Biomedical subjects

Paavo K J Kinnunen

Publications and source records attributed to Paavo K J Kinnunen.

At least 19 recordsLinked to original sources

PubMed2006

A versatile method for determining the molar ligand-membrane partition coefficient.

A novel method for the quantitative assessment of the membrane partitioning of a ligand from the aqueous phase is described, demonstrated here with the thoroughly studied antipsychotic chlorpromazine (CPZ). More specifically, collisional quenching of the fluorescence of a pyrene labeled fluorescent lipid analog 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) by CPZ was utilized, using 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and -serine (POPC and POPS) liposomes as model membranes. The molar partition coefficient is obtained from two series of titrations, one with constant [phospholipid] and increasing [drug] and the other with constant [drug] and varying total [phospholipid], the latter further comprising of large unilamellar vesicles (LUVs) of POPC/POPS/PPDPC at a constant concentration of 10 microM and indicated concentrations of POPC/POPS LUVs. Notably, the approach described is generic and can be employed in screening for the membrane partitioning of compounds, providing that a suitable fluorescence parameter can be incorporated into one population of liposomes utilized as model membranes.

Antipsychotic Agents↗

PubMed2006

Characteristics of fibers formed by cytochrome c and induced by anionic phospholipids.

Recent publications described the formation of millimeter-length fibers by diverse lipid-binding proteins (e.g., histone H1, cytochrome c, indolicidin, and endostatin) when they are mixed with 80:20 phosphatidylcholine/phosphatidylserine vesicles. Further, these fibers displayed amyloid characteristics when stained with Congo Red. In the study presented here, we found by FTIR the amide I absorption band to reveal significant variation in fibers formed by cytochrome c, with some consisting of cytochrome c in a nativelike conformation and some exhibiting strong amyloid (beta-sheet) characteristics. Protein structure also varied from amyloid to nearly native within single fibers. Fibers were frequently blue or bluish and sometimes iridescent, likely due to interference of light in the fibers. The amyloid-type amide I band was observed for blue fibers only. AFM shows that fibers consist of smaller 3-4 nm diameter fibers with 10 nm lateral spacing.

Amyloid↗

PubMed2006

Antimicrobial peptides temporins B and L induce formation of tubular lipid protrusions from supported phospholipid bilayers.

The binding of the antimicrobial peptides temporins B and L to supported lipid bilayer (SLB) model membranes composed of phosphatidylcholine and phosphatidylglycerol (4:1, mol/mol) caused the formation of fibrillar protrusions, visible by fluorescent microscopy of both a fluorescent lipid analog and a labeled peptide. Multicolor imaging at low peptide-to-lipid ratios (P/L < approximately 1:5) revealed an initial in-plane segregation of membrane-bound peptide and partial exclusion of lipid from the peptide-enriched areas. Subsequently, at higher P/L numerous flexible lipid fibrils were seen growing from the areas enriched in lipid. The fibrils have diameters <250 nm and lengths of up to approximately 1 mm. Fibril formation reduces the in-plane heterogeneity and results in a relatively even redistribution of bound peptide over the planar bilayer and the fibrils. Physical properties of the lipid fibrils suggest that they have a tubular structure. Our data demonstrate that the peptide-lipid interactions alone can provide a driving force for the spontaneous membrane shape transformations leading to tubule outgrowth and elongation. Further experiments revealed the importance of positive curvature strain in the tubulation process as well as the sufficient positive charge on the peptide (>/=+2). The observed membrane transformations could provide a simplified in vitro model for morphogenesis of intracellular tubular structures and intercellular connections.

Anti-Infective Agents↗

PubMed2006

Lack of enantiomeric specificity in the effects of anesthetic steroids on lipid bilayers.

