117 results on '"Heerklotz, H."'
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2. Pressure Perturbation Calorimetry
- Author
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Heerklotz, H., Winter, R., Royer, C., Seelig, J., and Winter, Roland, editor
- Published
- 2003
- Full Text
- View/download PDF
3. 5.5 Detergent Interactions with Lipid Bilayers and Membrane Proteins
- Author
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Heerklotz, H., primary and Blume, A., additional
- Published
- 2012
- Full Text
- View/download PDF
4. Surface area per molecule in lipid/C12E n membranes as seen by fluorescence resonance energy transfer
- Author
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Lantzsch, G., Binder, H., and Heerklotz, H.
- Published
- 1994
- Full Text
- View/download PDF
5. SANS study of mixed POPC/C 12E n aggregates
- Author
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Gutberlet, T, Kiselev, M, Heerklotz, H, and Klose, G
- Published
- 2000
- Full Text
- View/download PDF
6. Design and characterization of a multifunctional pH-triggered peptide C8 for selective anticancer activity
- Author
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Lu, S., Bennett, W.F.D., Ding, Y., Zhang, L., Fan, H.Y., Zhao, D., Zheng, T., Ouyang, P.K., Li, J., Wu, Y., Xu, W., Chu, D., Yuan, Y., Heerklotz, H., Karttunen, M., Chen, P., Lu, S., Bennett, W.F.D., Ding, Y., Zhang, L., Fan, H.Y., Zhao, D., Zheng, T., Ouyang, P.K., Li, J., Wu, Y., Xu, W., Chu, D., Yuan, Y., Heerklotz, H., Karttunen, M., and Chen, P.
- Abstract
Most drug delivery systems have been developed for efficient delivery to tumor sites via targeting and on-demand strategies, but the carriers rarely execute synergistic therapeutic actions. In this work, C8, a cationic, pH-triggered anticancer peptide, is developed by incorporating histidine-mediated pH-sensitivity, amphipathic helix, and amino acid pairing self-assembly design. We designed C8 to function as a pH-responsive nanostructure whose cytotoxicity can be switched on and off by its self-assembly: Noncytotoxic β-sheet fibers at high pH with neutral histidines, and positively charged monomers with membrane lytic activity at low pH. The selective activity of C8, tested for three different cancer cell lines and two noncancerous cell lines, is shown. Based on liposome leakage assays and multiscale computer simulations, its physical mechanisms of pore-forming action and selectivity are proposed, which originate from differences in the lipid composition of the cellular membrane and changes in hydrogen bonding. C8 is then investigated for its potential as a drug carrier. C8 forms a nanocomplex with ellipticine, a nonselective model anticancer drug. It selectively targets cancer cells in a pH-responsive manner, demonstrating enhanced efficacy and selectivity. This study provides a novel powerful strategy for the design and development of multifunctional self-assembling peptides for therapeutic and drug delivery applications.
- Published
- 2015
7. Coupled equilibria of a self-associating drug loaded into polymeric nanoparticles
- Author
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Fan, H.Y., primary, Raval, G., additional, Shalviri, A., additional, May, S., additional, Wu, X.Y., additional, and Heerklotz, H., additional
- Published
- 2015
- Full Text
- View/download PDF
8. Extension of ThermoML: The IUPAC standard for thermodynamic data communications (IUPAC Recommendations 2011)
- Author
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Frenkel, M., Chirico, R.D., Diky, V., Brown, P.L., Dymond, J.H., Goldberg, R.N., Goodwin, A.R.H., Heerklotz, H., Königsberger, E., Ladbury, J.E., Marsh, K.N., Remeta, D.P., Stein, S.E., Wakeham, W.A., Williams, P.A., Frenkel, M., Chirico, R.D., Diky, V., Brown, P.L., Dymond, J.H., Goldberg, R.N., Goodwin, A.R.H., Heerklotz, H., Königsberger, E., Ladbury, J.E., Marsh, K.N., Remeta, D.P., Stein, S.E., Wakeham, W.A., and Williams, P.A.
- Abstract
ThermoML is an XML-based approach for storage and exchange of experimental, predicted, and critically evaluated thermophysical and thermochemical property data. Extensions to the ThermoML schema for the representation of speciation, complex equilibria, and properties of biomaterials are described. The texts of 14 data files illustrating the new extensions are provided as Supplementary Information together with the complete text of the updated ThermoML schema.
- Published
- 2011
9. ThermoML: an XML-based approach for storage and exchange of experimental and critically evaluated thermophysical and thermochemical property data. 5. Speciation and complex equilibria
- Author
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Frenkel, M., Diky, V., Chirico, R.D., Goldberg, R.N., Heerklotz, H., Ladbury, J.E., Remeta, D.P., Dymond, J.H., Goodwin, A.R.H., Marsh, K.N., Wakeham, W.A., Stein, S.E., Brown, P.L., Königsberger, E., Williams, P.A., Frenkel, M., Diky, V., Chirico, R.D., Goldberg, R.N., Heerklotz, H., Ladbury, J.E., Remeta, D.P., Dymond, J.H., Goodwin, A.R.H., Marsh, K.N., Wakeham, W.A., Stein, S.E., Brown, P.L., Königsberger, E., and Williams, P.A.
- Abstract
ThermoML is an XML-based approach for storage and exchange of measured and critically evaluated thermophysical and thermochemical property data. Extensions to the ThermoML schema for the representation of speciation and complex equilibria are described. The text of ThermoML (xml) is provided to illustrate application of the new extensions. These are provided as Supporting Information together with the complete updated ThermoML schema.
- Published
- 2011
10. Surface areas and packing constraints in POPC C (12)EO (n) membranes. A time-resolved fluorescence study
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Lantzch, G., Binder, H., Heerklotz, H., Wendling, M., Klose, G., and Other departments
- Abstract
The surface area occupied by nonionic detergents of the type C(12)EO(n) (n = 1-8) in POPC C (12)EO (n) mixed membranes was studied by means of time-resolved resonance energy transfer (RET) between the fluorescent probe molecules NBD-PE and rhodamine-PE. The area data were interpreted within the frame of Israelachvili's concept of packing constraints yielding the critical packing parameter, f, as a measure of the asymmetry of the molecular shape of the membrane constituents. The asymmetry of the molecular shape of the detergent increases with the ethylene oxide chain length and correlates with the potency of the detergent to solubilize the bilayers and the reduction of the DPH order parameter. For n = 1-3, the membrane surface was found to expand by 0.25-0.30 nm(2) per incorporated C(12)EO(n) molecule. This value corresponds to the cross section of one hydrocarbon chain in liquid-crystalline phases. On increasing n from n = 4 to n = 8 the net area per detergent molecule increases from 0.43 nm(2) to 1.16 nm(2). These surface requirements are consistent with a disordered, coiled conformation of the EO-chains hydrated with up to two water molecules per ethylene oxide unit. For n > 5 the limiting mole fraction of the bilayer saturation was deduced from the f-data in the two-component bilayer. DPH and NBD-PE fluorescence lifetime data are discussed to give an indication of the accessibility of the probe environment to water molecules
- Published
- 1996
11. Triton Promotes Domain Formation in Lipid Raft Mixtures
- Author
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Heerklotz, H., primary
- Published
- 2002
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12. Titration calorimetry of surfactant–membrane partitioning and membrane solubilization
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Heerklotz, H, primary and Seelig, J, additional
- Published
- 2000
- Full Text
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13. Correlation of Membrane/Water Partition Coefficients of Detergents with the Critical Micelle Concentration
- Author
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Heerklotz, H., primary and Seelig, J., additional
- Published
- 2000
- Full Text
- View/download PDF
14. SANS study of mixed POPC/C12En aggregates
- Author
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Gutberlet, T, primary, Kiselev, M, additional, Heerklotz, H, additional, and Klose, G, additional
- Published
- 2000
- Full Text
- View/download PDF
15. Lipid/Detergent Interaction Thermodynamics as a Function of Molecular Shape
- Author
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Heerklotz, H., primary, Binder, H., additional, Lantzsch, G., additional, Klose, G., additional, and Blume, A., additional
- Published
- 1997
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16. Thermodynamic Characterization of Dilute Aqueous Lipid/Detergent Mixtures of POPC and C12EO8 by Means of Isothermal Titration Calorimetry
- Author
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Heerklotz, H., primary, Lantzsch, G., additional, Binder, H., additional, Klose, G., additional, and Blume, A., additional
- Published
- 1996
- Full Text
- View/download PDF
17. Surface areas and packing constraints in membranes. A time-resolved fluorescence study
- Author
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Lantzch, G., primary, Binder, H., additional, Heerklotz, H., additional, Wendling, M., additional, and Klose, G., additional
- Published
- 1996
- Full Text
- View/download PDF
18. Application of isothermal titration calorimetry for detecting lipid membrane solubilization
- Author
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Heerklotz, H., primary, Lantzsch, G., additional, Binder, H., additional, Klose, G., additional, and Blume, A., additional
- Published
- 1995
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19. Mechanistic studies on percutaneous penetration enhancement by N-(4-halobenzoyl)-S,S-dimethyliminosulfuranes
- Author
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Barrow, D. J., Chandrasekaran, S., Heerklotz, H. H., Henary, M. M., Michniak, B. B., Nguyen, P. M., Song, Y., Smith, J. C., and Strekowski, L.
