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1.
Initial structural and dynamic characterization of the M2 protein transmembrane and amphipathic helices in lipid bilayers
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Tian C Gao PF Pinto LH Lamb RA Cross TA 《Protein science : a publication of the Protein Society》2003,12(11):2597-2605
Amphipathic helices in membrane proteins that interact with the hydrophobic/hydrophilic interface of the lipid bilayer have been difficult to structurally characterize. Here, the backbone structure and orientation of an amphipathic helix in the full-length M2 protein from influenza A virus has been characterized. The protein has been studied in hydrated DMPC/DMPG lipid bilayers above the gel to liquid-crystalline phase transition temperature by solid-state NMR spectroscopy. Characteristic PISA (Polar Index Slant Angle) wheels reflecting helical wheels have been observed in uniformly aligned bilayer preparations of both uniformly 15N labeled and amino acid specific labeled M2 samples. Hydrogen/deuterium exchange studies have shown the very slow exchange of some residues in the amphipathic helix and more rapid exchange for the transmembrane helix. These latter results clearly suggest the presence of an aqueous pore. A variation in exchange rate about the transmembrane helical axis provides additional support for this claim and suggests that motions occur about the helical axes in this tetramer to expose the entire backbone to the pore. 相似文献
2.
《生物化学与生物物理学报:生物膜》2015,1848(7):1490-1501
The influence of maltose-modified poly(propylene imine) (PPI) dendrimers on dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) (3%) liposomes was studied. Fourth generation (G4) PPI dendrimers with primary amino surface groups were partially (open shell glycodendrimers — OS) or completely (dense shell glycodendrimers — DS) modified with maltose residues. As a model membrane, two types of 100 nm diameter liposomes were used to observe differences in the interactions between neutral DMPC and negatively charged DMPC/DMPG bilayers. Interactions were studied using fluorescence spectroscopy to evaluate the membrane fluidity of both the hydrophobic and hydrophilic parts of the lipid bilayer and using differential scanning calorimetry to investigate thermodynamic parameter changes. Pulsed-filed gradient NMR experiments were carried out to evaluate common diffusion coefficient of DMPG and DS PPI in D2O when using below critical micelle concentration of DMPG. Both OS and DS PPI G4 dendrimers show interactions with liposomes. Neutral DS dendrimers exhibit stronger changes in membrane fluidity compared to OS dendrimers. The bilayer structure seems more rigid in the case of anionic DMPC/DMPG liposomes in comparison to pure and neutral DMPC liposomes. Generally, interactions of dendrimers with anionic DMPC/DMPG and neutral DMPC liposomes were at the same level. Higher concentrations of positively charged OS dendrimers induced the aggregation process with negatively charged liposomes. For all types of experiments, the presence of NaCl decreased the strength of the interactions between glycodendrimers and liposomes. Based on NMR diffusion experiments we suggest that apart from electrostatic interactions for OS PPI hydrogen bonds play a major role in maltose-modified PPI dendrimer interactions with anionic and neutral model membranes where a contact surface is needed for undergoing multiple H-bond interactions between maltose shell of glycodendrimers and surface membrane of liposome. 相似文献
3.
The structures of seven synthetic transmembrane domains (TMDs) of the alpha-factor receptor (Ste2p) from Saccharomyces cerevisiae were studied in phospholipid multilayers by transmission Fourier transform infrared (FTIR) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopies. Peptide conformation assumed in multilayers depended on the method of sample preparation. Amide proton H/D exchange experiments showed that 60-80% of the NH bonds in these TMDs did not exchange with bulk water in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilayers. FTIR results showed that peptides corresponding to TMDs one, two, and seven were mostly alpha-helical in DMPC multilayers. Peptides corresponding to TMDs three and six assumed predominantly beta-sheet structures, whereas those corresponding to TMDs four and five were a mixture of alpha-helices and beta-sheets. ATR-FTIR showed that in DMPC the alpha-helices of TMDs two and five oriented with tilt angles of 34 degrees and 32 degrees, respectively, with respect to the multilayer normal. Similar results were obtained for six of the transmembrane domains in DMPC/DMPG (4:1) multilayers. In a mixture [POPC/POPE/POPS/PI/ergosterol (30:20:5:20:25)] which mimicked the lipid composition of the S. cerevisiae cell membrane, the percentage of alpha-helical structures found for TMDs one and five increased compared to those in DMPC and DMPC/DMPG (4:1) multilayers, and TMD six exhibited a mixture of beta-sheet ( approximately 60%) and alpha-helical ( approximately 40%) structure. These experiments provide biophysical evidence that peptides representing the seven transmembrane domains in Ste2p assume different structures and tilt angles within a membrane multilayer. 相似文献
4.