The most important target protein for many anesthetics, including volatile and steroid anesthetics, appears to be the type A gamma-amino butyric acid receptor (GABA(A)R), yet direct binding remains to be demonstrated. Hypotheses of lipid-mediated anesthesia suggest that lipid bilayer properties are changed by anesthetics and that this in turn affects the functions of proteins. While other data could equally well support direct or lipid-mediated action, enantiomeric specificity displayed by some anesthetics is not reflected in their interactions with lipids. In the present study, we studied the effects of two pairs of anesthetic steroid enantiomers on bilayers of several compositions, measuring potentially relevant physical properties. For one of the pairs, allopregnanolone and ent-allopregnanolone, the natural enantiomer is 300% more efficacious as an anesthetic, while for the other, pregnanolone and ent-pregnanolone, there is little difference in anesthetic potency. For each enantiomer pair, we could find no differences. This strongly favors the view that the effects of these anesthetics on lipid bilayers are not relevant for the main features of anesthesia. These steroids also provide tools to distinguish in general the direct binding of steroids to proteins from lipid-mediated effects.

Anesthetics↗

PubMed2006

Counterion-controlled transition of a cationic gemini from submicroscopic to giant vesicles.

While much is known about the self-assembly of lipids on nanoscale, our understanding of their biologically relevant mesoscale organization remains incomplete. Here, we show for a cationic gemini lipid a sharp and reversible transition from small vesicles with an average diameter of approximately 40 nm to giant vesicles (GVs) with an average diameter of approximately 11 microm. This transition is dependent on proper [NaCl] and specific temperature. Below this transition and in the vicinity of the air/water interface, a series of mesoscale morphological transitions was observed, revealing complex structures resembling biological membranes. On the basis of microscopy experiments, a tentative [NaCl] versus temperature shape/size phase diagram was constructed. To explain this unprecedented transition, we propose a novel mechanism whereby a specific interaction of Cl(-) counterion with the cationic gemini surfactant initiates the formation of a commensurate solute counterion lattice with low spontaneous curvature. In keeping with the high bending rigidity of NaCl crystal, this tightly associated ionic lattice enslaves membrane curvature and the mesoscale 3-D organization of this lipid.

Cations↗

PubMed2006

Interaction of fusidic acid with lipid membranes: Implications to the mechanism of antibiotic activity.

We have studied the effects of cholesterol and steroid-based antibiotic fusidic acid (FA) on the behavior of lipid bilayers using a variety of experimental techniques together with atomic-scale molecular dynamics simulations. Capillary electrophoretic measurements showed that FA was incorporated into fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes. Differential scanning calorimetry in turn showed that FA only slightly altered the thermodynamic properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, whereas cholesterol abolished all endotherms when the mole fraction of cholesterol (X(chol)) was >0.20. Fluorescence spectroscopy was then used to further characterize the influence of these two steroids on DPPC large unilamellar vesicles. In the case of FA, our result strongly suggested that FA was organized into lateral microdomains with increased water penetration into the membrane. For cholesterol/DPPC mixtures, fluorescence spectroscopy results were compatible with the formation of the liquid-ordered phase. A comparison of FA and cholesterol-induced effects on DPPC bilayers through atomistic molecular dynamics simulations showed that both FA and cholesterol tend to order neighboring lipid chains. However, the ordering effect of FA was slightly weaker than that of cholesterol, and especially for deprotonated FA the difference was significant. Summarizing, our results show that FA is readily incorporated into the lipid bilayer where it is likely to be enriched into lateral microdomains. These domains could facilitate the association of elongation factor-G into lipid rafts in living bacteria, enhancing markedly the antibiotic efficacy of FA.

Anti-Bacterial Agents↗

PubMed2006

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: implications for a novel mechanism of action.

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.

Acrylamide↗

PubMed2006

Mutational analysis and membrane-interactions of the beta-sheet-like N-terminal domain of the pediocin-like antimicrobial peptide sakacin P.