- Abstract
Halogen-substituted iminosulfuranes are transdermal penetration enhancers (TPEs) in permeation studies using hairless mouse or human cadaver skin. The interaction of N-(4-R-benzoyl)-S,S-dimethyliminosulfuranes 1-4, where R = H, Cl, Br, and I, with L-α-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) has been studied using differential scanning calorimetry, isothermal titration calorimetry, nuclear Overhauser effect spectroscopy (NOESY), and NMR spectroscopy, and by calculation of the iminosulfurane polarizabilities in order to elucidate the molecular basis of the TPE activity. The active compounds reduce the melting temperature of the gel-to-liquid-crystal phase transition and induce multiple components in the transition excess heat capacity profile. The partitioning of the bromo derivative 3, the most active compound, into DMPC is unique in that 3 may be trapped in the bilayer, affording an enhanced residence time and a reason for its high TPE activity. The entropy decrease associated with the transfer of 3 to the bilayer is much lower than that for the other compounds, indicating that 3 occupies or induces sites that afford it considerable local motional freedom. Correlations between the iminosulfurane TPE activities, the partition coefficients, and NOESY crosspeak volume were observed. Molecular polarizabilities are not consistent with a TPE mode of action involving interaction of these agents with protein side chains.
- Published
- 2005
20. Aggregation Behavior of the Antibiotic Moenomycin A in Aqueous Solution
- Author
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Lantzsch, G., Binder, H., Heerklotz, H., Welzel, P., and Klose, G.
- Abstract
The aggregation behavior of the antibiotic moenomycin A (MoA) in aqueous solution under the influence of pH and ionic strength has been investigated using the fluorescent probe pyrene. Changes of the cmc between 0.03 and 0.7 mM correlate with the effective charge of MoA originating from three ionizable groups, the pK
a of which have been determined by infrared spectroscopy. The dependence of the polarity-sensitive ratio of pyrene monomer fluorescence (I3 /I1 ) and the monomer-to-excimer ratio (Iex /Imo ) on the MoA concentration were analyzed in terms of a polydispersity model. This takes into account statistical and thermodynamic aspects of detergent aggregation, electrostatic interactions between charged groups in spherical micelles, fluorescence emission properties of the probe, and their partition between water and micelles. The mean aggregation number calculated was found to be compatible with the value obtained independently by means of time-resolved fluorescence measurements. The micellization of the nonionic detergent C12 E8 in water has been investigated in parallel as a reference. The fluorescent probe DPH has been found to be unsuitable to detect the cmc of MoA.- Published
- 1998
21. Surface areas and packing constraints in POPC/C~1~2EO~n membranes. A time-resolved fluorescence study
- Author
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Lantzsch, G., Binder, H., Heerklotz, H., Wendling, M., and Klose, G.
- Published
- 1996
- Full Text
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22. Surface area per molecule in lipid/C12En membranes as seen by fluorescence resonance energy transfer
- Author
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Lantzsch, G., Binder, H., and Heerklotz, H.
- Abstract
Fluorescence resonance energy transfer (FRET) from NBD-PE to rhodamine-PE has been used to measure the average area occupied by surfactant molecules in lipid bilayers. The Foerster radius has been determined from the spectral overlap of donor fluorescence and acceptor absorption (R
0 =4.6 nm). The results of steady-state as well as of time-resolved investigations have been compared. The analysis of time-resolved fluorescence data by means of nonexponential decay functions yields an average area per lipid of 0.65 nm2 in pure POPC bilayers. The area per surfactant in two-component C12 En /POPC-membranes (n=2, 4, 6) has been determined and compared with the results of X-ray investigations. The surfactant head group seems to adapt a predominantly disordered confirmation within the bilayer.- Published
- 1994
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23. Excess Enthalpies of Mixing in Phospholipid-Additive Membranes
- Author
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Heerklotz, H. H., Binder, H., and Schmiedel, H.
- Abstract
Isothermal titration calorimetry (ITC) allows the measurement of composition-dependent mixing heats of amphiphiles. A number of experimental protocols are now established to measure molecular transfer heats between, for example, micellar and lamellar aggregates. This study deals with the principle understanding of the physical effects contributing to the ITC data. The physical state of the mixture is described in terms of the molar excess enthalpy as a function of its composition h
E (X). A relation is derived between this system property and the observable heat per mole of titrant (qobs ) as qobs = (Xsyr − X)(∂hE /∂X) + hE (X) − hE (Xsyr ) with X and Xsyr being the mole fractions of one chosen component within the mixed aggregates in the sample cell and in the injection syringe, respectively. According to this differential equation, one may derive information about the second and further derivatives (i.e., the curvature) of the excess enthalpy function. This can serve to construct the hE (X) plot based on the ITC data. We emphasize that for aggregates mixing nonideally (which must be considered rather the rule than the exception) one has to carefully distinguish between observed mixing heats and enthalpic state of the mixture. The formalism is presented at the example of mixtures of the phospholipid POPC and detergents of the type C12 EOn with n = 3−6. For instance, the system C 12 EO3 /POPC was found to show an extremely asymmetric mixing enthalpy function with an attractive part (i.e., hE < 0) for low and a repulsive one for higher detergent contents in the mixed membranes. Such excess enthalpy functions could be modeled by a polynomial equation and discussed in terms of cooperative interactions between the molecules.- Published
- 1998
24. Statistics of individual leakage events to consistently interpret vesicle leakage
- Author
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Braun, S., Pokorna, S., Sachl, R., Martin Hof, Heerklotz, H., and Hoernke, M.