Bacteriophage M13 major coat protein has been incorporated at different lipid/protein ratios in lipid bilayers consisting of various ratios of dimyristoylphosphatidylcholine (DMPC) to dimyristoylphosphatidylglycerol (DMPG). Spin-label ESR experiments were performed with phospholipids labeled at the C-14 position of the sn-2 chain. For M13 coat protein recombinants with DMPC alone, the relative association constants were determined for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels and found to be 1.0, 1.0, and 2.1 relative to the background DMPC, respectively. The number of association sites for each phospholipid on the protein was found to be 4 per protein monomer. The intrinsic off-rates for lipid exchange at the intramembranous surface of the protein in DMPC alone at 30 degrees C were found to be 5 X 10(6), 6 X 10(6), and 2 X 10(6) s-1 for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels, respectively. Adding DMPG to the DMPC lipid system increased the exchange rates of the lipids on and off the protein. By gel filtration chromatography, it is found that protein aggregation is reduced after addition of DMPG to the lipid system. This is in agreement with measurements of tryptophan fluorescence, which show a decrease in quenching efficiency after introduction of DMPG in the lipid system. The results are interpreted in terms of a model relating the ESR data to the size of the protein-lipid aggregates. 相似文献
5.
Three-dimensional structure of the transmembrane domain of Vpu from HIV-1 in aligned phospholipid bicelles
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The three-dimensional backbone structure of the transmembrane domain of Vpu from HIV-1 was determined by solid-state NMR spectroscopy in two magnetically-aligned phospholipid bilayer environments (bicelles) that differed in their hydrophobic thickness. Isotopically labeled samples of Vpu(2-30+), a 36-residue polypeptide containing residues 2-30 from the N-terminus of Vpu, were incorporated into large (q = 3.2 or 3.0) phospholipid bicelles composed of long-chain ether-linked lipids (14-O-PC or 16-O-PC) and short-chain lipids (6-O-PC). The protein-containing bicelles are aligned in the static magnetic field of the NMR spectrometer. Wheel-like patterns of resonances characteristic of tilted transmembrane helices were observed in two-dimensional (1)H/(15)N PISEMA spectra of uniformly (15)N-labeled Vpu(2-30+) obtained on bicelle samples with their bilayer normals aligned perpendicular or parallel to the direction of the magnetic field. The NMR experiments were performed at a (1)H resonance frequency of 900 MHz, and this resulted in improved data compared to lower-resonance frequencies. Analysis of the polarity-index slant-angle wheels and dipolar waves demonstrates the presence of a transmembrane alpha-helix spanning residues 8-25 in both 14-O-PC and 16-O-PC bicelles, which is consistent with results obtained previously in micelles by solution NMR and mechanically aligned lipid bilayers by solid-state NMR. The three-dimensional backbone structures were obtained by structural fitting to the orientation-dependent (15)N chemical shift and (1)H-(15)N dipolar coupling frequencies. Tilt angles of 30 degrees and 21 degrees are observed in 14-O-PC and 16-O-PC bicelles, respectively, which are consistent with the values previously determined for the same polypeptide in mechanically-aligned DMPC and DOPC bilayers. The difference in tilt angle in C14 and C16 bilayer environments is also consistent with previous results indicating that the transmembrane helix of Vpu responds to hydrophobic mismatch by changing its tilt angle. The kink found in the middle of the helix in the longer-chain C18 bilayers aligned on glass plates was not found in either of these shorter-chain (C14 or C16) bilayers. 相似文献
6.
