Effect of lipid composition on buforin II structure and membrane entry

Buforin II is a 21-amino acid polycationic antimicrobial peptide derived from a peptide originally isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans. It is hypothesized to target a wide range of bacteria by translocating into cells without membrane permeabilization and binding to nucleic acids. Previous research found that the structure and membrane interactions of buforin II are related to lipid composition. In this study, we used molecular dynamics (MD) simulations along with lipid vesicle experiments to gain insight into how buforin II interacts differently with phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) lipids. Fluorescent spectroscopic measurements agreed with the previous assertion that buforin II does not interact with pure PC vesicles. Nonetheless, the reduced entry of the peptide into anionic PG membranes versus neutral PC membranes during simulations correlates with the experimentally observed reduction in BF2 translocation through pure PG membranes. Simulations showing membrane entry into PC also provide insight into how buforin II may initially penetrate cell membranes. Our MD simulations also allowed us to consider how neutral PE lipids affect the peptide differently than PC. In particular, the peptide had a more helical secondary structure in simulations with PE lipids. A change in structure was also apparent in circular dichroism measurements. PE also reduced membrane entry in simulations, which correlates with decreased translocation in the presence of PE observed in previous studies. Together, these results provide molecular-level insight into how lipid composition can affect buforin II structure and function and will be useful in efforts to design peptides with desired antimicrobial and cell-penetrating properties.     

The modulating effects of lipids on purified rat liver Golgi galactosyltransferase

Galactosyltransferase was purified from rat liver Golgi membranes. The Triton X-100, used to solubilize the enzyme was removed immediately prior to the lipid interaction studies. In lipid vesicles, prepared from a variety of phosphatidylcholines (PCs), including egg PC, DOPC, DMPC, DPPC and DSPC, the ability of the lipids to stimulate the enzyme decreased in the order egg PC greater than DOPC greater than DMPC greater than DPPC greater than DSPC, i.e. the lower the transition temperature (Tc) the greater the stimulation of the enzyme. A second, neutral lipid, phosphatidylethanolamine was used to permit a comparison of the effect of a different head group of the same net charge at neutral pH. The PEs included, egg PE, soy PE, Pl-PE, PE(PC) and DPPE in order of increasing Tc. The effect of the PEs was opposite to that of the PCs, i.e. the higher the Tc, the greater the stimulation of the enzyme. In fact egg PE and soy PE which have the lowest Tc values were inhibitory. Thus the modulation of the Golgi membrane galactosyltransferase by these lipids was different from that reported earlier for the bovine milk galactosyltransferase. The effects of two acidic lipids, egg phosphatidic acid (PA) and egg phosphatidylglycerol (PG) were studied also. Both totally inhibited the enzyme even at low concentrations of lipid, however, the PA was more effective than PG. In mixtures of neutral lipid (PC) and acidic lipid (PA or PG), the effect of the acidic lipid dominated. Even in the presence of excess PC, total inhibition of the enzyme was observed. It was concluded that the enzyme bound the acidic lipid preferentially to itself. The choice of the lipids allowed us to make several direct comparisons concerning the effect of the nature of the lipid head group on the activity of the enzyme. For example PE(PC), egg PA and egg PG would have fatty acid chains identical to egg PC since these three lipids are all prepared by modification of egg PC. As well, DPPE differs from DPPC only by nature of the head group. These comparisons indicated that not only the net charge but also chemical nature of the head group were important in the lipid modulation of Golgi galactosyltransferase.     