25. Time-resolved fluorescence of tryptophan characterizes membrane perturbation by cyclic lipopeptides.
- Author
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Carabadjac I, Steigenberger J, Geudens N, De Roo V, Muangkaew P, Madder A, Martins JC, and Heerklotz H
- Subjects
- Peptides, Cyclic chemistry, Peptides, Cyclic metabolism, Phosphatidylcholines chemistry, Lipopeptides chemistry, Lipopeptides metabolism, Cell Membrane metabolism, Cell Membrane chemistry, Time Factors, Liposomes chemistry, Liposomes metabolism, Tryptophan chemistry, Molecular Dynamics Simulation, Spectrometry, Fluorescence
- Abstract
Viscosin is a membrane-permeabilizing, cyclic lipopeptide (CLiP) produced by Pseudomonas species. Here, we have studied four synthetic analogs (L1W, V4W, L5W, and L7W), each with one leucine (Leu; L) or valine residue exchanged for tryptophan (Trp; W) by means of time-resolved fluorescence spectroscopy of Trp. To this end, we recorded the average fluorescence lifetime, rotational correlation time and limiting anisotropy, dipolar relaxation time and limiting extent of relaxation, rate constant of acrylamide quenching, effect of H
2 O-D2 O exchange, and time-resolved half-width of the spectrum in the absence and presence of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes. Structure, localization, and hydration of the peptides were described by molecular dynamics simulations. The combination of the parameters provides a good description of the molecular environments of the Trp positions and the behavior of viscosin as a whole. Of particular value for characterizing the impact of viscosin on the membrane is the dipolar relaxation of Trp4 in V4W, which is deeply embedded in the hydrophobic core. The limiting relaxation level represents the membrane perturbation-unlike typical membrane probes-at the site of the perturbant. Fractions of Trp4 relax at different rates; the one not in contact with water upon excitation relaxes via recruitment of a water molecule on the 10-ns timescale. This rate is sensitive to the concerted membrane perturbation by more than one lipopeptide, which appears at high lipopeptide concentration and is assumed a prerequisite for the final formation of a membrane-permeabilizing defect. Trp7 relaxes primarily with respect to neighboring Ser residues. Trp5 flips between a membrane-inserted and surface-exposed orientation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)- Published
- 2024
- Full Text
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26. After the gold rush: Getting far from the shallow in studying asymmetric membranes.
- Author
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Pabst G and Heerklotz H
- Subjects
- Cell Membrane metabolism, Cell Membrane chemistry
- Abstract
Competing Interests: Declaration of interests The authors declare no competing interests.
- Published
- 2024
- Full Text
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27. Transfer of ANS-Like Drugs from Micellar Drug Delivery Systems to Albumin Is Highly Favorable and Protected from Competition with Surfactant by "Reserved" Binding Sites.
- Author
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Carabadjac I, Vormittag LC, Muszer T, Wuth J, Ulbrich MH, and Heerklotz H
- Subjects
- Humans, Binding Sites, Spectrometry, Fluorescence, Anilino Naphthalenesulfonates chemistry, Protein Binding, Micelles, Drug Delivery Systems methods, Serum Albumin, Human chemistry, Serum Albumin, Human metabolism, Surface-Active Agents chemistry
- Abstract
Micellar drug delivery systems (MDDS) for the intravenous administration of poorly soluble drugs have great advantages over alternative formulations in terms of the safety of their excipients, storage stability, and straightforward production. A classic example is mixed micelles of glycocholate (GC) and lecithin, both endogenous substances in human blood. What limits the use of MDDS is the complexity of the transitions after injection. In particular, as the MDDS disintegrate partially or completely after injection, the drug has to be transferred safely to endogenous carriers in the blood, such as human serum albumin (HSA). If this transfer is compromised, the drug might precipitate─a process that needs to be excluded under all circumstances. The key question of this paper is whether the high local concentration of GC at the moment and site of MDDS dissolution might transiently saturate HSA binding sites and, hence, endanger quick drug transfer. To address this question, we have used a new approach, which is time-resolved fluorescence spectroscopy of the single tryptophan in HSA, Trp-214, to characterize the competitive binding of GC and the drug substitute anilinonaphthalenesulfonate (ANS) to HSA. Time-resolved fluorescence of Trp-214 showed important advantages over established methods for tackling this problem. ANS has been the standard "model drug" to study albumin binding for decades, given its structural similarity to the class of naphthalene-containing acidic drugs and the fact that it is displaced from HSA by numerous drugs (which presumably bind to the same sites). Our complex global fit uses the critical approximation that the average lifetimes behave similarly to a single lifetime, but the resulting errors are found to be moderate and the results provide a convincing explanation of the, at first glance, counterintuitive behavior. Accordingly, and largely in line with the literature, we observed two types of sites binding ANS at HSA: 3 type A, rather peripheral, and 2 type B, likely more central sites. The latter quench Trp-214 by Förster Resonance Energy Transfer (FRET) with a rate constant of ≈0.4 ns
-1 per ANS. Adding millimolar concentrations of GC displaces ANS from the A sites but not from B sites. At incomplete ANS saturation, this causes a GC-induced translocation of ANS from A to the more FRET-active B sites. This leads to the apparent paradox that the partial displacement of ANS from HSA increases its quenching effect on Trp-214. The most important conclusion is that (ANS-like) drugs cannot be displaced from the type-B sites, and consequently, drug transfer to these sites is not impaired by competitive binding of GC in the vicinity of a dissolving micelle. The second conclusion is that for unbound GC above the CMC (9 mM), ANS equilibrates between HSA and GC micelles but with a strong preference for free sites on HSA. That means that even persisting micelles would lose their cargo readily once exposed to HSA. For all MDDS sharing this property, targeted drug delivery approaches involving them as the nanocarrier would be pointless.- Published
- 2024
- Full Text
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28. Eutectic Resolves Lysolipid Paradox in Thermoresponsive Liposomes.
- Author
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Eckhardt D, Semeraro EF, Steigenberger J, Schnur J, Kalie L, Massing U, Pabst G, and Heerklotz H
- Subjects
- Scattering, Small Angle, Calorimetry, Differential Scanning, X-Ray Diffraction, 1,2-Dipalmitoylphosphatidylcholine chemistry, Liposomes chemistry, Lipid Bilayers chemistry
- Abstract
A better molecular understanding of the temperature-triggered drug release from lysolipid-based thermosensitive liposomes (LTSLs) is needed to overcome the recent setbacks in developing this important drug delivery system. Enhanced drug release was previously rationalized in terms of detergent-like effects of the lysolipid monostearyl lysophosphatidylcholine (MSPC), stabilizing local membrane defects upon LTSL lipid melting. This is highly surprising and here referred to as the 'lysolipid paradox,' because detergents usually induce the opposite effect─they cause leakage upon freezing, not melting. Here, we aim at better answers to (i) why lysolipid does not compromise drug retention upon storage of LTSLs in the gel phase, (ii) how lysolipids can enhance drug release from LTSLs upon lipid melting, and (iii) why LTSLs typically anneal after some time so that not all drug gets released. To this end, we studied the phase transitions of mixtures of dipalmitoylphosphatidylcholine (DPPC) and MSPC by a combination of differential scanning and pressure perturbation calorimetry and identified the phase structures with small- and wide-angle X-ray scattering (SAXS and WAXS). The key result is that LTSLs, which contain the standard amount of 10 mol % MSPC, are at a eutectic point when they release their cargo upon melting at about 41 °C. The eutectic present below 41 °C consists of a MSPC-depleted gel phase as well as small domains of a hydrocarbon chain interdigitated gel phase containing some 30 mol % MSPC. In these interdigitated domains, the lysolipid is stored safely without compromising membrane integrity. At the eutectic temperature, both the MSPC-depleted bilayer and interdigitated MSPC-rich domains melt at once to fluid bilayers, respectively. Intact, fluid membranes tolerate much less MSPC than interdigitated domains─where the latter have melted, the high local MSPC content causes transient pores. These pores allow for fast drug release. However, these pores disappear, and the membrane seals again as the MSPC distributes more evenly over the membrane so that its local concentration decreases below the pore-stabilizing threshold. We provide a pseudobinary phase diagram of the DPPC-MSPC system and structural and volumetric data for the interdigitated phase.