Solid-state NMR has been used to study the influence of lipid bilayer hydrophobic thickness on the tilt of a peptide (M2-TMP) representing the transmembrane portion of the M2 protein from influenza A. Using anisotropic (15)N chemical shifts as orientational constraints, single-site isotopically labeled M2-TMPs were studied in hydrated dioleoylphosphatidylcholine (DOPC) and dimyristoylphosphatidylcholine (DMPC) lipid bilayers oriented between thin glass plates. These chemical shifts provide orientational information for the molecular frame with respect to the magnetic field in the laboratory frame. When modeled as a uniform ideal alpha-helix, M2-TMP has a tilt of 37(+/-3) degrees in DMPC and 33(+/-3) degrees in DOPC with respect to the bilayer normal in these lipid environments. The difference in helix tilt between the two environments appears to be small. This lack of a substantial change in tilt further suggests that significant interactions occur between the helices, as in an oligomeric state, to prevent a change in tilt in thicker lipid bilayers. 相似文献
7.
W H Goldmann J M Teodoridis C P Sharma B Hu G Isenberg 《Biochemical and biophysical research communications》1999,259(1):108-112
Previous computer analyses suggested two possible lipid binding sites, residues 49-71 and 131-155, of the primary amino acid sequence on ABP-280 (filamin), which could facilitate membrane attachment/insertion. We expressed these regions as fusion proteins with schistosomal GST and investigated their interaction with mixtures of zwitterionic (dimyristoyl-l-alpha-phosphatidylcholine, DMPC) and anionic (dimyristoyl-l-alpha-phosphatidylglycerol, DMPG) phospholipids in reconstituted lipid bilayers by differential scanning calorimetry (DSC). Using vesicles of mixed DMPC/DMPG with increasing fusion protein concentrations, we established in calorimetric assays a decrease of the main chain transition enthalpy, DeltaH, and a shift in chain melting temperature. This is indicative of the insertion of these fragments into the hydrophobic region of lipid membranes. We confirmed these findings by the film balance technique using lipid monolayers (DMPG). The binding judged from both methods was of moderate affinity. 相似文献
8.
Magainin 2 and PGLa are among the best-studied cationic antimicrobial peptides. They bind preferentially to negatively charged membranes and apparently cause their disruption by the formation of transmembrane pores, whose detailed structure is still unclear. Here we report the results of 5–9 μs all-atom molecular dynamics simulations starting from tetrameric transmembrane helical bundles of these two peptides, as well as their stoichiometric mixture, and the analog MG-H2 in DMPC or 3:1 DMPC/DMPG membranes. The simulations produce pore structures that appear converged, although some effect of the starting peptide arrangement (parallel vs. antiparallel) is still observed on this timescale. The peptides remain mostly helical and adopt tilted orientations. The calculated tilt angles for PGLa are in excellent agreement with recent solid state NMR experiments. The antiparallel dimer structure in the magainin 2 simulations resembles previously determined NMR and crystal structures. More transmembrane orientations and a larger and more ordered pore are seen in the 1:1 heterotetramer with an antiparallel helix arrangement. Insights into the mechanism of synergy between these two peptides are obtained via implicit solvent modeling of homo- and heterodimers and analysis of interactions in the atomistic simulations. This analysis suggests stronger pairwise interactions in the heterodimer than in the two homodimers. 相似文献
9.
Galántai R Bárdos-Nagy I Módos K Kardos J Závodszky P Fidy J 《Archives of biochemistry and biophysics》2000,373(1):261-270
It is frequently observed in pharmaceutical practice that entrapped substances are lost rapidly when liposomes are used as carriers to introduce substances into cells. The reason for the loss is the interaction of serum components with liposomes. To elucidate the mechanism of this phenomenon the partition of mesoporphyrin (MP) was systematically studied in model systems composed of various lipids and human serum albumin (HSA). As surface charge is an important factor in the interaction, neutral (1, 2-dimyristoyl-sn-glycero-3-phosphatidylcoline, DMPC) and negatively charged (1,2-dimyristoyl-sn-glycero-3-phosphatidylcoline/1, 2-dimyristoyl-sn-glycero-3-phosphatidylglycerol, DMPC/DMPG = 19/1 w/w) lipids were compared. The liposome/apomyoglobin system was the negative control. The size distribution of sonicated samples was carefully analyzed by dynamic light scattering. Constants of association of MP to the proteins and to the liposomes were determined: K(p,1) = (2.5 +/- 0.7) x 10(7) M(-1), K(p,2) = (1.0 +/- 0.7) x 10(8) M(-1), K(L,1) = (1.3 +/- 0.3) x 10(5) M(-1), and K(L,2) = (3.2 +/- 0.6) x 10(4) M(-1) for HSA, apomyoglobin, DMPC, and DMPC/DMPG liposomes, respectively. These data were used to evaluate the partition experiments. The transfer of MP from the liposomes to the proteins was followed by fluorescence spectroscopy. In the case of apomyoglobin, the experimental points could be interpreted by ruling out the protein-liposome interaction. In the case of HSA, the efflux of MP from the liposomes was strongly inhibited above a critical HSA concentration range for negatively charged vesicles. This effect was interpreted as the result of HSA coat formation on the liposome surface. This direct interaction is significant for small liposomes. The interpretation is fully supported by differential scanning calorimetry experiments. 相似文献
10.
