Biomedical subjects

Anmin Tan

Publications and source records attributed to Anmin Tan.

9 recordsLinked to original sources

PubMed2006

Deletion of all cysteines in tachyplesin I abolishes hemolytic activity and retains antimicrobial activity and lipopolysaccharide selective binding.

Tachyplesin I is a cyclic beta-sheet antimicrobial peptide isolated from the hemocytes of Tachypleus tridentatus. The four cysteine residues in tachyplesin I play a structural role in imparting amphipathicity to the peptide which has been shown to be essential for its activity. We investigated the role of amphipathicity using an analogue of tachyplesin I (TP-I), CDT (KWFRVYRGIYRRR-NH(2)), in which all four cysteines were deleted. Like TP-I, CDT shows antimicrobial activity and disrupts Escherichia coli outer membrane and model membranes mimicking bacterial inner membranes at micromolar concentrations. The CDT peptide does not cause hemolysis up to 200 microg/mL while TP-I showed about 10% hemolysis at 100 microg/mL and about 25% hemolysis at 150 microg/mL. Peptide-into-lipid titrations under isothermal conditions reveal that the interaction of CDT with lipid membranes is an enthalpy-driven process. Binding assays performed using fluorometry demonstrate that the peptide CDT binds and inserts into only negatively charged membranes. The peptide-induced thermotropic phase transition of MLVs formed of DMPC and the DMPC/DMPG (7:3) mixture suggests specific lipid-peptide interactions. The circular dichroism study shows that the peptide exists as an unordered structure in an aqueous buffer and adopts a more ordered beta-structure upon binding to negatively charged membrane. The NMR data suggest that CDT binding to negatively charged bilayers induces a change in the lipid headgroup conformation with the lipid headgroup moving out of the bilayer surface toward the water phase, and therefore, a barrel stave mechanism of membrane disruption is unlikely as the peptide is located near the headgroup region of lipids. The lamellar phase (31)P chemical shift spectra observed at various concentrations of the peptide in bilayers suggest that the peptide may function neither via fragmentation of bilayers nor by promoting nonlamellar structures. NMR and fluorescence data suggest that the presence of cholesterol inhibits the peptide binding to the bilayers. These properties help to explain that cysteine residues may not contribute to antimicrobial activity and that the loss of hemolytic activity is due to lack of hydrophobicity and amphipathicity.

Anti-Infective Agents↗

PubMed2006

Solid-state NMR investigation of the membrane-disrupting mechanism of antimicrobial peptides MSI-78 and MSI-594 derived from magainin 2 and melittin.

The mechanism of membrane interaction of two amphipathic antimicrobial peptides, MSI-78 and MSI-594, derived from magainin-2 and melittin, is presented. Both the peptides show excellent antimicrobial activity. The 8-anilinonaphthalene-1-sulfonic acid uptake experiment using Escherichia coli cells suggests that the outer membrane permeabilization is mainly due to electrostatic interactions. The interaction of MSI-78 and MSI-594 with lipid membranes was studied using 31P and 2H solid-state NMR, circular dichroism, and differential scanning calorimetry techniques. The binding of MSI-78 and MSI-594 to the lipid membrane is associated with a random coil to alpha-helix structural transition. MSI-78 and MSI-594 also induce the release of entrapped dye from POPC/POPG (3:1) vesicles. Measurement of the phase-transition temperature of peptide-DiPoPE dispersions shows that both MSI-78 and MSI-594 repress the lamellar-to-inverted hexagonal phase transition by inducing positive curvature strain. 15N NMR data suggest that both the peptides are oriented nearly perpendicular to the bilayer normal, which infers that the peptides most likely do not function via a barrel-stave mechanism of membrane-disruption. Data obtained from 31P NMR measurements using peptide-incorporated POPC and POPG oriented lamellar bilayers show a disorder in the orientation of lipids up to a peptide/lipid ratio of 1:20, and the formation of nonbilayer structures at peptide/lipid ratio>1:8. 2H-NMR experiments with selectively deuterated lipids reveal peptide-induced disorder in the methylene units of the lipid acyl chains. These results are discussed in light of lipid-peptide interactions leading to the disruption of membrane via either a carpet or a toroidal-type mechanism.

Antimicrobial Cationic Peptides↗

PubMed2006

Cell selectivity correlates with membrane-specific interactions: a case study on the antimicrobial peptide G15 derived from granulysin.