To gain insight into how the N-terminal three-stranded beta-sheet-like domain in pediocin-like antimicrobial peptides positions itself on membranes, residues in the well-conserved (Y)YGNGV-motif in the domain were substituted and the effect of the substitutions on antimicrobial activity and binding of peptides to liposomes was determined. Peptide-liposome interactions were detected by measuring tryptophan-fluorescence upon exposing liposomes to peptides in which a tryptophan residue had been introduced in the N-terminal domain. The results revealed that the N-terminal domain associates readily with anionic liposomes, but not with neutral liposomes. The electrostatic interactions between peptides and liposomes facilitated the penetration of some of the peptide residues into the liposomes. Measuring the antimicrobial activity of the mutated peptides revealed that the Tyr2Leu and Tyr3Leu mutations resulted in about a 10-fold reduction in activity, whereas the Tyr2Trp, Tyr2Phe, Tyr3Trp and Tyr3Phe mutations were tolerated fairly well, especially the mutations in position 3. The Val7Ile mutation did not have a marked detrimental effect on the activity. The Gly6Ala mutation was highly detrimental, consistent with Gly6 being in one of the turns in the beta-sheet-like N-terminal domain, whereas the Gly4Ala mutation was tolerated fairly well. All mutations involving Asn5, including the conservative mutations Asn5Gln and Asn5Asp, were very deleterious. Thus, both the polar amide group on the side chain of Asn5 and its exact position in space were crucial for the peptides to be fully active. Taken together, the results are consistent with Val7 positioning itself in the hydrophobic core of target membranes, thus forcing most of the other residues in the N-terminal domain into the membrane interface region: Tyr3 and Asn5 in the lower half with their side chains pointing downward and approaching the hydrophobic core, Tyr2, Gly4 and His8 and 12 in the upper half, Lys1 near the middle of the interface region, and the side chain of Lys11 pointing out toward the membrane surface.

Acrylamide↗

PubMed2006

Characterization of two oxidatively modified phospholipids in mixed monolayers with DPPC.

The properties of two oxidatively modified phospholipids viz. 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) and 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), were investigated using a Langmuir balance, recording force-area (pi-A) isotherms and surface potential psi. In mixed monolayers with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) a progressive disappearance of the liquid expanded-liquid condensed transition and film expansion was observed with increasing content of the oxidized phospholipids. The above is in agreement with fluorescence microscopy of the monolayers, which revealed an increase in the liquid expanded region of DPPC monolayers. At a critical pressure pi(s) approximately 42 mN/m both Poxo- and PazePC induced a deflection in the pi-A isotherms, which could be rationalized in terms of reorientation of the oxidatively modified acyl chains into aqueous phase (adaptation of the so-called extended conformation), followed upon further film compression by solubilization of the oxidized phospholipids into the aqueous phase. Surface potential displayed a discontinuity at the same value of area/molecule, corresponding to the loss of the oxidized phospholipids from the monolayers. Our data support the view that lipid oxidation modifies both the small-scale structural dynamics of biological membranes as well as their more macroscopic lateral organization. Accordingly, oxidatively modified lipids can be expected to influence the organization and functions of membrane associated proteins.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2006

Cytochrome C interaction with cardiolipin/phosphatidylcholine model membranes: effect of cardiolipin protonation.

Resonance energy transfer between anthrylvinyl-labeled phosphatidylcholine as a donor and heme moiety of cytochrome c (cyt c) as an acceptor has been employed to explore the protein binding to model membranes, composed of phosphatidylcholine and cardiolipin (CL). The existence of two types of protein-lipid complexes has been hypothesized where either deprotonated or partially protonated CL molecules are responsible for cyt c attachment to bilayer surface. To quantitatively describe cyt c membrane binding, the adsorption model based on scaled particle and double layer theories has been employed, with potential-dependent association constants being treated as a function of acidic phospholipid mole fraction, degree of CL protonation, ionic strength, and surface coverage. Multiple arrays of resonance energy transfer data obtained under conditions of varying pH, ionic strength, CL content, and protein/lipid molar ratio have been analyzed in terms of the model of energy transfer in two-dimensional systems combined with the adsorption model allowing for area exclusion and electrostatic effects. The set of recovered model parameters included effective protein charge, intrinsic association constants, and heme distance from the bilayer midplane for both types of protein-lipid complexes. Upon increasing CL mole fraction from 10 to 20 mol % (the value close to that characteristic of the inner mitochondrial membrane), the binding equilibrium dramatically shifted toward cyt c association with partially protonated CL species. The estimates of heme distance from bilayer center suggest shallow bilayer location of cyt c at physiological pH, whereas at pH below 6.0, the protein tends to insert into membrane core.

Cardiolipins↗

PubMed2006

The role of lipid-protein interactions in amyloid-type protein fibril formation.