The influence of trehalose on the interaction of liposomes with porphyrins and with human serum albumin (HSA) was studied. Small unilamellar liposomes were prepared from 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and from DMPC/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPG) 19:1 w/w% and incorporated with mesoporphyrin IX (MP) or magnesium mesoporphyrin (MgMP). The fluorescence intensity and anisotropy of porphyrins were measured within the temperature range of 15-33 degrees C, in the presence and in the absence of 3x10(-2) M trehalose, to study the location of the porphyrins inside the liposomes and their partition between the liposomes and HSA. Based on the presented data and our earlier results (I. Bárdos-Nagy, R. Galántai, A.D. Kaposi, J. Fidy, Int. J. Pharm. 175 (1998) 255-267) we conclude that trehalose - even at this relatively low concentration - interacts with the head groups of the liposomes and that the presence of DMPG enhances the effect. This effect seems to hinder the binding of HSA to the liposome surface and influences the location of MgMP within the liposomes. In the case of MP, the porphyrin partition between the liposomes and HSA was affected by trehalose, while for MgMP, trehalose changed the structural conditions of porphyrin binding to the liposomes. The amount of trehalose used did not have a general trend to modify the association constants of porphyrin derivatives either to liposomes or to HSA. 相似文献
11.
Sathiah Thennarasu Anmin Tan Rajesh Penumatchu Deborah L. Heyl 《Biophysical journal》2010,98(2):248-257
A 21-residue peptide segment, LL7-27 (RKSKEKIGKEFKRIVQRIKDF), corresponding to residues 7-27 of the only human cathelicidin antimicrobial peptide, LL37, is shown to exhibit potent activity against microbes (particularly Gram-positive bacteria) but not against erythrocytes. The structure, membrane orientation, and target membrane selectivity of LL7-27 are characterized by differential scanning calorimetry, fluorescence, circular dichroism, and NMR experiments. An anilinonaphthalene-8-sulfonic acid uptake assay reveals two distinct modes of Escherichia coli outer membrane perturbation elicited by LL37 and LL7-27. The circular dichroism results show that conformational transitions are mediated by lipid-specific interactions in the case of LL7-27, unlike LL37. It folds into an α-helical conformation upon binding to anionic (but not zwitterionic) vesicles, and also does not induce dye leakage from zwitterionic lipid vesicles. Differential scanning calorimetry thermograms show that LL7-27 is completely integrated with DMPC/DMPG (3:1) liposomes, but induces peptide-rich and peptide-poor domains in DMPC liposomes. 15N NMR experiments on mechanically aligned lipid bilayers suggest that, like the full-length peptide LL37, the peptide LL7-27 is oriented close to the bilayer surface, indicating a carpet-type mechanism of action for the peptide. 31P NMR spectra obtained from POPC/POPG (3:1) bilayers containing LL7-27 show substantial disruption of the lipid bilayer structure and agree with the peptide's ability to induce dye leakage from POPC/POPG (3:1) vesicles. Cholesterol is shown to suppress peptide-induced disorder in the lipid bilayer structure. These results explain the susceptibility of bacteria and the resistance of erythrocytes to LL7-27, and may have implications for the design of membrane-selective therapeutic agents. 相似文献
12.