- Published
- 2024
- Full Text
- View/download PDF
29. Vesicle budding caused by lysolipid-induced asymmetry stress.
- Author
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Hua L, Kaiser M, Carabadjac I, Meister A, Hause G, and Heerklotz H
- Subjects
- Membrane Lipids, Fluorescence Polarization, Phosphatidylcholines chemistry, Lipid Bilayers chemistry, Liposomes, Unilamellar Liposomes chemistry
- Abstract
Lysolipids such as lauroyl, myristoyl, and palmitoyl lysophosphatidylcholine (LPC) insert into the outer leaflet of liposomes but do not flip to the inner leaflet over many hours. This way, they create asymmetry stress between the intrinsic areas of the two leaflets. We have studied how this stress is relaxed with particular emphasis on the budding and fission of small (diameter 20-30 nm) daughter vesicles (DVs). Asymmetric flow field-flow fractionation was utilized to quantify the extent of budding from large unilamellar vesicles after exposure to LPC. Budding starts at a low threshold of the order of 2 mol% LPC in the outer (and ≈0 mol% LPC in the inner) leaflet. We see reason to assume that the fractional fluorescence intensity from DVs is a good approximation for the fraction of membrane lipid, POPC, transferred into DVs. Accordingly, budding starts with a "budding power" of ≈6 POPC molecules budding off per LPC added, corresponding to a more than 10-fold accumulation of LPC in the outer leaflet of DVs to ≈24 mol%. As long as budding is possible, little strain is built up in the membranes, a claim supported by the lack of changes in limiting fluorescence anisotropy, rotational correlation time, and fluorescence lifetime of symmetrically and asymmetrically inserted TMA-DPH. At physiological osmolarity, budding is typically limited to 20-30% of budded fraction with some batch-to-batch variation, but independent of the LPC species. We hypothesize that the budding limit is determined by the excess area of the liposomes upon preparation, which is then used up upon budding given the larger area-to-volume ratio of smaller liposomes. As the mother vesicles approach ideal spheres, budding must stop. This is qualitatively supported by increased and decreased budding limits of osmotically predeflated and preinflated vesicles, respectively., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
30. Complex electrostatic effects on the selectivity of membrane-permeabilizing cyclic lipopeptides.
- Author
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Steigenberger J, Verleysen Y, Geudens N, Madder A, Martins JC, and Heerklotz H
- Subjects
- Static Electricity, Liposomes, Phosphatidylglycerols chemistry, Phosphatidylglycerols metabolism, Phosphatidylethanolamines, Peptides, Cyclic chemistry, Peptides, Cyclic metabolism, Cell Membrane Permeability
- Abstract
Cyclic lipopeptides (CLiPs) have many biological functions, including the selective permeabilization of target membranes, and technical and medical applications. We studied the anionic CLiP viscosin from Pseudomonas along with a neutral analog, pseudodesmin A, and the cationic viscosin-E2K to better understand electrostatic effects on target selectivity. Calcein leakage from liposomes of anionic phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) is measured in comparison with net-neutral phosphatidylcholine by time-resolved fluorescence. By contrast to the typical selectivity of cationic peptides against anionic membranes, we find viscosin more active against PG/PE at 30 μM lipid than viscosin-E2K. At very low lipid concentration, the selectivity is reversed. An equi-activity analysis reveals the reciprocal partition coefficients, 1/K, and the CLiP-to-lipid mole ratio within the membrane as leakage after 1 h reaches 50%, R
e 50 . As expected, 1/K to PG/PE is much lower (higher affinity) for viscosin-E2K (3 μM) than viscosin (15 μM). However, the local damage to the PG/PE membrane caused by a viscosin molecule is much stronger than that of viscosin-E2K. This can be explained by the strong membrane expansion due to PG/viscosin repulsion inducing asymmetry stress between the two leaflets and, ultimately, transient limited leakage at Re 50 = 0.08. PG/viscosin-E2K attraction opposes expansion and leakage starts only as the PG charges in the outer leaflet are essentially compensated by the cationic peptide (Re 50 = 0.32). In the high-lipid regime (at lipid concentrations cL ≫ 1/K), virtually all CLiP is membrane bound anyway and Re 50 governs selectivity, favoring viscosin. In the low-lipid regime at cL ≪ 1/K, virtually all CLiP is in solution, 1/K becomes important and the "cation attacks anionic membrane" selectivity gets restored. Overall, activity and selectivity data can only properly be interpreted if the lipid regime is known and predictions for other lipid concentrations or cell counts require knowledge of 1/K and Re 50 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)- Published
- 2023
- Full Text
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31. A Guide to Your Desired Lipid-Asymmetric Vesicles.
- Author
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Krompers M and Heerklotz H
- Abstract
Liposomes are prevalent model systems for studies on biological membranes. Recently, increasing attention has been paid to models also representing the lipid asymmetry of biological membranes. Here, we review in-vitro methods that have been established to prepare free-floating vesicles containing different compositions of the classic two-chain glycero- or sphingolipids in their outer and inner leaflet. In total, 72 reports are listed and assigned to four general strategies that are (A) enzymatic conversion of outer leaflet lipids, (B) re-sorting of lipids between leaflets, (C) assembly from different monolayers and (D) exchange of outer leaflet lipids. To guide the reader through this broad field of available techniques, we attempt to draw a road map that leads to the lipid-asymmetric vesicles that suit a given purpose. Of each method, we discuss advantages and limitations. In addition, various verification strategies of asymmetry as well as the role of cholesterol are briefly discussed. The ability to specifically induce lipid asymmetry in model membranes offers insights into the biological functions of asymmetry and may also benefit the technical applications of liposomes.
- Published
- 2023
- Full Text
- View/download PDF
32. Tailoring the Lamellarity of Liposomes Prepared by Dual Centrifugation.
- Author
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Koehler JK, Gedda L, Wurster L, Schnur J, Edwards K, Heerklotz H, and Massing U
- Abstract
Dual centrifugation (DC) is a new and versatile technique for the preparation of liposomes by in-vial homogenization of lipid-water mixtures. Size, size distribution, and entrapping efficiencies are strongly dependent on the lipid concentration during DC-homogenization. In this study, we investigated the detailed structure of DC-made liposomes. To do so, an assay to determine the ratio of inner to total membrane surfaces of liposomes (inaccessible surface) was developed based on either time-resolved or steady-state fluorescence spectroscopy. In addition, cryogenic electron microscopy (cryo-EM) was used to confirm the lamellarity results and learn more about liposome morphology. One striking result leads to the possibility of producing a novel type of liposome-small multilamellar vesicles (SMVs) with low PDI, sizes of the order of 100 nm, and almost completely filled with bilayers. A second particularly important finding is that VPGs can be prepared to contain open bilayer structures that will close spontaneously when, after storage, more aqueous phase is added and liposomes are formed. Through this process, a drug can effectively be entrapped immediately before application. In addition, dual centrifugation at lower lipid concentrations is found to produce predominantly unilamellar vesicles.
