Size and orientation of the lipid II headgroup as revealed by AFM imaging.

In this study, we investigated the size and orientation of the bacterial Lipid II (L II) headgroup when the L II molecule is present in liquid-crystalline domains of DOPC in a supported DPPC bilayer. Using atomic force microscopy, we detected that L II causes the appearance of a 1.9 nm thick layer, situated over the DOPC headgroup region. With an increased scanning force, this layer can be penetrated by the AFM tip down to the level of the DOPC bilayer. Using different L II precursor molecules, we demonstrated that the detected layer consists of the headgroups of L II and that the MurNAc-pentapeptide unit of the headgroup is responsible for the measured 1.9 nm height of that layer. Monolayer experiments provided information about the in-plane dimensions of the L II headgroup. On the basis of these results and considerations of the molecular dimensions of L II headgroup constituents, we propose a model for the orientation of the L II headgroup in the membrane. In this model, the pentapeptide of the L II headgroup is rather extended and points away from the bilayer surface, which could be important for biological processes, in which L II is involved.

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.

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.

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.

Apocytochrome c requires the TOM complex for translocation across the mitochondrial outer membrane.

The import of proteins into the mitochondrial intermembrane space differs in various aspects from the classical import pathway into the matrix. Apocytochrome c defines one of several pathways known to reach the intermembrane space, yet the components and pathways involved in outer membrane translocation are poorly defined. Here, we report the reconstitution of the apocytochrome c import reaction using proteoliposomes harbouring purified components. Import specifically requires the protease-resistant part of the TOM complex and is driven by interactions of the apoprotein with internal parts of the complex (involving Tom40) and the 'trans-side receptor' cytochrome c haem lyase. Despite the necessity of TOM complex function, the translocation pathway of apocytochrome c does not overlap with that of presequence-containing preproteins. We conclude that the TOM complex is a universal preprotein translocase that mediates membrane passage of apocytochrome c and other preproteins along distinct pathways. Apocytochrome c may provide a paradigm for the import of other small proteins into the intermembrane space such as factors used in apoptosis and protection from stress.

Effect of nonbilayer lipids on membrane binding and insertion of the catalytic domain of leader peptidase.

Biological membranes contain a substantial amount of "nonbilayer lipids", which have a tendency to form nonlamellar phases. In this study the hypothesis was tested that the presence of nonbilayer lipids in a membrane, due to their overall small headgroup, results in a lower packing density in the headgroup region, which might facilitate the interfacial insertion of proteins. Using the catalytic domain of leader peptidase (delta2-75) from Escherichia coli as a model protein, we studied the lipid class dependence of its insertion and binding. In both lipid monolayers and vesicles, the membrane binding of (catalytically active) delta2-75 was much higher for the nonbilayer lipid DOPE compared to the bilayer lipid DOPC. For the nonbilayer lipids DOG and MGDG a similar effect was observed as for DOPE, strongly suggesting that no specific interactions are involved but that the small headgroups create hydrophobic interfacial insertion sites. On the basis of the results of the monolayer experiments, calculations were performed to estimate the space between the lipid headgroups accessible to the protein. We estimate a maximal size of the insertion sites of 15 +/- 7 A2/lipid molecule for DOPE, relative to DOPC. The size of the insertion sites decreases with an increase in headgroup size. These results show that nonbilayer lipids stimulate the membrane insertion of delta2-75 and support the idea that such lipids create insertion sites by reducing the packing density at the membrane-water interface. It is suggested that PE in the bacterial membrane facilitates membrane insertion of the catalytic domain of leader peptidase, allowing the protein to reach the cleavage site in preproteins.

Imaging domains in model membranes with atomic force microscopy.

Lateral segregation in biomembranes can lead to the formation of biologically functional domains. This paper reviews atomic force microscopy studies on domain formation in model membranes, with special emphasis on transbilayer asymmetry, and on lateral domains induced by lipid-lipid interactions or by peptide-lipid interactions.

Membrane-spanning peptides induce phospholipid flop: a model for phospholipid translocation across the inner membrane of E. coli.