Phospholamban is a cardiac regulatory protein that, in its monomeric form, inhibits the Ca(2+)-ATPase. Lipid-protein interactions with a synthetic variant of phospholamban, in which all cysteine residues are replaced with alanine, have been studied by spin-label electron spin resonance (ESR) in different lipid host membranes. Both the stoichiometry and selectivity of lipid interactions were determined from the two-component ESR spectra of phospholipid species spin-labeled on the 14 C atom of the sn-2 chain. The lipid stoichiometry is determined by the oligomeric state of the protein and the selectivity by the membrane disposition of the positively charged residues in the N-terminal section of the protein. In dimyristoylphosphatidylcholine (DMPC) membranes, the stoichiometry (N(b)) is 7 lipids/monomer for the full-length protein and 4 for the transmembrane section (residues 26-52). These stoichiometries correspond to the dimeric and pentameric forms, respectively. In palmitoyloleoylphosphatidylcholine, N(b) = 4 for both the whole protein and the transmembrane peptide. In negatively charged membranes of dimyristoylphosphatidylglycerol (DMPG), the lipid stoichiometry is N(b) = 10-11 per monomer for both the full-length protein and the transmembrane peptide. This stoichiometry corresponds to monomeric dispersion of the protein in the negatively charged lipid. The sequence of lipid selectivity is as follows: stearic acid > phosphatidic acid > phosphatidylserine = phosphatidylglycerol = phosphatidylcholine > phosphatidylethanolamine for both the full-length protein and the transmembrane peptide in DMPC. Absolute selectivities are, however, lower for the transmembrane peptide. A similar pattern of lipid selectivity is obtained in DMPG, but the absolute selectivities are reduced considerably. The results are discussed in terms of the integration of the regulatory species in the lipid membrane. 相似文献
13.
UV-visible and dichroic spectrum analysis and electron microscopy have been used to characterize a new amphotericin B (AmB) lipid formulation prepared by a solvent displacement process. The composition was dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) in molar ratio DMPC/DMPG/AmB 7:3:5, a similar composition to that of Abelcet®. Although the latter has a “ribbon-like” structure, our process gave a thin disc-like structure. Analysis of circular dichroism (CD) and UV-visible spectra of formulations containing different percentages of AmB revealed that a minimum of AmB self-association was observed with 7:3:5 molar ratio. Varying the lipid ratio (DMPC/DMPG) while maintaining the fixed ratio of AmB yielded similar results when DMPC was in excess (DMPC/DMPG from 10:0 to 6:4). However, when the ratio was between 5:5 to 3:7, AmB self-aggregation increased. For compositions rich in DMPG (2:8 and 0:10), inversion of the CD spectrum was observed. The influence of the lipid composition on the morphology of the complex was also evident in electron microscopy. DMPC/DMPG/AmB (10:0:5) gave large unfracturable lamellae. The presence of DMPG shortened the lamellae, which often appeared as disc-like structures. AmB content, the presence of DMPG and the preparation process all contribute to generating these original structures with particular CD spectra. 相似文献
14.
Bacteriophage M13 major coat protein has been isolated with cholate and reconstituted in dimyristoyl- and dioleoylphosphatidylcholine (DMPC and DOPC, respectively) bilayers by dialysis. Fourier transform infrared spectra of DMPC/coat protein recombinants confirmed that, whereas the protein isolated by phenol extraction was predominantly in a beta-sheet conformation, the cholate-isolated coat protein contained a higher proportion of the alpha-helical conformation [cf. Spruijt, R. B., Wolfs, C. J. A. M., & Hemminga, M. A. (1989) Biochemistry 28, 9158-9165]. The cholate-isolated coat protein/lipid recombinants gave different electron spin resonance (ESR) spectral line shapes of incorporated lipid spin labels, as compared with those from recombinants with the phenol-extracted protein that were studied previously [Wolfs, C. J. A. M., Horváth, L. I., Marsh, D., Watts, A., & Hemminga, M. A. (1989) Biochemistry 28, 9995-10001]. Plots of the ratio of the fluid/motionally restricted components in the ESR spectra of spin-labeled phosphatidylglycerol were linear with respect to the lipid/protein ratio in the recombinants up to 20 mol/mol. The corresponding values of the relative association constants, Kr, and number of association sites, N1, on the protein were Kr approximately 1 and N1 approximately 4 for DMPC recombinants and Kr approximately 1 and N1 approximately 5 for DOPC recombinants. Simulation of the two-component lipid spin label ESR spectra with the exchange-coupled Bloch equations gave values for the off-rate of the lipids leaving the protein surface of 2.0 x 10(7) s-1 at 27 degrees C in DMPC recombinants and 3.0 x 10(7) s-1 at 24 degrees C in DOPC recombinants.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
15.