- Published
- 2023
- Full Text
- View/download PDF
33. The effect of membrane thickness on the membrane permeabilizing activity of the cyclic lipopeptide tolaasin II.
- Author
-
Steigenberger J, Mergen C, De Roo V, Geudens N, Martins JC, and Heerklotz H
- Abstract
Tolaasin II is an amphiphilic, membrane-active, cyclic lipopeptide produced by Pseudomonas tolaasii and is responsible for brown blotch disease in mushroom. To better understand the mode of action and membrane selectivity of tolaasin II and related lipopeptides, its permeabilizing effect on liposomes of different membrane thickness was characterized. An equi-activity analysis served to distinguish between the effects of membrane partitioning and the intrinsic activity of the membrane-bound peptide. It was found that thicker membranes require higher local peptide concentrations to become leaky. More specifically, the mole ratio of membrane-bound peptide per lipid needed to induce 50% leakage of calcein within 1 h, R
e 50 , increased monotonically with membrane thickness from 0.0016 for the 14:1 to 0.0070 for the 20:1 lipid-chains. Moreover, fast but limited leakage kinetics in the low-lipid regime were observed implying a mode of action based on membrane asymmetry stress in this time and concentration window. While the assembly of the peptide to oligomeric pores of defined length along the bilayer z -axis can in principle explain inhibition by increasing membrane thickness, it cannot account for the observed limited leakage. Therefore, reduced intrinsic membrane-permeabilizing activity with increasing membrane thickness is attributed here to the increased mechanical strength and order of thicker membranes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Steigenberger, Mergen, De Roo, Geudens, Martins and Heerklotz.)- Published
- 2022
- Full Text
- View/download PDF
34. Extending the Pseudo-Phase Model of Detergent-Lipid Dispersions by a Detergent-Binding Protein.
- Author
-
Vormittag LC and Heerklotz H
- Subjects
- Micelles, Temperature, Thermodynamics, Lipid Bilayers chemistry, Detergents chemistry
- Abstract
Mixed micellar drug delivery systems for poorly soluble active pharmaceutical ingredients (APIs) are easy to produce and long-term stable, because they represent equilibrium structures. However, their fate after intravenous injection is still largely unknown. Once injected into the bloodstream, they can potentially convert to vesicles or disappear altogether, with both API and excipients being picked up by blood components. Our study aimed at reducing the gap between the good, quantitative understanding of aqueous glycocholate (GC)-lecithin dispersions alone and the highly complex situation in the blood. To this end, we extended the pseudophase model previously established for lipid-detergent dispersions to include the detergent-binding protein albumin as another component. The model predicted a quaternary phase diagram with planar phase boundaries defined by key parameters of the ternary subsystems, which were then determined by isothermal titration calorimetry. They include the aqueous GC concentration upon bilayer-micelle coexistence, 5.2 mM, the GC-to-lipid mole ratios in coexisting bilayers ( R
e sat = 0.2) and micelles ( Re sol = 0.7), as well as the capacity of the albumin to bind 0.1 GC molecules with a dissociation constant of KD = 0.1 mM and 6 GC molecules with KD = 0.7 mM. Subsequent measurements in the quaternary system showed phase boundaries in good agreement with the model predictions. In addition, the critical micelle concentration of GC shows a minimal value (midpoint of transition) of 9.1 mM at the temperature of 24 °C where the demicellization enthalpy is zero. The demicellization process is accompanied by a heat capacity change of 29 cal/mol K. The model improves the understanding of the mixed micellar drug delivery systems. The success of the approach encourages including even more blood components, like lipoproteins, to a quantitative treatment.- Published
- 2022
- Full Text
- View/download PDF
35. Designed membrane protein heterodimers and control of their affinity by binding domain and membrane linker properties.
- Author
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Lan C, Stulz A, Barthes NPF, Lauw S, Salavei P, Jung M, Heerklotz H, and Ulbrich MH
- Subjects
- Cell Membrane, Dimerization, Membranes, Membrane Proteins
- Abstract
Many membrane proteins utilize dimerization to transmit signals across the cell membrane via regulation of the lateral binding affinity. The complexity of natural membrane proteins hampers the understanding of this regulation on a biophysical level. We designed simplified membrane proteins from well-defined soluble dimerization domains with tunable affinities, flexible linkers, and an inert membrane anchor. Live-cell single-molecule imaging demonstrates that their dimerization affinity indeed depends on the strength of their binding domains. We confirm that as predicted, the 2-dimensional affinity increases with the 3-dimensional binding affinity of the binding domains and decreases with linker lengths. Models of extended and coiled linkers delineate an expected range of 2-dimensional affinities, and our observations for proteins with medium binding strength agree well with the models. Our work helps in understanding the function of membrane proteins and has important implications for the design of synthetic receptors.
- Published
- 2021
- Full Text
- View/download PDF
36. Screening for Optimal Liposome Preparation Conditions by Using Dual Centrifugation and Time-Resolved Fluorescence Measurements.
- Author
-
Koehler JK, Schnur J, Heerklotz H, and Massing U
- Abstract
Dual centrifugation (DC) is a novel in-vial homogenization technique for the preparation of liposomes in small batch sizes under gentle and sterile conditions which allows encapsulation efficiencies ( EE ) for water soluble compounds of >50%. Since liposome size, size distribution (PDI), and EE depend on the lipid concentration used in the DC process, a screening method to find optimal lipid concentrations for a defined lipid composition was developed. Four lipid mixtures consisting of cholesterol, hydrogenated or non-hydrogenated egg PC, and/or PEG-DSPE were screened and suitable concentration ranges could be identified for optimal DC homogenization. In addition to the very fast and parallel liposome preparation of up to 40 samples, the screening process was further accelerated by the finding that DC generates homogeneously mixed liposomes from a macroscopic lipid mixture without the need to initially prepare a molecularly mixed lipid film from an organic solution of all components. This much simpler procedure even works for cholesterol containing lipid blends, which could be explained by a nano-milling of the cholesterol crystals during DC homogenization. Furthermore, EE determination was performed by time-resolved fluorescence measurements of calcein-loaded liposomes without removing the non-entrapped calcein. The new strategy allows the rapid characterization of a certain lipid composition for the preparation of liposomes within a working day.
- Published
- 2021
- Full Text
- View/download PDF
37. Membrane-water partitioning - Tackling the challenges of poorly soluble drugs using chaotropic co-solvents.
- Author
-
Naßwetter LC, Fischer M, Scheidt HA, and Heerklotz H
- Subjects
- Lapatinib chemistry, Protein Kinase Inhibitors chemistry, Protein Kinase Inhibitors pharmacology, Calorimetry, Solubility, Water chemistry, Solvents chemistry, Dimethyl Sulfoxide chemistry
- Abstract
Many newly developed drugs suffer from poor water solubility and low bioavailability and hence, need special formulation vehicles like vesicular or micellar drug delivery systems. The knowledge of their membrane-water partition coefficient K becomes critical as is governs drug loading and release from the vehicle, as well as absorption into the body. The dilemma is that measuring K is particularly challenging for these very compounds. Here we establish a strategy to resolve this problem. We added DMSO to shift K and solubility into a convenient range and extrapolated these results back to zero-DMSO. Isothermal titration calorimetry revealed that logK of the kinase inhibitor Lapatinib decreased proportionally to DMSO content (2.5 - 20v%) with a slope of -1/20v% (m value = 28 kJ/mol). This implies a K of 84 mM
-1 in DMSO-free buffer. This strategy should be transferable to other poorly soluble drugs and further detection methods., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2021
- Full Text
- View/download PDF
38. The Optimal Lipid Chain Length of a Membrane-Permeabilizing Lipopeptide Results From the Balance of Membrane Partitioning and Local Damage.
