 2H NMR. At low temperature monoglyceride-water systems form the coagel or crystalline phase, which transforms with increasing temperature subsequently into the gel, liquid crystalline and cubic phase. The 2H NMR spectra exhibit characteristic features representative of these phases. The gel phase is metastable and gradually transforms into the coagel at temperatures below 40 degrees C. The undercooled cubic phase transforms into the liquid crystalline phase during days. In the liquid crystalline phase, the chain order profile indicates an increase of the chain flexibility towards the methyl group. In the liquid crystalline phase, bilayers spontaneously align in a magnetic field with their normal perpendicular to the field. The results demonstrate that 2H NMR can serve as a convenient tool to study both structure and dynamics of different monoglyceride-water phases.

Crystallization↗
PubMed1999

Interaction mode specific reorganization of gel phase monoglyceride bilayers by beta-lactoglobulin.

The interaction between beta-lactoglobulin and sonicated aqueous dispersions of the gel phase forming monoglyceride monostearoylglycerol were studied using isothermal titration calorimetry, direct binding experiments, differential scanning calorimetry, leakage of a fluorescent dye and solid-state (31)P- and (2)H-NMR. In the absence of a charged amphiphile, monostearoylglycerol forms a precipitate. Under these conditions, no interaction with beta-lactoglobulin was observed. In the presence of the negatively charged amphiphile dicetylphosphate, the gel phase monostearoylglycerol formed stable and closed, probably unilamellar, vesicles with an average diameter of 465 nm. beta-Lactoglobulin interacts with these bilayer structures at pH 4, where the protein is positively charged, as well as at pH 7 where the protein is negatively charged. Under both conditions of pH, the binding affinity of beta-lactoglobulin is in the micromolar range as observed with ITC and the direct binding assay. At pH 4, two binding modes were found, one of which is determined with ITC while the direct binding assay determines the net result of both. The first binding mode is observed with ITC and is characterized by a large binding enthalpy, a decreased enthalpy of the MSG L(beta) to L(alpha) phase transition and leakage of a fluorescent dye. These characteristics are explained by a beta-lactoglobulin induced partial L(beta) to coagel phase transition that results from a specific electrostatic interaction between the protein and the charged amphiphile. This explanation is confirmed by solid-state (2)H-NMR using 1-monostearoylglycerol with a fully deuterated acyl chain. Upon interaction with beta-lactoglobulin, the isotropic signal in the (2)H-NMR spectrum of the monostearoylglycerol-dicetylphosphate mixture partially transforms into a broad anisotropic signal which could be assigned to coagel formation. The second binding mode probably results from an aspecific electrostatic attraction between the negatively charged bilayer and the positively charged protein and causes the precipitation of the dispersion. At pH 7, only the first binding mode is observed.

Animals↗
PubMed1996

Secondary structure and topology of a mitochondrial presequence peptide associated with negatively charged micelles. A 2D H-NMR study.

In this study the secondary structure and topology of the peptide, corresponding to the presequence of cytochrome oxidase subunit IV (p25) in a negatively charged membrane-mimetic environment, were assessed by circular dichroism and two-dimensional nuclear magnetic resonance. The micelles used consisted of dodecylphosphoglycol (DPG), a mild anionic detergent with a headgroup resembling that of phosphatidylglycerol. The secondary structure was analyzed by interresidue nuclear Overhauser enhancement measurements and chemical shifts of backbone protons. The data revealed alpha-helix formation of the peptide upon interaction with the micelles, both in the N- and in the C-terminal halves, which are separated from each other by the proline residue at position 13. The topology of the peptide was studied by determining the effect of spin-labeled 12-doxylstearate on the line widths of the peptide proton resonances. This method revealed the insertion of hydrophobic residues of both the N- and the C-terminal halves of p25 into the hydrophobic environment of the micelles, demonstrating the orientation of the amphiphilic helix.

Amino Acid Sequence↗
PubMed1995

Molecular basis of glycoalkaloid induced membrane disruption.

In this study the interaction between the glycoalkaloids alpha-chaconine, alpha-solanine and alpha-tomatine and sterols in model membranes was analysed systematically using techniques like membrane leakage, binding experiments, detergent extraction, electron microscopy, NMR and molecular modelling. The most important properties for sterols to interact with glycoalkaloids turned out to be a planer ring structure and a 3 beta-OH group, whereas for alpha-chaconine the 5-6 double bond and the 10-methyl group were also of importance. The importance of sugar-sugar interactions was illustrated by the high synergistic effect between alpha-chaconine and alpha-solanine, the leakage enhancing effect of glycolipids, and the almost complete loss of activity after deleting one or more mono-saccharides from the glycoalkaloids. The formed complexes which were resistant against detergent extraction existed of glycoalkaloid/sterol in a 1:1 ratio and formed tubular structures (alpha-chaconine) with an inner monolayer of phospholipids, whereas with alpha-tomatine also spherical structures were formed. Based on the results a molecular model for glycoalkaloid induced membrane disruption is presented.

Carbohydrate Sequence↗
PubMed1995

Cardiolipin modulates the secondary structure of the presequence peptide of cytochrome oxidase subunit IV: a 2D 1H-NMR study.

The secondary structure of the presequence of cytochrome oxidase subunit IV (p25) was studied by circular dichroism and 2D nuclear magnetic resonance in micelles of dodecylphosphocholine (DPC) and mixed micelles of DPC and mitochondrial cardiolipin (CL). In both systems, alpha-helix formation was observed. The alpha-helix stretches from the N- to the C-terminus with a break at the proline residue at position 13. Upon introduction of CL in the DPC micellar system, an increased stability of the helix was observed around proline13 and in the C-terminal half. This observation, together with reported results on specific interactions between CL and p25, led to the proposal of a two-state equilibrium of the alpha-helical conformation of p25, modulated by CL.