Aisenbrey C Harzer U Bauer-Manz G Bär G Chotimah IN Bertani P Sizun C Kuhn A Bechinger B 《The FEBS journal》2006,273(4):817-828
The coat proteins of filamentous phage are first synthesized as transmembrane proteins and then assembled onto the extruding viral particles. We investigated the transmembrane conformation of the Pseudomonas aeruginosa Pf3 phage coat protein using proton-decoupled 15N and 31P solid-state NMR spectroscopy. The protein was either biochemically purified and uniformly labelled with 15N or synthesized chemically and labelled at specific sites. The proteins were then reconstituted into oriented phospholipid bilayers and the resulting samples analysed. The data suggest a model in which the protein adopts a tilted helix with an angle of approximately 30 degrees and an N-terminal 'swinging arm' at the membrane surface. 相似文献
16.
The partition coefficients (K(p)) between lipid bilayers of dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) unilamellar liposomes and water were determined using derivative spectrophotometry for chlordiazepoxide (benzodiazepine), isoniazid and rifampicin (tuberculostatic drugs) and dibucaine (local anaesthetic). A comparison of the K(p) values in water/DMPG with those in water/DMPC (dimyristoyl-L-alpha-phosphatidylcholine) revealed that for chlordiazepoxide and isoniazid, neutral drugs at physiological pH, the partition coefficients are similar in anionic (DMPG) and zwitterionic (DMPC) liposomes. However, for ionised drugs at physiological pH, the electrostatic interactions are different with DMPG and DMPC, with the cationic dibucaine having a stronger interaction with DMPG, and the anionic rifampicin having a much larger K(p) in zwitterionic DMPC. These results show that liposomes are a better model membrane than an isotropic two-phase solvent system, such as water-octanol, to predict drug-membrane partition coefficients, as they mimic better the hydrophobic part and the outer polar charged surface of the phospholipids of natural membranes. 相似文献
17.
Buffy JJ Traaseth NJ Mascioni A Gor'kov PL Chekmenev EY Brey WW Veglia G 《Biochemistry》2006,45(36):10939-10946
Sarcolipin (SLN), a 31 amino acid integral membrane protein, regulates SERCA1a and SERCA2a, two isoforms of the sarco(endo)plasmic Ca-ATPase, by lowering their apparent Ca(2+) affinity and thereby enabling muscle relaxation. SLN is expressed in both fast-twitch and slow-twitch muscle fibers with significant expression levels also found in the cardiac muscle. SLN shares approximately 30% identity with the transmembrane domain of phospholamban (PLN), and recent solution NMR studies carried out in detergent micelles indicate that the two polypeptides bind to SERCA in a similar manner. Previous 1D solid-state NMR experiments on selectively (15)N-labeled sites showed that SLN crosses the lipid bilayer with an orientation nearly parallel to the bilayer normal. With a view toward the characterization of SLN structure and its interactions with both lipids and SERCA, herein we report our initial structural and topological assignments of SLN in mechanically oriented DOPC/DOPE lipid bilayers as mapped by 2D (15)N PISEMA experiments. The PISEMA spectra obtained on uniformly (15)N-labeled protein as well as (15)N-Leu, (15)N-Ile and (15)N-Val map the secondary structure of SLN and, simultaneously, reveal that SLN exists in two distinct topologies. Both the major and the minor populations assume an orientation with the helix axis tilted by approximately 23 degrees with respect to the lipid bilayer normal, but vary in the rotation angle about the helix axis by approximately 5 degrees . The existence of the multiple populations in model membranes may be a significant requirement for SLN interaction with SERCA. 相似文献
18.
Maculatin 1.1 is an antimicrobial peptide isolated from the Australian tree frog Litoria genimaculata that adopts an amphipathic, alpha-helical structure in solution. Its orientation and conformation when incorporated to pre-formed DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol) and DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) vesicles was determined using polarised Fourier transform infrared-attenuated total reflection infrared and deuterium exchange experiments. For DMPG membranes, our results show insertion of 70% of the maculatin 1.1 molecules, with an angle of insertion of approximately 35 degrees to the membrane normal and with a predominant alpha-helical structure. These results suggest that maculatin 1.1 acts through a pore-forming mechanism to lyse bacterial membranes. A similar degree of insertion in DMPG (65%) and alpha-helical structure was observed for a biologically inactive, less amphipathic maculatin 1.1 analogue, P15A, although the helix tilt was found to be greater (46 degrees) than for maculatin 1.1. Similar experiments performed using DMPC liposomes showed poor insertion, less than 5%, for both maculatin 1.1 and its analogue. In addition, the shape of the amide I band in these samples is consistent with alpha-helix, beta-structure and disordered structures being present in similar proportion. These results clearly show that maculatin 1.1 inserts preferentially in negatively charged membranes (DMPG) which mimic the negatively charged membrane of Gram-positive bacteria. We attribute the high percentage of insertion of the biologically inactive analogue in DMPG to the fact that its concentration on the membrane surface in our experiments is likely to be much higher than that found in physiological conditions. 相似文献
19.