The mechanism by which phospholipids translocate (flop) across the E. coli inner membrane remains to be elucidated. We tested the hypothesis that the membrane-spanning domains of proteins catalyze phospholipid flop by their mere presence in the membrane. As a model, peptides mimicking the transmembrane stretches of proteins, with the amino acid sequence GXXL(AL)(n)XXA (with X = K, H, or W and n = 8 or 12), were incorporated in large unilamellar vesicles composed of E. coli phospholipids. Phospholipid flop was measured by assaying the increase in accessibility to dithionite of a 2,6-(7-nitro-2,1,3-benzoxadiazol-4-yl)aminocaproyl (C(6)NBD)-labeled phospholipid analogue, initially exclusively present in the inner leaflet of the vesicle membrane. Fast flop of C(6)NBD-phosphatidylglycerol (C(6)NBD-PG) was observed in vesicles in which GKKL(AL)(12)KKA was incorporated, with the apparent first-order flop rate constant (K(flop)) linearly increasing with peptide:phospholipid molar ratios, reaching a translocation half-time of approximately 10 min at a 1:250 peptide:phospholipid molar ratio at 25 degrees C. The peptides of the series GXXL(AL)(8)XXA also induced flop of C(6)NBD-PG, supporting the hypothesis that transmembrane parts of proteins mediate phospholipid translocation. In this series, K(flop) decreased in the order X = K > H > W, indicating that peptide-lipid interactions in the interfacial region of the membrane modulate the efficiency of a peptide to cause flop. For the peptides tested, flop of C(6)NBD-phosphatidylethanolamine (C(6)NBD-PE) was substantially slower than that of C(6)NBD-PG. In vesicles without peptide, flop was negligible both for C(6)NBD-PG and for C(6)NBD-PE. A model for peptide-induced flop is proposed, which takes into account the observed peptide and lipid specificity.

The N-terminal portion of the preToc75 transit peptide interacts with membrane lipids and inhibits binding and import of precursor proteins into isolated chloroplasts.

Toc75 is an outer envelope membrane protein of chloroplasts. It is unusual among the outer membrane proteins in that its precursor form has a bipartite transit peptide. The N-terminal portion of the Toc75 transit peptide is sufficient to target the protein to the stromal space of chloroplasts. We prepared a 45 amino-acid peptide containing the stromal targeting domain of the Toc75 transit peptide in Escherichia coli, using the intein-mediated system, and purified it by reverse-phase HPLC. Its identity was confirmed by N-terminal amino-acid sequencing and matrix assisted laser desorption ionization mass spectrometry. In monolayer experiments, the peptide inserted into the chloroplastic membrane lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol and into a nonchloroplastic lipid phosphatidylethanolamine. However, it did not insert into other chloroplastic lipids, such as mono- and digalactosyl diacylglycerol, and phosphatidylcholine. Furthermore, the peptide significantly inhibited binding of radiolabeled precursors of Toc75 and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase to intact chloroplasts as effectively as did a bacterially produced precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase. The peptide also inhibited import of radiolabeled precursors into isolated chloroplasts, however, to a lesser extent than did nonlabeled precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase.

Visualizing detergent resistant domains in model membranes with atomic force microscopy.

Evidence is accumulating that in cell membranes microdomains exist, also referred to as rafts or detergent resistant membranes. In this study, atomic force microscopy is used to study supported lipid bilayers, consisting of a fluid phosphatidylcholine, sphingomyelin and cholesterol. Domains were visualized of which the morphology and size depended on the cholesterol concentration. The presence of cholesterol was found to induce bilayer coupling. At 30 mol% cholesterol, a change in percolation phase was observed, and at 50 mol%, when both fluid lipids and solid lipids are saturated with cholesterol, phase separation was still observed. In addition, we were able to directly visualize the resistance of domains against non-ionic detergent.

Biophysical characterization of the influence of salt on tetrameric SecB.

