Heiko Heerklotz

University of Toronto, Toronto, Ontario, Canada

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Publications (89)379.38 Total impact

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    ABSTRACT: Most antimicrobial peptides act upon target microorganisms by permeabilizing their membranes. The mode of action is often assessed by vesicle leakage experiments that use model membranes, with the assumption that biological activity correlates with the permeabilization of the lipid bilayer. The current work aims to extend the interpretation of vesicle leakage results and examine the correlation between vesicle leakage and antimicrobial activity. To this end, we used a lifetime-based leakage assay with calcein-loaded vesicles to study the membrane permeabilizing properties of a novel antifungal polymer poly-NM, two of its analogs, and a series of detergents. In conjunction, the biological activities of these compounds against Candida albicans were assessed and correlated with data from vesicle leakage. Poly-NM induces all-or-none leakage in polar yeast lipid vesicles at the polymer's MIC, 3 μg mL(-1). At this and higher concentrations, complete leakage after an initial lag time was observed. Concerted activity tests imply that this polymer acts independently of the detergent octyl glucoside (OG) for both vesicle leakage and activity against C. albicans spheroplasts. In addition, poly-NM was found to have negligible activity against zwitterionic vesicles and red blood cells. Our results provide a consistent, detailed picture of the mode of action of poly-NM: this polymer induces membrane leakage by electrostatic lipid clustering. In contrast, poly-MM:CO, a nylon-3 polymer comprised of both cationic and hydrophobic segments, seems to act by a different mechanism that involves membrane asymmetry stress. Vesicle leakage for this polymer is transient (limited to <100%) and graded, non-specific among zwitterionic and polar yeast lipid vesicles, additive with detergent action, and correlates poorly with biological activity. Based on these results, we conclude that comprehensive leakage experiments can provide a detailed description of the mode of action of membrane permeabilizing compounds. Without this thorough approach, it would have been logical to assume that the two nylon-3 polymers we examined act via similar mechanisms; it is surprising that their mechanisms are so distinct. Some, but not all mechanisms of vesicle permeabilization allow for antimicrobial activity.
    Soft Matter 08/2015; DOI:10.1039/c5sm01521a · 4.15 Impact Factor
  • Sebastian Fiedler · Quang Huynh · Hiren Patel · Heiko Heerklotz
    Biophysical Journal 01/2015; 108(2):549a. DOI:10.1016/j.bpj.2014.11.3013 · 3.97 Impact Factor
  • Helen Y. Fan · Dar’ya S. Redka · Heiko Heerklotz
    Biophysical Journal 01/2015; 108(2):543a. DOI:10.1016/j.bpj.2014.11.2978 · 3.97 Impact Factor
  • Sara Hovakeemian · Runhui Liu · Samuel H. Gellman · Heiko Heerklotz
    Biophysical Journal 01/2015; 108(2):551a. DOI:10.1016/j.bpj.2014.11.3021 · 3.97 Impact Factor
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    Alekos D Tsamaloukas · Sandro Keller · Heiko Heerklotz
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    ABSTRACT: The effective charge of membrane-active molecules such as the fungicidal lipopeptide surfactin (SF) is a crucial property governing solubility, membrane partitioning, and membrane permeability. We present zeta potential measurements of liposomes to measure the effective charge as well as membrane partitioning of SF by utilizing what we call an equi-activity analysis of several series of samples with different lipid concentrations. We observe an effective charge of -1.0 for SF at pH 8.5 and insignificantly lower at pH 7.4, illustrating that the effective charge may deviate strongly from the nominal value (-2 for 1 Asp, 1 Glu). The apparent partition coefficient decreases from roughly 100 to 20/mM with increasing membrane content of SF in agreement with the literature. Finally, by comparing zeta potentials measured soon after addition of peptide to liposomes with those measured after a heat treatment to induce transmembrane equilibration of SF, we quantified the asymmetry of partitioning between outer and inner leaflet. At very low concentration, SF binds exclusively to the outer leaflet. The onset of partial translocation to the inner leaflet occurs at about 5 mol-% SF in the membrane. This article is part of a Special Issue entitled: Interfacially active peptides and proteins.
    Biochimica et Biophysica Acta 09/2014; 2014. DOI:10.1016/j.bbamem.2014.02.018 · 4.66 Impact Factor
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    Hiren Patel · Quang Huynh · Dominik Bärlehner · Heiko Heerklotz
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    ABSTRACT: Certain antibiotic peptides are thought to permeabilize membranes of pathogens by effects that are also observed for simple detergents, such as membrane thinning and disordering, asymmetric bilayer expansion, toroidal pore formation, and micellization. Here we test the hypothesis that such peptides act additively with detergents when applied in parallel. Additivity is defined analogously to a fractional inhibitory concentration index of unity, and the extent and mechanism of leakage is measured by the fluorescence lifetime-based vesicle leakage assay using calcein-loaded vesicles. Good additivity was found for the concerted action of magainin 2, the fungicidal lipopeptide class of surfactins from Bacillus subtilis QST713, and the detergent octyl glucoside, respectively, with the detergent C12EO8. Synergistic or superadditive action was observed for fengycins from B. subtilis, as well as the detergent CHAPS, when combined with C12EO8. The results illustrate two mechanisms of synergistic action: First, maximal leakage requires an optimum degree of heterogeneity in the system that may be achieved by mixing a graded with an all-or-none permeabilizer. (The optimal perturbation should be focused to certain defect structures, yet not to the extent that some vesicles are not affected at all.) Second, a cosurfactant may enhance the bioavailability of a poorly soluble peptide. The results are important for understanding the concerted action of membrane-permeabilizing compounds in biology as well as for optimizing formulations of such antimicrobials for medical applications or crop protection.