- Author
-
Steigenberger J, Verleysen Y, Geudens N, Martins JC, and Heerklotz H
- Abstract
Pseudodesmin A (PSD) is a cyclic lipodepsipeptide produced by Pseudomonas that kills certain bacteria at MIC
1/2 in the single micromolar range, probably by permeabilizing their cellular membranes. Synthetic PSD variants, where the native decanoic (C10) acyl chain is varied in length from C4 to C8 and C12 to C14 carbons, were described to be not or less active against a panel of gram-positive strains, as compared to native PSD-C10. Here, we test the membrane-permeabilizing activity of PSD-C4 through PSD-C14 in terms of calcein release from liposomes, which is characterized in detail by the fluorescence-lifetime based leakage assay. Antagonistic concentrations and their chain length dependence agree well for liposome leakage and antimicrobial activity. The optimal chain length is governed by a balance between membrane partitioning (favoring longer chains) and the local perturbation or "damage" inflicted by a membrane-bound molecule (weakening for longer chains). Local perturbation, in turn, may involve at least two modes of action. Asymmetry stress between outer and inner leaflet builds up as the lipopeptides enter the outer leaflet and when it reaches a system-specific stability threshold, it causes a transient membrane failure that allows for the flip of some molecules from the outer to the inner leaflet. This cracking-in may be accompanied by transient, incomplete leakage from the aqueous cores of the liposomes observed, typically, for some seconds or less. The mismatch of the lipopeptide with the lipid leaflet geometry, expressed for example in terms of a spontaneous curvature, has two effects. First, it affects the threshold for transient leakage as described. Second, it controls the rate of equilibrium leakage proceeding as the lipopeptide has reached sufficient local concentrations in both leaflets to form quasi-toroidal defects or pores. Both modes of action, transient and equilibrium leakage, synergize for intermediate chain lengths such as the native, i.e., for PSD-C10. These mechanisms may also account for the reported chain-length dependent specificities of antibiotic action against the target bacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Steigenberger, Verleysen, Geudens, Martins and Heerklotz.)- Published
- 2021
- Full Text
- View/download PDF
39. Complex Micellization Behavior of the Polysorbates Tween 20 and Tween 80.
- Author
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Knoch H, Ulbrich MH, Mittag JJ, Buske J, Garidel P, and Heerklotz H
- Subjects
- Calorimetry methods, Chemistry, Pharmaceutical methods, Drug Compounding methods, Drug Stability, Dynamic Light Scattering methods, Esters chemistry, Excipients chemistry, Fatty Acids chemistry, Hot Temperature, Hydrophobic and Hydrophilic Interactions, Lauric Acids chemistry, Oleic Acid chemistry, Protein Stability, Solubility, Micelles, Polysorbates chemistry, Surface-Active Agents chemistry
- Abstract
Polysorbates (PSs, Tweens) are widely used surfactant products consisting of a sorbitan ring connecting up to four ethylene oxide (EO) chains of variable lengths, one or more of which are esterified with fatty acids of variable lengths and saturation degrees. Pharmaceutical applications include the stabilization of biologicals in solutions and the solubilization of poorly water soluble, active ingredients. This study characterizes the complex association behavior of compendial PSs PS20 and PS80, which is fundamentally different from that of single-component surfactants. To this end, a series of demicellization experiments of isothermal titration calorimetry with different PS concentrations are evaluated. Their experiment-dependent heats of titration are converted into a common function of the state of a sample, the micellar enthalpy Q
m ( c ). These functions demonstrate that initial micelles are already present at the lowest concentrations investigated, 2 μM for PS20 and 10 μM for PS80. Initial micelles consist primarily of the surfactant species with the lowest individual critical micelle concentration (cmc). With increasing concentration, the other PS species gradually enter these micelles in the sequence of increasing individual cmc's and hydrophilic-lipophilic balance. Concentration ranges with pronounced slopes of Qm ( c ) can be tentatively assigned to the uptake of the major components of the PS products. Micellization and the variation of the micelle properties progress up to at least 10 mM PS. That means the published cmc values or ranges of PS20 and PS80 may be related to certain, major components being incorporated into and forming specific micelles but must not be interpreted in terms of an absence of micelles below and constant properties, e.g., the surface activity, of the micelles above these ranges. The micellization enthalpy curves differ quite substantially between PS20 and PS80 and, in a subtler fashion, between individual quality grades such as high purity, pure lauric acid/pure oleic acid, super-refined, and China grade.- Published
- 2021
- Full Text
- View/download PDF
40. Determining critical parameters that influence in vitro performance characteristics of a thermosensitive liposome formulation of vinorelbine.
- Author
-
Regenold M, Steigenberger J, Siniscalchi E, Dunne M, Casettari L, Heerklotz H, and Allen C
- Subjects
- Antibiotics, Antineoplastic, Doxorubicin, Drug Delivery Systems, Vinorelbine, Antineoplastic Agents therapeutic use, Liposomes
- Abstract
Studies have demonstrated the advantages associated with heat-triggered drug delivery via thermosensitive liposomes for the treatment of localized cancer. Challenges that traditional liposomal systems face such as limited drug release and homogeneous distribution throughout the region of interest can potentially be overcome when triggering intravascular drug release. The most prominent example is a thermosensitive liposome formulation of doxorubicin known as ThermoDox®. Many other drugs may benefit from the same targeted and localized delivery approach using thermosensitive liposomes as it can result in a significant improvement in the therapeutic index. Vinorelbine is a semi-synthetic vinca alkaloid which has shown to be active in a broad range of cancers. Several liposome formulations encapsulating vinorelbine have been developed as a means to reduce systemic drug exposure. The present study takes a systematic approach in exploring formulation and drug loading parameters and their influence on performance characteristics of a rapidly releasing thermosensitive liposome formulation of vinorelbine. More broadly, this study shows that trends observed for non-thermosensitive liposome formulations of specific drugs (i.e. vinorelbine) can not be easily translated to their thermosensitive counterparts. The profound impact of the presence of albumin on stability and in vitro release is also highlighted. This is of significance given that a number of recent reports examine drug release in the absence of biologically relevant components. As a result, a strong recommendation emanating from this is a thorough challenge of the liposome formulation in vitro in order to gain a better understanding of its likely behaviour in vivo as well as potential for future clinical translation., (Copyright © 2020 Elsevier B.V. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
41. Lipid Scrambling Induced by Membrane-Active Substances.
- Author
-
Dietel L, Kalie L, and Heerklotz H
- Subjects
- Calorimetry, Lipid Bilayers, Micelles, Phosphatidylcholines, Lipids, Liposomes
- Abstract
The functional roles of the lipid asymmetry of biomembranes are attracting increasing attention. This study characterizes the activity of surfactants to induce transmembrane flip-flop of lipids and thus "scramble" this asymmetry. Detergent-induced lipid scrambling of liposomes mimicking the charge asymmetry of bacterial membranes with 20 mol % of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol in the outer leaflet only was quantified by ζ-potential measurements for octaethylene glycol dodecyl ether (C
12 EO8 ), octyl glucoside (OG), and dodecyl maltoside. Membrane leakage was separately measured by the fluorescence lifetime-based calcein leakage assay and the onset of the membrane-to-micelle transition by isothermal titration calorimetry. Partition coefficients and partial molar areas were obtained as well. For the quickly membrane-permeant C12 EO8 and OG, leakage proceeds at a rather sharp threshold content in the membrane, which is well below the onset of solubilization and little dependent on incubation time; it is accompanied by fast lipid scrambling. However, unlike leakage, flip-flop is a relaxation process that speeds up gradually from taking weeks in the detergent-free membrane to minutes or less in the leaking membrane. Hence, after 24 h of incubation, 10 mol % of C12 EO8 or 50 mol % of OG in the membrane suffice for virtually complete lipid scrambling, whereas leakage remains below 10% for up to 14 mol % of C12 EO8 and 88 mol % of OG. There is thus a concentration window in which lipid scrambling proceeds without leakage. This implies that lipid scrambling must be considered a possible mode of action of antimicrobial peptides and other membrane-active drugs or biomolecules. A related, detergent-based protocol for scrambling the lipid asymmetry of liposomes and maybe cells without compromising their overall integrity would be a very valuable tool to study functions of lipid asymmetry., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)- Published