Amino Acid Sequence↗
PubMed1995

PhoE signal peptide inserts into micelles as a dynamic helix-break-helix structure, which is modulated by the environment. A two-dimensional 1H NMR study.

Proteins that are destined for export out of the cytoplasm of Escherichia coli cells are synthesized as precursor proteins with N-terminal extensions or signal sequences, which are essential for translocation of the protein across the inner membrane. Signal sequences contain very little primary sequence homology, and therefore recognition of these sequences is thought to involve specific folding. To assess the conformational flexibility of signal sequences, we have studied the signal peptide of PhoE (MKKSTLALVVMGIVASASVQA) by two-dimensional nuclear magnetic resonance and circular dichroism in different membrane mimetic environments. The secondary structure of the PhoE signal peptide was analyzed via interresidue nuclear Overhauser enhancement measurements, chemical shifts of backbone protons, and by measuring amide proton exchange. The membrane mimetic environments studied were trifluoroethanol (TFE) and micelles of sodium dodecyl sulfate (SDS) or dodecylphosphocholine (DPC). In all systems alpha-helix formation was observed. In TFE, the alpha-helix stretches from the positively charged N-terminus to Ser18. In SDS and DPC micelles, the N- and C-terminal alpha-helical half are separated from each other by a kink at the Gly12 position, with the helical content being higher at the N-terminus and lower at the C-terminus. In zwitterionic DPC micelles, the C-terminal region has a less regular or more flexible structure compared to SDS. The insertion of the PhoE signal peptide into the hydrophobic environment of the micelles was demonstrated by the effect of spin-labeled 12-doxylstearate on the line widths of the peptide proton resonances.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

The transit sequence of a chloroplast precursor protein reorients the lipids in monogalactosyl diglyceride containing bilayers.

The interaction of the chloroplast precursor protein of ferredoxin with mixed model membranes composed of 2H chain labeled monogalactosyl diacylglycerol and phosphatidylcholine was studied by 2H and 31P NMR. The bilayers were found to have special chain packing properties which most likely are the result of a specific arrangement of head groups at the interface. The precursor and not the corresponding apoprotein induced a bilayer-->isotropic transition in lipid organization as a result of the transit sequence-lipid interaction. The implications of these observations for proteins import into chloroplasts are indicated.

Biological Transport, Active↗
PubMed1994

A general method for the preparation of mixed micelles of hydrophobic peptides and sodium dodecyl sulphate.

A new method is reported for the incorporation of hydrophobic peptides into sodium dodecyl sulphate (SDS) micelles. First, a homogeneous solution of peptide and detergent is obtained by adding the peptide in trifluoroethanol to an equal volume of an aqueous solution of SDS. Upon subsequent addition of excess water, mixed peptide-SDS micelles are formed. Next, all solvent is removed by lyophilization and an appropriate amount of water is added to the dry powder. For various hydrophobic peptides this was shown to yield clear and stable solutions that are highly concentrated and suitable for characterization by spectroscopic techniques.

Amino Acid Sequence↗
PubMed1993

The membrane potential has no detectable effect on the phosphocholine headgroup conformation in large unilamellar phosphatidylcholine vesicles as determined by 2H-NMR.

In this study the effect of a transmembrane electrical potential on the phospholipid headgroup conformation was investigated using the 2H-NMR technique. Large unilamellar vesicles were prepared of dioleoylphosphatidylcholine, specifically 2H-labeled at the alpha- or beta-position of the choline group. No conformational change of the phosphocholine headgroup could be detected after induction of a valinomycin-induced K(+)-diffusion potential across the bilayer. However, this method could be used to measure the redistribution of tetraphenylphosphonium across the bilayer in response to delta psi, which reorients the phosphocholine headgroups in the opposite bilayer-water interfaces.

Benzothiazoles↗
PubMed1987

2H-nuclear magnetic resonance investigations on phospholipid acyl chain order and dynamics in the gramicidin-induced hexagonal HII phase.

The following results are reported in this paper: The interaction of gramicidin with [11,11-2H2]dioleoylphosphatidylcholine (DOPC) and [11,11-2H2]dioleoylphosphatidylethanolamine (DOPE) at different stages of hydration was studied by 2H- and 31P-nuclear magnetic resonance. In the L alpha phase in excess water the acyl chains of phosphatidylethanolamine (PE) are more ordered than phosphatidylcholine (PC) most likely as the result of the lower headgroup hydration of the former lipid. In excess water gramicidin incorporation above 5 mol % in DOPC causes a bilayer----hexagonal HII phase change. In the HII phase acyl chain order is virtually unaffected by gramicidin but the peptide restricts the fast chain motions. At low water content gramicidin cannot induce the HII phase but it markedly decreases chain order in the DOPC bilayer. Increasing water content results in separation between a gramicidin-poor and a gramicidin-rich L alpha phase with decreased order of the entire lipid molecule. Further increase in hydration reverts at low gramicidin contents the phase separation and at high gramicidin contents results in a direct change of the disordered lamellar to the hexagonal HII phase. Gramicidin also promotes HII phase formation in the PE system but interacts much less strongly with PE than with PC. The results support our hypothesis that gramicidin, by a combination of strong intermolecular attraction forces and its pronounced cone shape, both involving the four tryptophans at the COOH-terminus, has a strong tendency to organize, with the appropriate lipid, in intramembranous cylindrical structures such as is found in the HII phase.

Deuterium↗