Effect of drastic sequence alteration and D-amino acid incorporation on the membrane binding behavior of lytic peptides

The amphipathic alpha-helix is a common motif found in many cell lytic peptides including antimicrobial peptides. We have recently shown that significantly altering the amphipathic structure of a lytic peptide by reshuffling its sequence and/or replacing a few l-amino acids with their D-enantiomers did not significantly affect the antimicrobial activity of the peptides nor their ability to bind and permeate negatively charged (PE/PG) membranes. However, a pronounced effect was observed regarding their hemolytic activity and their ability to bind and permeate zwitterionic (PC/Cho) membranes. To shed light on these findings, here we used surface plasmon resonance (SPR) with mono- and bilayer membranes. We found that the L-amino acid (aa) peptides bound 10-25-fold stronger to PC/Cho bilayers compared with monolayers, whereas the diastereomers bound similarly to both membranes. A two-state reaction model analysis of the data indicated that this difference is due to the insertion of the L-aa peptides into the PC/Cho bilayers, whereas the diastereomers are surface-localized. In contrast, only an approximately 2-fold difference was found with negatively charged membranes. Changes in the amphipathicity markedly affected only the insertion of the L-aa peptides into PC/Cho bilayers. Furthermore, whereas the all-L-aa peptides bound similarly to the PC/Cho and PE/PG membranes, the diastereomers bound approximately 100-fold better to PE/PG compared with PC/Cho membranes, and selectivity was determined only in the first binding step. The effect of the peptides on the lipid order determined by using ATR-FTIR studies supported these findings. Besides shedding light on the mode of action of these peptides, the present study demonstrates SPR as a powerful tool to differentiate between non-cell-selective compared with bacteria-selective peptides, based on differences in their membrane binding behavior.     

Activity and characterization of a pH-sensitive antimicrobial peptide

Antimicrobial peptides (AMPs) have been an area of great interest, due to the high selectivity of these molecules toward bacterial targets over host cells and the limited development of bacterial resistance to these molecules throughout evolution. Previous work showed that when Histidine was incorporated into the peptide C18G it lost antimicrobial activity. The role of pH on activity and biophysical properties of the peptide was investigated to explain this phenomenon. Minimal inhibitory concentration (MIC) results demonstrated that decreased media pH increased antimicrobial activity. Trichloroethanol (TCE) quenching and red-edge excitation spectroscopy (REES) showed a clear pH dependence on peptide aggregation in solution. Trp fluorescence was used to monitor binding to lipid vesicles and demonstrated the peptide binds to anionic bilayers at all pH values tested, however, binding to zwitterionic bilayers was enhanced at pH 7 and 8 (above the His pKa). Dual Quencher Analysis (DQA) confirmed the peptide inserted more deeply in PC:PG and PE:PG membranes, but could insert into PC bilayers at pH conditions above the His pKa. Bacterial membrane permeabilization assays which showed enhanced membrane permeabilization at pH 5 and 6 but vesicle leakage assays indicate enhanced permeabilization of PC and PC:PG bilayers at neutral pH. The results indicate the ionization of the His side chain affects the aggregation state of the peptide in solution and the conformation the peptide adopts when bound to bilayers, but there are likely more subtle influences of lipid composition and properties that impact the ability of the peptide to form pores in membranes.     

On the role of anionic lipids in charged protein interactions with membranes

We investigate the role of anionic lipids in the binding to, and subsequent movement of charged protein groups in lipid membranes, to help understand the role of membrane composition in all membrane-active protein sequences. We demonstrate a small effect of phosphatidylglycerol (PG) lipids on the ability of an arginine (Arg) side chain to bind to, and cross a lipid membrane, despite possessing a neutralizing charge. We observe similar membrane deformations in lipid bilayers composed of phosphatidylcholine (PC) and PC/PG mixtures, with comparable numbers of water and lipid head groups pulled into the bilayer hydrocarbon core, and prohibitively large ~20 kcal/mol barriers for Arg transfer across each bilayer, dropping by just 2-3 kcal/mol due to the binding of PG lipids. We explore the causes of this small effect of introducing PG lipids and offer an explanation in terms of the limited membrane interaction for the choline groups of PC lipids bound to the translocating ion. Our calculations reveal a surprising lack of preference for Arg binding to PG lipids themselves, but a small increase in interfacial binding affinity for lipid bilayers containing PG lipids. These results help to explain the nature of competitive lipid binding to charged protein sequences, with implications for a wide range of membrane binding domains and cell perturbing peptides.     