SecB is a tetrameric chaperone, with a monomeric molecular mass of 17 kDa, that is involved in protein translocation in Escherichia coli. It has been hypothesized that SecB undergoes a conformational change as a function of the salt concentration. To gain more insight into the salt-dependent behavior of SecB, we studied the protein in solution by dynamic light scattering, size exclusion chromatography, analytical ultracentrifugation, and small angle neutron scattering. The results clearly demonstrate the large influence of the salt concentration on the behavior of SecB. At high salt concentration, SecB is a non-spherical protein with a radius of gyration of 3.4 nm. At low salt concentration the hydrodynamic radius of the protein is apparently decreased, whereas the ratio of the frictional coefficients is increased. The protein solution behaves in a non-ideal way at low salt concentrations, as was shown by the analytical ultracentrifugation data and a pronounced interparticle effect observed by small angle neutron scattering. A possible explanation is a change in surface charge distribution dependent on the salt concentration in the solvent. We summarize our data in a model for the salt-dependent conformation of tetrameric SecB.

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.

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.

Membrane activity of the peptide antibiotic clavanin and the importance of its glycine residues.

The peptide antibiotic clavanin A (VFQFLGKIIHHVGNFVHGFSHVF-NH(2)) is rich in histidine and glycine residues. In this study the antimicrobial activity and membrane activity of wild-type clavanin A and seven Gly --> Ala mutants thereof were investigated. Clavanin A effectively killed the test microorganism Micrococcus flavus and permeabilized its cytoplasmic membrane in the micromolar concentration range, suggesting that the membrane is the target for this molecule. Consistent with this suggestion, it was observed that clavanin A efficiently inserted into different phospholipid monolayers mainly via hydrophobic interactions. Bilayer permeabilization was observed for both low and high molecular mass fluorophores enclosed in unilamellar vesicles and occurred at the same concentration as the antimicrobial activity. It is therefore suggested that the loss of barrier function does not involve specific receptors in the target membrane. Circular dichroism spectroscopy indicated that under membrane mimicking conditions a random coil --> helical transition was induced for all clavanin derivatives tested. Observed differences in peptide-membrane interaction and biological activity between the various clavanin derivatives demonstrated the functional importance of Gly at the positions 6 and 13. These two glycines may act as flexible hinges that facilitate the hydrophobic N-terminal end of clavanin to deeply insert into the bilayer. On the contrary, no such role is evident for Gly 18, as its substitution by Ala actually stimulated membrane interaction and biological activity. This study suggests that the combined hydrophobicity, overall state of charge, and conformational flexibility of the peptide determine the (membrane) activity of clavanin A and its Gly --> Ala mutants.

Anionic lipids stimulate Sec-independent insertion of a membrane protein lacking charged amino acid side chains.

We have investigated the influence of the different lipid classes of Escherichia coli on Sec-independent membrane protein insertion, using an assay in which a mutant of the single-spanning Pf3 coat protein is biosynthetically inserted into liposomes. It was found that phosphatidylethanolamine and other non-bilayer lipids do not have a significant effect on insertion. Surprisingly, the anionic lipids phosphatidylglycerol and cardiolipin stimulate N-terminal translocation of the protein, even though it has no charged amino acid side chains. This novel effect is general for anionic lipids and depends on the amount of charge on the lipid headgroup. Since the N-terminus of the protein is at least partially positively charged due to a helix dipole moment, apparently negatively charged lipids can stimulate translocation of slightly positively charged protein segments in a direction opposite to the positive-inside rule. A mechanism is proposed to explain these results.

Sensitivity of single membrane-spanning alpha-helical peptides to hydrophobic mismatch with a lipid bilayer: effects on backbone structure, orientation, and extent of membrane incorporation.