A 15-residue peptide dimer G15 derived from the cell lytic protein granulysin has been shown to exert potent activity against microbes, including E. coli, but not against human Jurkat cells [Z. Wang, E. Choice, A. Kaspar, D. Hanson, S. Okada, S.C. Lyu, A.M. Krensky, C. Clayberger, Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin. J. Immunol. 165 (2000) 1486-1490]. We investigated the target membrane selectivity of G15 using fluorescence, circular dichroism and 31P NMR methods. The ANS uptake assay shows that the extent of E. coli outer membrane disruption depends on G15 concentration. 31P NMR spectra obtained from E. coli total lipid bilayers incorporated with G15 show disruption of lipid bilayers. Fluorescence binding studies on the interaction of G15 with synthetic liposomes formed of E. coli lipids suggest a tight binding of the peptide at the membrane interface. The peptide also binds to negatively charged POPC/POPG (3:1) lipid vesicles but fails to insert deep into the membrane interior. These results are supported by the peptide-induced changes in the measured isotropic chemical shift and T1 values of POPG in 3:1 POPC:POPG multilamellar vesicles while neither a non-lamellar phase nor a fragmentation of bilayers was observed from NMR studies. The circular dichroism studies reveal that the peptide exists as a random coil in solution but folds into a less ordered conformation upon binding to POPC/POPG (3:1) vesicles. However, G15 does not bind to lipid vesicles made of POPC/POPG/Chl (9:1:1) mixture, mimicking tumor cell membrane. These results explain the susceptibility of E. coli and the resistance of human Jurkat cells to G15, and may have implications in designing membrane-selective therapeutic agents.

Amino Acid Sequence↗

PubMed2005

Solution structure and interaction of the antimicrobial polyphemusins with lipid membranes.

The horseshoe crab cationic antimicrobial peptide polyphemusin I is highly active in vitro but not protective in mouse models of bacterial and LPS challenge, while a synthetic polyphemusin variant, PV5, was previously shown to be protective in vivo. In this study, we investigated the interaction of these peptides with lipid membranes in an effort to propose a mechanism of interaction. The solution structure of PV5 was determined by proton NMR in the absence and presence of dodecylphosphocholine (DPC) micelles. Like polyphemusin I, PV5 is a beta-hairpin but appeared less amphipathic in solution. Upon association with DPC micelles, PV5 underwent side chain rearrangements which resulted in an increased amphipathic conformation. Using fluorescence spectroscopy, both peptides were found to have limited affinity for neutral vesicles composed of phosphatidylcholine (PC). Incorporation of 25 mol % cholesterol or phosphatidylethanolamine into PC vesicles produced little change in the partitioning of either peptide. Incorporation of 25 mol % phosphatidylglycerol (PG) into PC vesicles, a simple prokaryotic model, resulted in a large increase in the affinity for both peptides, but the partition coefficient for PV5 was almost twice that of polyphemusin I. Differential scanning calorimetry studies supported the partitioning data and demonstrated that neither peptide interacted readily with neutral PC vesicles. Both peptides showed affinity for negatively charged membranes incorporating PG. The affinity of PV5 was much greater as the pretransition peak was absent at low peptide to lipid ratios (1:400) and the reduction in enthalpy of the main transition was greater than that produced by polyphemusin I. Both peptides decreased the lamellar to inverted hexagonal phase transition temperature of PE indicating the induction of negative curvature strain. These results, combined with previous findings that polyphemusin I promotes lipid flip-flop but does not induce significant vesicle leakage, ruled out the torroidal pore and carpet mechanisms of antimicrobial action for these polyphemusins.

Animals↗

PubMed2005

Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843.

Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH2) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts alpha-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. 2H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. 31P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from 31P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and 31P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843.

Anti-Bacterial Agents↗

PubMed2004

Calorimetric measurement of phospholipid interaction with methyl-beta-cyclodextrin.

Cyclodextrins are able to bind hydrophobic molecules in their interior cavity and as such have received a great deal of attention as carriers of cholesterol, lipophilic drugs, and other sparingly soluble compounds. Despite the importance of these biochemical applications, relatively little is known about the interactions of cyclodextrins with phospholipid membranes. Here we characterize the binding of randomly methylated beta-cyclodextrin (m beta CD) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using right-angle light scattering and isothermal titration calorimetry. Existing models of lipophile-membrane interactions are inadequate to describe the observed binding; we introduce a modified chemical reaction model in which the chemical activity of the phospholipid is independent of its concentration. We find that an average of four m beta CD molecules bind to each POPC molecule with an enthalpy of reaction of 46 kJ mol(-1) and an equilibrium constant of 90 M(-3). These results are consistent with earlier qualitative observations and suggest that disruption of phospholipid membranes may be minimized if the concentration of m beta CD is kept below about 15 mM.

Binding Sites↗

PubMed2004

Investigation of the interaction of myelin basic protein with phospholipid bilayers using solid-state NMR spectroscopy.