Exploring peptide membrane interaction using surface plasmon resonance: differentiation between pore formation versus membrane disruption by lytic peptides

Lytic peptides comprise a large group of membrane-active peptides used in the defensive and offensive systems of all organisms. Differentiating between their modes of interaction with membranes is crucial for understanding how these peptides select their target cells. Here we utilized SPR to study the interaction between lytic peptides and lipid bilayers (L1 sensor chip). Using studies also on hybrid monolayers (HPA sensor chip) revealed that SPR is a powerful tool for obtaining a real-time monitoring of the steps involved in the mode of action of membrane-active peptides, some of which previously could not be detected directly by other techniques and reported here for the first time. We investigated the mode of action of peptides that represent two major families: (i) the bee venom, melittin, as a model of a non-cell-selective peptide that forms transmembrane pores and (ii) magainin and a diastereomer of melittin (four amino acids were replaced by their D enantiomers), as models of bacteria-selective non-pore-forming peptides. Fitting the SPR data to different interaction models allows differentiating between two major steps: membrane binding and membrane insertion. Melittin binds to PC/cholesterol approximately 450-fold better than its diastereomer and magainin, mainly because it is inserted into the inner leaflet (2/3 of the binding energy), whereas the other two are not. In contrast, there is only a slight difference in the binding of all the peptides to negatively charged PE/PG mono- and bilayer membranes (in the first and second steps), indicating that the inner leaflet contributes only slightly to their binding to PE/PG bilayers. Furthermore, the 100-fold stronger binding of the cell-selective peptides to PE/PG as compared with PC/cholesterol resulted only from electrostatic attraction to the negatively charged headgroups of the outer leaflet. These results clearly differentiate between the two general mechanisms: pore formation by melittin only in zwitterionic membranes and a detergent-like effect (carpet mechanism) for all the peptides in negatively charged membranes, in agreement with their biological function.     

Improved model systems for bacterial membranes from differing species: The importance of varying composition in PE/PG/cardiolipin ternary mixtures

Abstract

Steady-state fluorescence anisotropy and dynamic light scattering (DLS) were used to determine the thermotropic properties of lipid systems that act as models for bacterial membranes of Yersinia kristensenii and Proteus mirabilis. Lipid proportions of PE:PG:CL of 0.60:0.20:0.20 and 0.80:0.15:0.05, were used in order to mimic these two membranes respectively. We observed that the introduction of cardiolipin (CL) as a third lipid component of any PE:PG mixture, changes the system's properties considerably. The results obtained by these two techniques show that the main transition temperatures obtained are undoubtedly CL-dependent. Additionally AFM experiments were performed and these results show that even at small concentration CL produces important changes not only in the membrane thermotropic properties, but also in the bilayer structure. In summary, we were able to compare how low and high CL concentration affect bacterial membrane model system properties which can provide a further explanation for the different antibiotic susceptibilities reported for Y. kristensenii and P. mirabilis.     

Biophysical Investigation of the Membrane-Disrupting Mechanism of the Antimicrobial and Amyloid-Like Peptide Dermaseptin S9