The extent of matching of membrane hydrophobic thickness with the hydrophobic length of transmembrane protein segments potentially constitutes a major director of membrane organization. Therefore, the extent of mismatch that can be compensated, and the types of membrane rearrangements that result, can provide valuable insight into membrane functionality. In the present study, a large family of synthetic peptides and lipids is used to investigate a range of mismatch situations. Peptide conformation, orientation, and extent of incorporation are assessed by infrared spectroscopy, tryptophan fluorescence, circular dichroism, and sucrose gradient centrifugation. It is shown that peptide backbone structure is not significantly affected by mismatch, even when the extent of mismatch is large. Instead, this study demonstrates that for tryptophan-flanked peptides the dominant response of a membrane to large mismatch is that the extent of incorporation is reduced, when the peptide is both too short and too long. With increasing mismatch, a smaller fraction of peptide is incorporated into the lipid bilayer, and a larger fraction is present in extramembranous aggregates. Relatively long peptides that remain incorporated in the bilayer have a small tilt angle with respect to the membrane normal. The observed effects depend on the nature of the flanking residues: long tryptophan-flanked peptides do not associate well with thin bilayers, while equisized lysine-flanked peptides associate completely, thus supporting the notion that tryptophan and lysine interact differently with membrane-water interfaces. The different properties that aromatic and charged flanking residues impart on transmembrane protein segments are discussed in relation to protein incorporation in biological systems.

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.

Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity.

Unlike numerous pore-forming amphiphilic peptide antibiotics, the lantibiotic nisin is active in nanomolar concentrations, which results from its ability to use the lipid-bound cell wall precursor lipid II as a docking molecule for subsequent pore formation. Here we use genetically engineered nisin variants to identify the structural requirements for the interaction of the peptide with lipid II. Mutations affecting the conformation of the N-terminal part of nisin comprising rings A through C, e.g. [S3T]nisin, led to reduced binding and increased the peptide concentration necessary for pore formation. The binding constant for the S3T mutant was 0.043 x 10(7) m(-1) compared with 2 x 10(7) m(-1) for the wild-type peptide, and the minimum concentration for pore formation increased from the 1 nm to the 50 nm range. In contrast, peptides mutated in the flexible hinge region, e.g. [DeltaN20/DeltaM21]nisin, were completely inactive in the pore formation assay, but were reduced to some extent in their in vivo activity. We found the remaining in vivo activity to result from the unaltered capacity of the mutated peptide to bind to lipid II and thus to inhibit its incorporation into the peptidoglycan network. Therefore, through interaction with the membrane-bound cell wall precursor lipid II, nisin inhibits peptidoglycan synthesis and forms highly specific pores. The combination of two killing mechanisms in one molecule potentiates antibiotic activity and results in nanomolar MIC values, a strategy that may well be worth considering for the construction of novel antibiotics.

The high affinity ATP binding site modulates the SecA-precursor interaction.

SecA is the central component of the protein-translocation machinery of Escherichia coli. It is able to interact with the precursor protein, the chaperone SecB, the integral membrane protein complex SecYEG, acidic phospholipids and its own mRNA. We studied the interaction between prePhoE and SecA by using a site-specific photocrosslinking strategy. We found that SecA is able to interact with both the signal sequence and the mature domain of prePhoE. Furthermore, this interaction was dependent on the type of nucleotide bound. SecA in the ADP-bound conformation was unable to crosslink with the precursor, whereas the ATP-bound conformation was active in precursor crosslinking. The SecA-precursor interaction was maintained in the presence of E. coli phospholipids but was loosened by the presence of phosphatidylglycerol bilayers. Examining SecA ATP binding site mutants demonstrated that ATP hydrolysis at the N-terminal high affinity binding site is responsible for the changed interaction with the preprotein.

Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase.

Recombinant membrane proteins in Escherichia coli are either expressed at relatively low level in the cytoplasmic membrane or they accumulate as inclusion bodies. Here, we report that the abundant over-production of subunit b of E. coli F(1)F(o) ATP synthase in the mutant host strains E. coli C41(DE3) and C43(DE3) is accompanied by the proliferation of intracellular membranes without formation of inclusion bodies. Maximal levels of proliferation of intracellular membranes were observed in C43(DE3) cells over-producing subunit b. The new proliferated membranes contained all the over-expressed protein and could be recovered by a single centrifugation step. Recombinant subunit b represented up to 80% of the protein content of the membranes. The lipid:protein ratios and phospholipid compositions of the intracellular membranes differ from those of bacterial cytoplasmic membranes, and they are particularly rich in cardiolipin.