Interaction of bovine myelin basic protein and its constituent charge isomers (C1-C3) with phospholipid bilayers was studied using solid-state NMR experiments on model membranes. 31P NMR experiments on multilamellar vesicles and mechanically aligned bilayers were used to measure the degree of protein-induced disorder in the lipid headgroup region while 2H NMR data provided the disorder caused by the protein in the hydrophobic core of the bilayers. Our results suggest that MBP and its charge isomers neither fragment nor significantly disrupt DMPC, POPC, POPC:POPG, and POPE bilayers. These results demonstrate that the MBP-induced fragmentation of POPC bilayers is due to the freeze-thaw cycles used in the preparation of multilamellar vesicles and not due to intrinsic protein-lipid interactions.

Lipid Bilayers↗

PubMed2003

Distinguishing between different pathways of bilayer disruption by the related antimicrobial peptides cecropin B, B1 and B3.

Different pathways of bilayer disruption by the structurally related antimicrobial peptides cecropin B, B1 and B3, revealed by surface plasma resonance analysis of immobilized liposomes, differential scanning calorimetry of peptide-large unilamellar vesicle interactions, and light microscopic analysis of peptide-treated giant unilamellar vesicles, have been identified in this study. Natural cecropin B (CB) has one amphipathic and one hydrophobic alpha-helix, whereas cecropins B1 (CB1) and B3 (CB3), which are custom-designed, chimaeric analogues of CB, possess either two amphipathic or two hydrophobic alpha-helices, respectively. Surface plasma resonance analysis of unilamellar vesicles immobilized through a biotin-avidin interaction showed that both CB and CB1 bind to the lipid bilayers at high concentration (>10 microm); in contrast, CB3 induces disintegration of the vesicles at all concentrations tested. Differential scanning calorimetry showed the concentration-dependent effect of bilayer disruption, based on the different thermotrophic phase behaviours and the shapes of the thermal phase-transition curves obtained. The kinetics of the lysis of giant unilamellar vesicles observed by microscopy demonstrated that both CB and CB1 effect a continuous process involving loss of integrity followed by coalescence and resolution into smaller vesicles, whereas CB3 induces rapid formation of irregular-shaped, nonlamellar structures which rapidly disintegrate into twisted, microtubule-containing debris before being completely destroyed. On the basis of these observations, models by which CB, CB1 and CB3 induce lysis of lipid bilayers are discussed.

Amino Acid Sequence↗

PubMed2002

Thermodynamics of sodium dodecyl sulfate partitioning into lipid membranes.

The partition equilibria of sodium dodecyl sulfate (SDS) and lithium dodecyl sulfate between water and bilayer membranes were investigated with isothermal titration calorimetry and spectroscopic methods (light scattering, (31)P-nuclear magnetic resonance) in the temperature range of 28 degrees C to 56 degrees C. The partitioning of the dodecyl sulfate anion (DS(-)) into the bilayer membrane is energetically favored by an exothermic partition enthalpy of Delta H(O)(D) = -6.0 kcal/mol at 28 degrees C. This is in contrast to nonionic detergents where Delta H(O)(D) is usually positive. The partition enthalpy decreases linearly with increasing temperature and the molar heat capacity is Delta C(O)(P) = -50 +/- 3 cal mol(-1) K(-1). The partition isotherm is nonlinear if the bound detergent is plotted versus the free detergent concentration in bulk solution. This is caused by the electrostatic repulsion between the DS(-) ions inserted into the membrane and those free in solution near the membrane surface. The surface concentration of DS(-) immediately above the plane of binding was hence calculated with the Gouy-Chapman theory, and a strictly linear relationship was obtained between the surface concentration and the extent of DS(-) partitioning. The surface partition constant K describes the chemical equilibrium in the absence of electrostatic effects. For the SDS-membrane equilibrium K was found to be 1.2 x 10(4) M(-1) to 6 x 10(4) M(-1) for the various systems and conditions investigated, very similar to data available for nonionic detergents of the same chain length. The membrane-micelle phase diagram was also studied. Complete membrane solubilization requires a ratio of 2.2 mol SDS bound per mole of total lipid at 56 degrees C. The corresponding equilibrium concentration of SDS free in solution is C (sat)(D,F) approximately 1.7 mM and is slightly below the critical micelles concentration (CMC) = 2.1 mM (at 56 degrees C and 0.11 M buffer). Membrane saturation occurs at approximately 0.3 mol SDS per mol lipid and the equilibrium SDS concentration is C (sat)(D,F)approximately equal 2.2 mM +/- 0.6 mM. SDS translocation across the bilayer is slow at ambient temperature but increases at high temperatures.

Anisotropy↗