Dermaseptin S9 (Drs S9) is an atypical cationic antimicrobial peptide with a long hydrophobic core and with a propensity to form amyloid-like fibrils. Here we investigated its membrane interaction using a variety of biophysical techniques. Rather surprisingly, we found that Drs S9 induces efficient permeabilisation in zwitterionic phosphatidylcholine (PC) vesicles, but not in anionic phosphatidylglycerol (PG) vesicles. We also found that the peptide inserts more efficiently in PC than in PG monolayers. Therefore, electrostatic interactions between the cationic Drs S9 and anionic membranes cannot explain the selectivity of the peptide towards bacterial membranes. CD spectroscopy, electron microscopy and ThT fluorescence experiments showed that the peptide adopts slightly more β-sheet and has a higher tendency to form amyloid-like fibrils in the presence of PC membranes as compared to PG membranes. Thus, induction of leakage may be related to peptide aggregation. The use of a pre-incorporation protocol to reduce peptide/peptide interactions characteristic of aggregates in solution resulted in more α-helix formation and a more pronounced effect on the cooperativity of the gel-fluid lipid phase transition in all lipid systems tested. Calorimetric data together with ²H- and ³¹P-NMR experiments indicated that the peptide has a significant impact on the dynamic organization of lipid bilayers, albeit slightly less for zwitterionic than for anionic membranes. Taken together, our data suggest that in particular in membranes of zwitterionic lipids the peptide binds in an aggregated state resulting in membrane leakage. We propose that also the antimicrobial activity of Drs S9 may be a result of binding of the peptide in an aggregated state, but that specific binding and aggregation to bacterial membranes is regulated not by anionic lipids but by as yet unknown factors.     

The cytoplasmic domains of phospholamban and phospholemman associate with phospholipid membrane surfaces

Phospholamban (PLB) and phospholemman (PLM, also called FXYD1) are small transmembrane proteins that interact with P-type ATPases and regulate ion transport in cardiac cells and other tissues. This work has investigated the hypothesis that the cytoplasmic domains of PLB and PLM, when not interacting with their regulatory targets, are stabilized through associations with the surface of the phospholipid membrane. Peptides representing the 35 C-terminal cytoplasmic residues of PLM (PLM(37-72)), the 23 N-terminal cytoplasmic residues of PLB (PLB(1-23)), and the same sequence phosphorylated at Ser-16 (P-PLB(1-23)) were synthesized to examine their interactions with model membranes composed of zwitterionic phosphatidylcholine (PC) lipids alone or in admixture with anionic phosphatidylglycerol (PG) lipids. Wide-line 2H NMR spectra of PC/PG membranes, with PC deuterated in the choline moiety, indicated that all three peptides interacted with the membrane surface and perturbed the orientation of the choline headgroups. Fluorescence and 31P magic-angle spinning (MAS) NMR measurements indicated that PLB(1-23) and P-PLB(1-23) had a higher affinity for PC/PG membranes, which carry an overall negative surface charge, than for PC membranes, which have no net surface charge. The 31P MAS NMR spectra of the PC/PG membranes in the presence of PLM(37-72), PLB(1-23), and P-PLB(1-23) indicated that all three peptides induced clustering of the lipids into PC-enriched and PG-enriched regions. These findings support the theory that the cytoplasmic domains of PLB and PLM are stabilized by interacting with lipid headgroups at the membrane surface, and it is speculated that such interactions may modulate the functional properties of biological membranes.     

Structural and functional consequences of galactolipids on thylakoid membrane organization

Photosynthetic membranes of higher plant chloroplasts are composed primarily of polar, but uncharged, galactolipids unlike most mammalian membranes which contain large amounts of phosphatidylcholine. It is unclear what role(s) the galactolipids play in maintaining the differentiated thylakoid membranes, or in stabilizing the photosynthetically active enzyme complexes. Some of the membrane complexes show no lipid selectivity for maintaining structural or functional integrity. Others are poisoned or dissociated in the presence of high concentrations of a trace lipid class. The efficiency of energy transfer and the reconstitution of protein complexes into liposomes are dependent on the lipid class employed. The lipids are asymmetrically arranged along and across the thylakoid membranes but not as distinctly as the proteins.Abbreviations DGDG digalactosyldiglyceride - MGDG monogalactosyldiglyceride - SQDG sulfoquinovosyldiglyceride - PG phosphatidylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - PSI photosystem I - PSII photosystem II - LHC chlorophylla/b lightharvesting complex - cytb ₆ f cytochromeb ₆ f complex - CF₀/CF₁ coupling factor ATPase - DCIP 2,6-dichlorophenolindophenol - LRa galactolipase fromRhizopus arrhis     