Structural transition of polypeptide chains into the beta-sheet state followed by amyloid fibril formation is the key characteristic of a number of the so-called conformational diseases. The multistep process of protein fibrillization can be modulated by a variety of factors, in particular by lipid-protein interactions. A wealth of experimental evidence provides support to the notion that amyloid fibril assembly and the toxicity of pre-fibrillar aggregates are closely related and are both intimately membrane associated phenomena. The present review summarizes the principal factors responsible for the enhancement of fibril formation in a membrane environment, viz. (i) structural transformation of polypeptide chain into a partially folded conformation, (ii) increase of the local concentration of a protein upon its membrane binding, (iii) aggregation-favoring orientation of the bound protein, and (iv) variation in the depth of bilayer penetration affecting the nucleation propensity of the membrane associated protein. The molecular mechanisms of membrane-mediated protein fibrillization are discussed. Importantly, the toxicity of lipid-induced pre-fibrillar aggregates is likely to have presented a very strong negative selection pressure in the evolution of amino acid sequences.

Amino Acid Sequence↗

PubMed2006

Enantiomers of neuroactive steroids support a specific interaction with the GABA-C receptor as the mechanism of steroid action.

Neuroactive steroids can either potentiate or inhibit a variety of membrane channels. Most studies have suggested that the effects are mediated by specific association of the steroid with the affected channel. However, a recent study of the rho1 (GABA-C) receptor (Mol Pharmacol 66:56-69, 2004) concluded that the actions were consistent with an action of the steroid in the lipid bilayer to alter the lateral pressure profile in the membrane. The enantiomers of an optically active compound are expected to have identical physical properties, including interactions with hydrophobic portions of the cell membrane. We have used two pairs of enantiomers (pregnanolone and ent-pregnanolone, allopregnanolone and ent-allopregnanolone) and show that the ability to potentiate (allopregnanolone) or inhibit (pregnanolone) the rho1 receptor is enantioselective. Therefore, these results strongly suggest that the actions of these neuroactive steroids are mediated by interactions with chiral regions of the target protein, rather than by a change in membrane properties (including lateral pressure).

Animals↗

PubMed2006

Impact of reductive cleavage of an intramolecular disulfide bond containing cationic gemini surfactant in monolayers and bilayers.

The properties of a novel disulfide-bond-containing gemini surfactant bis[N,N-dimethyl-N-hexadecyl-N-(2-mercaptoethyl)ammonium bromide] disulfide (DSP) were studied using a Langmuir balance, supported monolayers, differential scanning calorimetry, giant vesicles, and LUVs. In 150 mM NaCl the cmc for DSP was 7.5 microM whereas that of the monomer N,N-dimethyl-N-hexadecyl-N-(2-mercaptoethyl)ammonium bromide (MSP) was 12.1 microM. Both surfactants exhibited single endotherms upon DSC, with peak temperatures Tm at 21.7 and 20.1 degrees C for DSP and MSP, respectively. The endotherm for MSP was significantly broader indicating less cooperative melting. Both in monolayers and in vesicles reductive cleavage of the disulfide bond of DSP could be obtained by glutathione (GSH). For Langmuir films of DSP the addition of GSH into the subphase led to a decrease in surface pressure pi as well as surface dipole potential psi. Although the cleavage by GSH was significantly slower in the presence of a charge saturating concentration of DNA, it did not prevent the reaction. The resulting monomers detached from supported monolayers, leading to loss of affinity of the surface for DNA. Disruption of giant vesicles containing DSP within approximately 30 s following a local injection of GSH was observed, revealing membrane destabilization.

Calorimetry, Differential Scanning↗

PubMed2005

Observation of the main phase transition of dinervonoylphosphocholine giant liposomes by fluorescence microscopy.