With molecular dynamics simulations of phospholipid membranes becoming a reality, there is a growing need for experiments that provide the molecular details necessary to test these computational results. Pyridine is used here to explore the interaction of planar aromatic groups with the water-lipid interface of membranes. It is shown by magic angle spinning 13C nuclear magnetic resonance (NMR) to bind between the glycerol and choline groups of dimyristoylphosphatidylcholine (DMPC) liposomes. The axial pattern for the 31P NMR spectrum of DMPC liposomes is preserved even with more than half of the interfacial sites occupied, indicating that pyridine does not disrupt the lamellar phase of this lipid. 2H NMR experiments of liposomes in deuterium oxide demonstrate that pyridine might promote greater penetration of water into restricted regions in the interface. Magnetically oriented DMPC/surfactant micelles were investigated as a means for improving resolution and sensitivity in NMR studies of species bound to bilayers. The quadrupolar splittings in the 2H NMR spectra of d5-pyridine in DMPC liposomes and magnetically oriented DMPC/Trixon X-100 micelles indicate a common bound state for the two bilayer systems. The well resolved quadrupolar splittings of d5-pyridine in oriented micelles were used to establish the tilt of the pyridine ring relative to the bilayer plane. 相似文献
20.
Physical biochemistry of a liposomal amphotericin B mixture used for patient treatment 总被引:1,自引:0,他引:1
There seems little doubt now that intravenous liposomal amphotericin B can be a useful treatment modality for the management of immunocompromised patients with suspected or proven disseminated fungal infections. Interestingly, the very significant reduction in toxicity reported when amphotericin B is part of a bilayer membrane is closely tied to the physical characteristics of the liposomes involved, although these are poorly understood at the molecular level. We record here an examination by spectroscopy and freeze-etch electron microscopy of unsonicated amphotericin B multilamellar vesicles prepared along the lines that we and others have followed for samples used in clinical trials and preclinical in vivo or in vitro studies. Our study has focussed on liposomes of 7:3 dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bearing 0-25 mol% amphotericin B, since this lipid mixture has been the choice for the first clinical trials. Phase transition behaviour of these liposomes was examined by electron paramagnetic resonance (EPR) spectroscopy of a nitroxide spin label partitioning into the bilayers. The same experiments were then performed on similarly prepared liposomes of the disaturated species, dipalmitoylphosphatidylcholine (DPPC), and the diunsaturated species, dielaidoylphosphatidylcholine (DEPC). Partial phase diagrams were constructed for each of the lipid/drug mixtures. Melting curves and derived phase diagrams showed evidence that amphotericin B is relatively immiscible with the solid phase of bilayer membranes. The phase diagram for DEPC/amphotericin B was very similar to that of DPPC/amphotericin B, and both exhibited less extensive temperature ranges of phase separation than did the 7:3 DMPC/DMPG mixture with amphotericin B. Between 25 and 37 degrees C the measured fluidity of the 7:3 DMPC/DMPG liposomes was similar to that of the (unsaturated fatty acid) DEPC liposomes, and considerably higher than that seen for (saturated fatty acid) DPPC liposomes. Preparations of 7:3 DMPC/DMPG, DPPC, and DEPC containing 0-25 mol% amphotericin B were examined by freeze-etch electron microscopy at 35 and 22 degrees C (to cover the temperature range of the mammalian body core and periphery). The same liposome features were present in all three liposome types studied. The appearance of individual liposomes at x 100,000 magnification reflected their molecular characteristics, which were found to be significantly heterogeneous within each batch. The lipid/drug structures were bilayer in nature, although liposomes showing considerable disruption were common, particularly at the highest drug concentrations.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献