Molecular basis for membrane selectivity of NK-2, a potent peptide antibiotic derived from NK-lysin

Increasing resistance of pathogenic bacteria against antibiotics is a severe problem in health care. Natural antimicrobial peptides and derivatives thereof have emerged as promising candidates for “new antibiotics”. In contrast to classical antibiotics, these peptides act by direct physical destabilization of the target cell membrane. Nevertheless, they exhibit a high specificity for bacteria over mammalian cells. However, the precise mechanism of action and the molecular basis for membrane selectivity are still a matter of debate. We have designed a new peptide antibiotic (NK-2) with enhanced antimicrobial activity based on an effector protein of mammalian immune cells (NK-lysin). Here we describe the interaction of this α-helical synthetic peptide with membrane mimetic systems, designed to mimic the lipid compositions of mammalian and bacterial cytoplasmic membranes. Utilizing fluorescence and biosensor assays, we could show that on one hand, NK-2 strongly interacts with negatively charged membranes; on the other hand, NK-2 is able to discriminate, without the necessity of negative charges, between the zwitterionic phospholipids phosphatidylethanolamine (PE) and phosphatidylcholine (PC), the major constituents of the outer leaflet of the cytoplasmic membranes of bacteria and mammalian cells, respectively.     

Adaptation of the bacterial membrane to changing environments using aminoacylated phospholipids

Fine-tuning of the biophysical properties of biological membranes is essential for adaptation of cells to changing environments. For instance, to lower the negative charge of the lipid bilayer, certain bacteria add lysine to phosphatidylglycerol (PG) converting the net negative charge of PG (−1) to a net positive charge in Lys-PG (+1). Reducing the net negative charge of the bacterial cell wall is a common strategy used by bacteria to resist cationic antimicrobial peptides (CAMPs) secreted by other microbes or produced by the innate immune system of a host organism. The article by Klein et al . in the current issue of Molecular Microbiology reports a new modification of the bacterial membrane, addition of alanine to PG, in Pseudomonas aeruginosa . In spite of the neutral charge of Ala-PG, this modified lipid was found to be linked to several resistance phenotypes in P. aeruginosa . For instance, Ala-PG increases resistance to two positively charged antibacterial agents, a β-lactam and high concentrations of lactate. These findings shed light on the mechanisms by which bacteria fine-tune the properties of their cell membranes by adding various amino acids on the polar head group of phospholipids.     

NMR structural studies of the antibiotic lipopeptide daptomycin in DHPC micelles

Daptomycin is a cyclic anionic lipopeptide that exerts its rapid bactericidal effect by perturbing the bacterial cell membrane, a mode of action different from most other currently commercially available antibiotics (except e.g. polymyxin and gramicidin). Recent work has shown that daptomycin requires calcium in the form of Ca2+ to form a micellar structure in solution and to bind to bacterial model membranes. This evidence sheds light on the initial steps in the mechanism of action of this novel antibiotic. To understand how daptomycin goes on to perturb bacterial membranes, its three-dimensional structure has been determined in the presence of 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micelles. NMR spectra of daptomycin in DHPC were obtained under two conditions, namely in the presence of Ca2+ as used by Jung et al. [D. Jung, A. Rozek, M. Okon, R.E.W. Hancock, Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin, Chem. Biol. 11 (2004) 949-57] to solve the calcium-conjugated structure of daptomycin in solution and in a phosphate buffer as used by Rotondi and Gierasch [K.S. Rotondi, L.M. Gierasch, A well-defined amphipathic conformation for the calcium-free cyclic lipopeptide antibiotic, daptomycin, in aqueous solution, Biopolymers 80 (2005) 374-85] to solve the structure of apo-daptomycin. The structures were calculated using molecular dynamics time-averaged refinement. The different sample conditions used to obtain the NMR spectra are discussed in light of fluorescence data, lipid flip-flop and calcein release assays in PC liposomes, in the presence and absence of Ca2+ [D. Jung, A. Rozek, M. Okon, R.E.W. Hancock, Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin, Chem. Biol. 11 (2004) 949-57]. The implications of these results for the membrane perturbation mechanism of daptomycin are discussed.     