The phase heterogeneity of giant unilamellar dinervonoylphosphocholine (DNPC) vesicles in the course of the main phase transition was investigated by confocal fluorescence microscopy observing the fluorescence from the membrane incorporated lipid analog, 1-palmitoyl-2-(N-4-nitrobenz-2-oxa-1,3-diazol)aminocaproyl-sn-glycero-3-phosphocholine (NBDPC). These data were supplemented by differential scanning calorimetry (DSC) of DNPC large unilamellar vesicles (LUV, diameter approximately 0.1 and 0.2 microm) and multilamellar vesicles (MLV). The present data collected upon cooling reveal a lack of micron-scale gel and fluid phase coexistence in DNPC GUVs above the temperature of 20.5 degrees C, this temperature corresponding closely to the heat capacity maxima (T(em)) of DNPC MLVs and LUVs (T(em) approximately 21 degrees C), measured upon DSC cooling scans. This is in keeping with the model for phospholipid main transition inferred from our previous fluorescence spectroscopy data for DMPC, DPPC, and DNPC LUVs. More specifically, the current experiments provide further support for the phospholipid main transition involving a first-order process, with the characteristic two-phase coexistence converting into an intermediate phase in the proximity of T(em). This at least macroscopically homogenous intermediate phase would then transform into the liquid crystalline state by a second-order process, with further increase in acyl chain trans-->gauche isomerization.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2005

Increasing surface charge density induces interdigitation in vesicles of cationic amphiphile and phosphatidylcholine.

Binary vesicles of cationic lipid dihexadecyldimethylammoniumbromide (DHAB) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were examined by differential scanning calorimetry, fluorescence spectroscopy, and Fourier transform infrared spectroscopy. DHAB/DMPC vesicles demonstrate a complex dependence of the main-transition temperature (T(m)) on their mole proportion of DHAB, with a maximum of 42 degrees C at X(DHAB) = 0.4. An increase of T(m) at X(DHAB) < 0.4 is explained by reorientation of P(-)-N(+) dipoles of the phosphocholine headgroup, resulting in tighter packing of the acyl chains, which increases the thermal energy required for trans --> gauche isomerization. At X(DHAB) > 0.4, Coulombic repulsion between the cationic DHAB headgroups expands the bilayer evident as a decrease in T(m) until a plateau of approximately 28 degrees C at 0.7 < or = X(DHAB) > or = 0.9 is reached, followed by an increment of T(m) to approximately 30 degrees C at X(DHAB) > 0.9. The quenching of DPH-PC fluorescence emission and the decrease in the ratio of peak height intensities of symmetric and antisymmetric -CH(2)- stretching modes suggest an interdigitated phase to form at X(DHAB) > 0.6. Interdigitation allows the membrane to accommodate the augmented Coulombic repulsion between DHAB headgroups because of increasing cationic surface charge density while simultaneously causing tighter packing of the acyl chains evident first as a plateau at 0.7 < or = X(DHAB) > or = 0.9 and subsequently as an increase in T(m) at X(DHAB) > 0.9. Screening of the membrane charges by NaCl abolishes the quenching of DPH emission and decreases T(m), thus revealing electrostatic repulsion as the driving force for interdigitation.

Cations↗

PubMed2005

Binding of endostatin to phosphatidylserine-containing membranes and formation of amyloid-like fibers.

Endostatin, the 20-kDa C-terminal NC1 domain of collagen XVIII, is an endogenous inhibitor of tumor angiogenesis and tumor growth. A major problem in reconciling the many reported in vitro effects of endostatin is the lack of a high-affinity receptor, and a search for the latter continues. In accordance with the above, the molecular mechanisms of action of endostatin remain elusive. We show here that endostatin binds to membranes containing acidic phospholipids, phosphatidylserine (PS) or phosphatidylglycerol (PG). More specifically, a red shift in the fluorescence emission of Trp of endostatin in the presence of liposomes containing these anionic lipids was evident, revealing the average environment of Trps to become less hydrophobic. This shift was not observed for phosphatidylcholine (PC) liposomes, demonstrating the acidic lipid to be required. Quenching by endostatin of the fluorescence of a pyrene-labeled phospholipid analogue in PS containing membranes was seen, while there was no effect for PC liposomes. Resonance energy transfer from the Trp residues of endostatin to a dansyl-labeled phospholipid further confirmed the association of endostatin with PS-containing membranes, whereas there was no binding to PC liposomes. Intriguingly, the association of endostatin with PS-containing liposomes triggered the formation of fibers, with Congo red staining producing green birefringence characteristic for amyloid. Lipid was incorporated into these fibers, as shown by staining when a trace amount (X = 0.02) of fluorescent phospholipid analogues was present in the liposomes. No fiber formation was seen when endostatin was added to liposomes composed of PC only. Because PS has been reported to be exposed in the outer surface of the plasma membrane of cancer cells and vascular endothelial cells, our results suggest that this lipid could represent a target for endostatin in the cancer cell surface and tumors, thus suggesting a novel mechanism of its action. More specifically, analogous to a number of other cytotoxic proteins interacting with negatively charged lipids, PS-triggered fiber formation by endostatin on the surface of cancer cells would impair the permeability barrier function of the plasma membrane, resulting in cell death.