- 2020
- Full Text
- View/download PDF
42. Primary and Secondary Binding of Exenatide to Liposomes.
- Author
-
Stulz A, Breitsamer M, Winter G, and Heerklotz H
- Subjects
- Calorimetry, Exenatide, Peptides, Phospholipids, Liposomes, Phosphatidylglycerols
- Abstract
The interactions of exenatide, a Trp-containing peptide used as a drug to treat diabetes, with liposomes were studied by isothermal titration calorimetry (ITC), tryptophan (Trp) fluorescence, and microscale thermophoresis measurements. The results are not only important for better understanding the release of this specific drug from vesicular phospholipid gel formulations but describe a general scenario as described before for various systems. This study introduces a model to fit these data on the basis of primary and secondary peptide-lipid interactions. Finally, resolving apparent inconsistencies between different methods aids the design and critical interpretation of binding experiments in general. Our results show that the net cationic exenatide adsorbs electrostatically to liposomes containing anionic diacyl phosphatidylglycerol lipids (PG); however, the ITC data could not properly be fitted by any established model. The combination of electrostatic adsorption of exenatide to the membrane surface and its self-association (K
d = 46 μM) suggested the possibility of secondary binding of peptide to the first, primarily (i.e., lipid-) bound peptide layer. A global fit of the ITC data validated this model and suggested one peptide to bind primarily per five PG molecules with a Kd ≈ 0.2 μM for PC/PG 1:1 and 0.6 μM for PC/PG 7:3 liposomes. Secondary binding shows a weaker affinity and a less exothermic or even endothermic enthalpy change. Depending on the concentration of liposomes, secondary binding may also lead to liposomal aggregation as detected by dynamic light-scattering measurements. ITC quantifies primary and secondary binding separately, whereas microscale thermophoresis and Trp fluorescence represent a summary or average of both effects, possibly with the fluorescence data showing somewhat greater weighting of primary binding. Systems with secondary peptide-peptide association within the membrane are mathematically analogous to the adsorption discussed here., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)- Published
- 2020
- Full Text
- View/download PDF
43. Calcium affects CHP1 and CHP2 conformation and their interaction with sodium/proton exchanger 1.
- Author
-
Liang S, Fuchs S, Mymrikov EV, Stulz A, Kaiser M, Heerklotz H, and Hunte C
- Subjects
- Binding Sites, Calcium-Binding Proteins metabolism, Humans, Hydrophobic and Hydrophilic Interactions, Protein Binding, Sodium-Hydrogen Exchanger 1 chemistry, Calcium metabolism, Calcium-Binding Proteins chemistry, Sodium-Hydrogen Exchanger 1 metabolism
- Abstract
Calcineurin B homologous proteins (CHPs) belong to the EF-hand Ca
2+ -binding protein (EFCaBP) family. They have multiple important functions including the regulation of the Na+ /H+ exchanger 1 (NHE1). The human isoforms CHP1 and CHP2 share high sequence similarity, but have distinct expression profiles with CHP2 levels for instance increased in malignant cells. These CHPs bind Ca2+ with high affinity. Biochemical data indicated that Ca2+ can regulate their functions. Experimental evidence for Ca2+ -modulated structural changes was lacking. With a newly established fluorescent probe hydrophobicity (FPH) assay, we detected Ca2+ -induced conformational changes in both CHPs. These changes are in line with an opening of their hydrophobic pocket that binds the CHP-binding region (CBD) of NHE1. Whereas the pocket is closed in the absence of Ca2+ in CHP2, it is still accessible for the dye in CHP1. Both CHPs interacted with CBD in the presence and absence of Ca2+ . Isothermal titration calorimetry (ITC) analysis revealed high binding affinity for both CHPs to CBD with equilibrium dissociation constants (KD s) in the nanomolar range. The KD for CHP1:CBD was not affected by Ca2+ , whereas Ca2+ -depletion increased the KD 7-fold for CHP2:CBD showing a decreased affinity. The data indicate an isoform specific regulatory interaction of CHP1 and CHP2 with NHE1., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals, Inc. on behalf of Federation of American Societies for Experimental Biology.)- Published
- 2020
- Full Text
- View/download PDF
44. Stairway to Asymmetry: Five Steps to Lipid-Asymmetric Proteoliposomes.
- Author
-
Markones M, Fippel A, Kaiser M, Drechsler C, Hunte C, and Heerklotz H
- Subjects
- Proteolipids metabolism, Salmonella typhimurium cytology, Unilamellar Liposomes chemistry, Unilamellar Liposomes metabolism, Lipids chemistry, Proteolipids chemistry
- Abstract
Membrane proteins are embedded in a complex lipid environment that influences their structure and function. One key feature of nearly all biological membranes is a distinct lipid asymmetry. However, the influence of membrane asymmetry on proteins is poorly understood, and novel asymmetric proteoliposome systems are beneficial. To our knowledge, we present the first study on a multispanning protein incorporated in large unilamellar liposomes showing a stable lipid asymmetry. These asymmetric proteoliposomes contain the Na
+ /H+ antiporter NhaA from Salmonella Typhimurium. Asymmetry was introduced by partial, outside-only exchange of anionic phosphatidylglycerol (PG), mimicking this key asymmetry of bacterial membranes. Outer-leaflet and total fractions of PG were determined via ζ-potential (ζ) measurements after lipid exchange and after scrambling of asymmetry. ζ-Values were in good agreement with exclusive outside localization of PG. The electrogenic Na+ /H+ antiporter was active in asymmetric liposomes, and it can be concluded that reconstitution and generation of asymmetry were successful. Lipid asymmetry was stable for more than 7 days at 23°C and thus enabled characterization of the Na+ /H+ antiporter in an asymmetric lipid environment. We present and validate a simple five-step protocol that addresses key steps to be taken and pitfalls to be avoided for the preparation of asymmetric proteoliposomes: 1) optimization of desired lipid composition, 2) detergent-mediated protein reconstitution with subsequent detergent removal, 3) generation of lipid asymmetry by partial exchange of outer-leaflet lipid, 4) verification of lipid asymmetry and stability, and 5) determination of protein activity in the asymmetric lipid environment. This work offers guidance in designing asymmetric proteoliposomes that will enable researchers to compare functional and structural properties of membrane proteins in symmetric and asymmetric lipid environments., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)- Published