Profiling membrane lipids in plant stress responses. Role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis

A sensitive approach based on electrospray ionization tandem mass spectrometry has been employed to profile membrane lipid molecular species in Arabidopsis undergoing cold and freezing stresses. Freezing at a sublethal temperature induced a decline in many molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but induced an increase in phosphatidic acid (PA) and lysophospholipids. To probe the metabolic steps generating these changes, lipids of Arabidopsis deficient in the most abundant phospholipase D, PLD alpha, were analyzed. The PC content dropped only half as much, and PA levels rose only half as high in the PLD alpha-deficient plants as in wild-type plants. In contrast, neither PE nor PG levels decreased significantly more in wild-type plants than in PLD alpha-deficient plants. These data suggest that PC, rather than PE and PG, is the major in vivo substrate of PLD alpha. The action of PLD alpha during freezing is of special interest because Arabidopsis plants that are deficient in PLD alpha have improved tolerance to freezing. The greater loss of PC and increase in PA in wild-type plants as compared with PLD alpha-deficient plants may be responsible for destabilizing membrane bilayer structure, resulting in a greater propensity toward membrane fusion and cell death in wild-type plants.     

The effect of salinity on the phase behaviour of total lipid extracts and binary mixtures of the major phospholipids isolated from a moderately halophilic eubacterium

The effects of molar NaCl concentrations on the phase behaviour of the total lipid extracts and binary mixtures of the major phospholipids, namely phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), isolated from the moderately halophilic eubacterium, Vibrio costicola, grown in 1 M and 3 M NaCl containing media have been studied using X-ray diffraction and freeze-fracture electron microscopy. The effect of both the PE/PG ratio and alterations in fatty acid composition were examined by using binary mixtures which mimicked the PE/PG ratio found in the native bacterial membranes. We show that the samples exhibited complex phase behaviour, including the formation of non-bilayer phases, which depend upon the salinity of both the bacterial culture medium and the suspending solution. The total lipid from bacteria cultured in 1 M NaCl-containing medium and dispersed in 1 M NaCl exhibited a mixture of L alpha and hexagonal-II phases at the optimum growth temperature of the organism (i.e., 30 degrees C), whereas the same lipid dispersed in 3 M NaCl showed only a hexagonal-II phase down to a temperature of +3 degrees C. The total lipid extracted from 3 M NaCl cultures showed only lamellar phases over the temperature range studied (+50 degrees C to -50 degrees C), but the phase transition temperatures of the various lamellar phases were generally higher when the lipid was dispersed in 3 M compared with 1 M NaCl. The phase behaviour of the binary mixtures was similar but not identical to that of the corresponding total lipid extracts and it is suggested that the minor lipid components (diphosphatidylglycerol, lysophosphatidylethanolamine and lysophosphatidylglycerol) play a part in determining the phase behaviour of the native membranes. These results show that the PE/PG ratio and fatty acid composition of the individual phospholipids, which are normally regulated by Vibrio costicola in vivo in response to culture medium salinity, are both important in maintaining a stable bilayer structure within the membrane.     

膜脂对菠菜细胞色素b6f复合体电子传递活性的影响

利用从菠菜(Spinacia oleracea L.)叶绿体分离、纯化出的缺失膜脂的细胞色素b6f蛋白复合体(Cyt b6f)制剂与从菠菜类囊体分离、纯化的膜脂进行体外重组,检测了不同膜脂对Cyt b6f催化电子传递活性的影响.结果表明:被检测的5种膜脂,即单半乳糖基甘油二酯(MGDG)、双半乳糖基甘油二酯(DGDG)、磷脂酰胆碱(PC)、磷脂酰甘油(PG)和硫代异鼠李糖基甘油二酯(SQDG)对Cyt b6f催化电子传递的活性均有明显的促进作用,但促进的程度各不相同,这可能与这些膜脂分子的带电性质密切相关.不带电荷的MGDG和DGDG及分子整体呈电中性的PC对促进Cyt b6f催化电子传递的活性非常有效,可分别使其活性提高89%、75%和77%;而带负电荷的PG和SQDG对活性的促进作用则相对较弱,仅可使其活性分别提高43%和26%.     