Amyloid↗

PubMed2005

Modification of permeability transition pore arginine(s) by phenylglyoxal derivatives in isolated mitochondria and mammalian cells. Structure-function relationship of arginine ligands.

Methylglyoxal and synthetic glyoxal derivatives react covalently with arginine residue(s) on the mitochondrial permeability transition pore (PTP). In this study, we have investigated how the binding of a panel of synthetic phenylglyoxal derivatives influences the opening and closing of the PTP. Using both isolated mitochondria and mammalian cells, we demonstrate that the resulting arginine-phenylglyoxal adduct can lead to either suppression or induction of permeability transition, depending on the net charge and hydrogen bonding capacity of the adduct. We report that phenylglyoxal derivatives that possess a net negative charge and/or are capable of forming hydrogen bonds induced permeability transition. Derivatives that were overall electroneutral and cannot form hydrogen bonds suppressed permeability transition. When mammalian cells were incubated with low concentrations of negatively charged phenylglyoxal derivatives, the addition of oligomycin caused a depolarization of the mitochondrial membrane potential. This depolarization was completely blocked by cyclosporin A, a PTP opening inhibitor, indicating that the depolarization was due to PTP opening. Collectively, these findings highlight that the target arginine(s) is functionally linked with the opening/closing mechanism of the PTP and that the electric charge and hydrogen bonding of the resulting arginine adduct influences the conformation of the PTP. These results are consistent with a model where the target arginine plays a role as a voltage sensor.

Animals↗

PubMed2005

Intermolecular interactions of lysobisphosphatidic acid with phosphatidylcholine in mixed bilayers.

Lysobisphosphatidic acid (LBPA) can be regarded to represent a unique derivative of phosphatidylglycerol. This lipid is highly enriched in late endosomes where it can comprise up to 10-15 mol% of all lipids and in these membranes, LBPA appears to be segregated into microdomains. We studied the thermotropic behavior of pure dioleoyl-LBPA mono- and bilayers using Langmuir-lipid monolayers, electron microscopy, differential scanning calorimetry (DSC), and fluorescence spectroscopy. LBPA formed metastable, liquid-expanded monolayers at an air/buffer interface, and its compression isotherms lacked any indication for structural phase transitions. Neat LBPA formed multilamellar vesicles with no structural transitions or phase transitions between 10 and 80 degrees C at a pH range of 3.0-7.4. We then proceeded to study mixed LBPA/dipalmitoylphosphatidylcholine (DPPC) bilayers by DSC and fluorescence spectroscopy. Incorporating increasing amounts of LBPA (up to X(LBPA) (molar fraction)=0.10) decreased the co-operativity of the main transition for DPPC, and a decrease in the main phase transition as well as pretransition temperature of DPPC was observed yet with no effect on the enthalpy of this transition. In keeping with the DSC data for DPPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC)/LBPA mixed bilayers were more fluid, and no evidence for lateral phase segregation was observed. These results were confirmed using fluorescence microscopy of Langmuir-lipid films composed of POPC and LBPA up to X(LBPA)=0.50 with no evidence for lateral phase separation. As late endosomes are eminently acidic, we examined the effect of lowering pH on lateral organization of mixed PC/LBPA bilayers by DSC and fluorescence spectroscopy. Even at pH 3.0, we find no evidence of LBPA-induced microdomain formation at LBPA contents found in cellular organelles.

Calorimetry, Differential Scanning↗