- 2020
- Full Text
- View/download PDF
45. Kiss and Run Asymmetric Vesicles to Investigate Coupling.
- Author
-
Heerklotz H and London E
- Subjects
- Exocytosis, Synaptic Vesicles
- Published
- 2019
- Full Text
- View/download PDF
46. Preparation of Asymmetric Liposomes Using a Phosphatidylserine Decarboxylase.
- Author
-
Drechsler C, Markones M, Choi JY, Frieling N, Fiedler S, Voelker DR, Schubert R, and Heerklotz H
- Subjects
- Cell Membrane chemistry, Liposomes metabolism, Phosphatidylethanolamines metabolism, Bacterial Proteins metabolism, Carboxy-Lyases metabolism, Cell Membrane metabolism, Lipid Bilayers metabolism, Liposomes chemistry, Phosphatidylserines metabolism, Plasmodium knowlesi enzymology
- Abstract
Lipid asymmetries between the outer and inner leaflet of the lipid bilayer exist in nearly all biological membranes. Although living cells spend great effort to adjust and maintain these asymmetries, little is known about the biophysical phenomena within asymmetric membranes and their role in cellular function. One reason for this lack of insight into such a fundamental membrane property is the fact that the majority of model-membrane studies have been performed on symmetric membranes. Our aim is to overcome this problem by employing a targeted, enzymatic reaction to prepare asymmetric liposomes with phosphatidylserine (PS) primarily in the inner leaflet. To achieve this goal, we use a recombinant version of a water soluble PS decarboxylase from Plasmodium knowlesi, which selectively decarboxylates PS in the outer leaflet, converting it to phosphatidylethanolamine. The extent of decarboxylation is quantified using high-performance thin-layer chromatography, and the local concentration of anionic PS in the outer leaflet is monitored in terms of the ζ potential. Starting, for example, with 21 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine sodium salt, the assay leads to liposomes with 21 mol % in the inner and 6 mol % PS in the outer leaflet. This asymmetry persists virtually unchanged for at least 4 days at 20°C and at least 2 days at 40°C. The use of a highly specific enzyme carries the advantage that a minor component such as PS can be adjusted without affecting or being affected by the other lipid species present in the model membrane. The phenomena governing the residual outside PS content are addressed but warrant further study., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
47. Calcineurin B homologous protein 3 binds with high affinity to the CHP binding domain of the human sodium/proton exchanger NHE1.
- Author
-
Fuchs S, Hansen SC, Markones M, Mymrikov EV, Heerklotz H, and Hunte C
- Subjects
- Binding Sites, Calcium-Binding Proteins isolation & purification, Calmodulin metabolism, Calorimetry, Chromatography, Gel, Humans, Kinetics, Protein Binding, Protein Interaction Mapping, Sodium-Hydrogen Exchanger 1 isolation & purification, Calcium-Binding Proteins metabolism, Sodium-Hydrogen Exchanger 1 metabolism
- Abstract
The Na
+ /H+ exchanger NHE1 is critical for cell vitality as it controls intracellular pH and cell volume. Its functionality is influenced by calcineurin B homologous proteins (CHPs). The human isoform CHP3 is important for transport of NHE1 to the plasma membrane and for its activity. Here, we characterized the binding interaction of human CHP3 with the regulatory domain of NHE1. The exact binding site of CHP3 was previously debated. CHP3 as well as both regions of NHE1 in question were produced and purified. CHP3 specifically formed stable complexes with the CHP-binding region (CBD) of NHE1 (residues 503-545) in size-exclusion chromatography (SEC), but not with the C-terminal region (CTD, residues 633-815). CTD was functional as shown by Ca2+ -dependent binding of calmodulin in SEC analysis. CHP3 bound with high affinity to CBD with an equilibrium dissociation constant (KD ) of 56 nM determined by microscale thermophoresis. The high affinity was substantiated by isothermal calorimetry analysis (KD = 3 nM), which also revealed that the interaction with CBD is strongly exothermic (ΔG° = -48.6 kJ/mol, ΔH = -75.3 kJ/mol, -TΔS° = 26.7 kJ/mol). The data provide insights in the molecular mechanisms that underlie the regulatory interaction of CHP3 and NHE1 and more general of calcineurin homologous proteins with their target proteins.- Published
- 2018
- Full Text
- View/download PDF
48. Engineering Asymmetric Lipid Vesicles: Accurate and Convenient Control of the Outer Leaflet Lipid Composition.
- Author
-
Markones M, Drechsler C, Kaiser M, Kalie L, Heerklotz H, and Fiedler S
- Subjects
- Models, Molecular, Molecular Conformation, Engineering, Membrane Lipids chemistry, Unilamellar Liposomes chemistry
- Abstract
The asymmetric distribution of lipids between the two bilayer leaflets represents a typical feature of biological membranes. The loss of this asymmetry, for example the exposure of negatively charged lipids on the extracellular membrane leaflet of mammalian cells, is involved in apoptosis and occurs in tumor cells. Thus, the controlled production of asymmetric liposomes helps to better understand such crucial cellular processes. Here, we present an approach that allows us to design asymmetric model-membrane experiments on a rational basis and predict the fraction of exchanged lipid. In addition, we developed a label-free and nondestructive assay to quantify the asymmetric uptake of negatively charged lipids in terms of the zeta potential. This significantly enhances the applicability, impact, and predictive power of model membranes.
- Published
- 2018
- Full Text
- View/download PDF
49. Biomembrane Permeabilization: Statistics of Individual Leakage Events Harmonize the Interpretation of Vesicle Leakage.
- Author
-
Braun S, Pokorná Š, Šachl R, Hof M, Heerklotz H, and Hoernke M
- Subjects
- Cell Membrane Permeability, Microscopy, Fluorescence, Particle Size, Permeability, Spectrometry, Fluorescence, Fluoresceins metabolism, Fluorescent Dyes metabolism, Liposomes metabolism
- Abstract
The mode of action of membrane-active molecules, such as antimicrobial, anticancer, cell penetrating, and fusion peptides and their synthetic mimics, transfection agents, drug permeation enhancers, and biological signaling molecules (e.g., quorum sensing), involves either the general or local destabilization of the target membrane or the formation of defined, rather stable pores. Some effects aim at killing the cell, while others need to be limited in space and time to avoid serious damage. Biological tests reveal translocation of compounds and cell death but do not provide a detailed, mechanistic, and quantitative understanding of the modes of action and their molecular basis. Model membrane studies of membrane leakage have been used for decades to tackle this issue, but their interpretation in terms of biology has remained challenging and often quite limited. Here we compare two recent, powerful protocols to study model membrane leakage: the microscopic detection of dye influx into giant liposomes and time-correlated single photon counting experiments to characterize dye efflux from large unilamellar vesicles. A statistical treatment of both data sets does not only harmonize apparent discrepancies but also makes us aware of principal issues that have been confusing the interpretation of model membrane leakage data so far. Moreover, our study reveals a fundamental difference between nano- and microscale systems that needs to be taken into account when conclusions about microscale objects, such as cells, are drawn from nanoscale models.
- Published
- 2018
- Full Text
- View/download PDF
50. Digitonin does not flip across cholesterol-poor membranes.
- Author
-
Fan HY and Heerklotz H
- Subjects
- Lipid Bilayers chemistry, Micelles, Thermodynamics, Cholesterol chemistry, Digitonin chemistry, Liposomes chemistry, Phosphatidylcholines chemistry, Phosphatidylserines chemistry
- Abstract
Digitonin is commonly used to permeabilize cell membranes and solubilize membrane components. It interacts specifically with cholesterol in the membrane which leads to the formation of pores. Thus far, the mechanism by which digitonin interacts with the membrane has only been described qualitatively. We investigated this interaction in model membranes that contain little or no cholesterol with a combination of isothermal titration calorimetry, dynamic light scattering, and zeta potential measurements. Digitonin partitions fully asymmetrically into large unilamellar vesicles of phosphocholine (PC) lipid at 20°C (remaining in the outer leaflet only), with a partition coefficient of 0.22±0.04mM
-1 and ΔH of partitioning of 23.3±1.6kJmol-1 . Beyond a digitonin/lipid ratio of ∼0.1 in the outer leaflet, digitonin micelles coexist with vesicles without solubilizing them-even at high digitonin concentrations. This "staying out" of digitonin was also observed with phosphoserine (PS), PC/PS, and PC/PS/cholesterol vesicles. The mechanism by which digitonin perturbs and solubilizes the membrane is very different when the membrane contains little or no cholesterol as opposed to 20-30mol% cholesterol. The role of digitonin should thus be carefully considered in the design of preparative protocols and experiments in studies of cellular processes and membrane proteins., (Copyright © 2017 Elsevier Inc. All rights reserved.)- Published
- 2017
- Full Text
- View/download PDF
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