Role of phosphatidylglycerols in the stability of bacterial membranes

An extensive 100-ns molecular dynamics simulation of lipid bilayer composed of mixture of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) was performed to elucidate the role of PGs to the stability of bacterial membranes. In addition, a control simulation of pure PE over 150 ns was performed. We observed that PGs decrease both the PE headgroup protrusions into the water phase, and the PE headgroup motion along bilayer normal. The above effects are caused by stronger inter-lipid interactions in the mixed bilayer: the number of hydrogen bonds created by PEs is 34% higher in the mixed than in the pure bilayer. Another contribution is due to the numerous ion-mediated inter-lipid links, which strongly enhance interface stability. That provides a plausible mechanism for preventing lipid desorption from the membrane, for example, under the influence of an organic solvent. A more compact and less dynamic interface structure also decreases membrane permeability. That provides a possible mechanism for stabilizing, e.g., bacterial membranes.     

NMR structural studies of the antibiotic lipopeptide daptomycin in DHPC micelles

Daptomycin is a cyclic anionic lipopeptide that exerts its rapid bactericidal effect by perturbing the bacterial cell membrane, a mode of action different from most other currently commercially available antibiotics (except e.g. polymyxin and gramicidin). Recent work has shown that daptomycin requires calcium in the form of Ca²⁺ to form a micellar structure in solution and to bind to bacterial model membranes. This evidence sheds light on the initial steps in the mechanism of action of this novel antibiotic. To understand how daptomycin goes on to perturb bacterial membranes, its three-dimensional structure has been determined in the presence of 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micelles. NMR spectra of daptomycin in DHPC were obtained under two conditions, namely in the presence of Ca²⁺ as used by Jung et al. [D. Jung, A. Rozek, M. Okon, R.E.W. Hancock, Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin, Chem. Biol. 11 (2004) 949-57] to solve the calcium-conjugated structure of daptomycin in solution and in a phosphate buffer as used by Rotondi and Gierasch [K.S. Rotondi, L.M. Gierasch, A well-defined amphipathic conformation for the calcium-free cyclic lipopeptide antibiotic, daptomycin, in aqueous solution, Biopolymers 80 (2005) 374-85] to solve the structure of apo-daptomycin. The structures were calculated using molecular dynamics time-averaged refinement. The different sample conditions used to obtain the NMR spectra are discussed in light of fluorescence data, lipid flip-flop and calcein release assays in PC liposomes, in the presence and absence of Ca²⁺ [D. Jung, A. Rozek, M. Okon, R.E.W. Hancock, Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin, Chem. Biol. 11 (2004) 949-57]. The implications of these results for the membrane perturbation mechanism of daptomycin are discussed.     

Interaction of the K+ channel KcsA with membrane phospholipids as studied by ESI mass spectrometry

In this study we have used electrospray ionization mass spectrometry (ESI-MS) to investigate interactions between the bacterial K(+) channel KcsA and membrane phospholipids. KcsA was reconstituted into lipid vesicles of variable lipid composition. These vesicles were directly analyzed by ESI-MS or mixed with trifluoroethanol (TFE) before analysis. In the resulting mass spectra, non-covalent complexes of KcsA and phospholipids were observed with an interesting lipid specificity. The anionic phosphatidylglycerol (PG), and, to a lesser extent, the zwitterionic phosphatidylethanolamine (PE), which both are abundant bacterial lipids, were found to preferentially associate with KcsA as compared to the zwitterionic phosphatidylcholine (PC). These preferred interactions may reflect the differences in affinity of these phospholipids for KcsA in the membrane.