    Biophysical Journal 05/2014; 106(10):2115–2125. DOI:10.1016/j.bpj.2014.04.006 · 3.97 Impact Factor
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    ABSTRACT: Guanine-rich DNA sequences that may form G-quadruplexes are located in strategic DNA loci with the ability to regulate biological events. G-quadruplexes have been under intensive scrutiny owing to their potential to serve as novel drug targets in emerging anticancer strategies. Thermodynamic characterization G-quadruplexes is an important and necessary step in developing predictive algorithms for evaluating the conformational preferences of G-rich sequences in the presence or the absence of their complementary C-rich strands. We use a combination of spectroscopic, calorimetric, and volumetric techniques to characterize the folding/unfolding transitions of the 26-meric human telomeric sequence d[A3 G3 (T2 AG3 )3 A2 ]. In the presence of K(+) ions, the latter adopts the hybrid-1 G-quadruplex conformation, a tightly packed structure with an unusually small number of solvent-exposed atomic groups. The K(+) -induced folding of the G-quadruplex at room temperature is a slow process that involves significant accumulation of an intermediate at the early stages of the transition. The G-quadruplex state of the oligomeric sequence is characterized by a larger volume and compressibility and a smaller expansibility than the coil state. These results are in qualitative agreement with each other all suggesting significant dehydration to accompany the G-quadruplex formation. Based on our volume data, 432 ± 19 water molecules become released to the bulk upon the G-quadruplex formation. This large number is consistent with a picture in which DNA dehydration is not limited to water molecules in direct contact with the regions that become buried but involves a general decrease in solute-solvent interactions all over the surface of the folded structure.
    Biopolymers 03/2014; 101(3). DOI:10.1002/bip.22317 · 2.29 Impact Factor
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    Hiren Patel · Quang Huynh · Dominik Bärlehner · Heiko Heerklotz
    Biophysical Journal 01/2014; 106(2):293a. DOI:10.1016/j.bpj.2013.11.1711 · 3.97 Impact Factor
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    Helen Y. Fan · Dar'ya S. Redka · Heiko Heerklotz
    Biophysical Journal 01/2014; 106(2):700a. DOI:10.1016/j.bpj.2013.11.3872 · 3.97 Impact Factor
  • Biophysical Journal 01/2014; 106(2):700a. DOI:10.1016/j.bpj.2013.11.3873 · 3.97 Impact Factor
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    Heiko Heerklotz · Sandro Keller
    Biophysical Journal 12/2013; 105(12):2607-10. DOI:10.1016/j.bpj.2013.10.031 · 3.97 Impact Factor
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    Dar'ya S Redka · Heiko Heerklotz · James W Wells
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    ABSTRACT: Muscarinic and other G protein-coupled receptors exhibit an agonist-specific heterogeneity that tracks efficacy and commonly is attributed to an effect of the G protein on an otherwise homogeneous population of sites. To examine this notion, M2 muscarinic receptors were purified from Sf9 cells as monomers devoid of G protein and reconstituted as tetramers in phospholipid vesicles. In assays with N-[(3)H]methylscopolamine, seven agonists revealed a dispersion of affinities indicative of two or more classes of sites. Unlabeled N-methylscopolamine and the antagonist quinuclidinylbenzilate recognized one class of sites; atropine recognized two classes with a preference opposite to that of agonists, as indicated by the effects of N-ethylmaleimide. The data were inconsistent with an explicit model of constitutive asymmetry within a tetramer, and the fit improved markedly upon the introduction of cooperative interactions (P < 0.00001). Purified monomers appeared homogeneous or nearly so to all ligands except the partial agonists pilocarpine and McN-A-343, where heterogeneity emerged from intramolecular cooperativity between the orthosteric site and an allosteric site. The breadth of each dispersion was quantified empirically as the area between the fitted curve for two classes of sites and the theoretical curve for a single class of lower affinity, which approximates the expected effect of GTP if a G protein were present. The areas measured for ten ligands at reconstituted tetramers correlated with similar measures of heterogeneity and with intrinsic activities reported previously for binding and response in natural membranes (P ≤ 0.00002). The data suggest that the GTP-sensitive heterogeneity typically revealed by agonists at M2 receptors is intrinsic to the receptor in its tetrameric state. It exists independently of the G protein, and it appears to arise at least in part from cooperativity between linked orthosteric sites.
    Biochemistry 09/2013; 52(42). DOI:10.1021/bi4003869 · 3.01 Impact Factor
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    Mozhgan Nazari · Gaurav Raval · Zubeir Khan · Hiren Patel · Heiko Heerklotz
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    ABSTRACT: We demonstrate the use of zeta potential measurements of liposomes to address membrane binding of peptides and surfactants, membrane-induced protonation and counterion binding effects, membrane asymmetry and permeation, and membrane domain formation. Instead of estimating membrane binding from the surface charge density by guessing the effective charge per molecule, we used what we refer to as an equi-activity evaluation to correct for binding and, hence, measure the effective charge. To this end, zeta potentials were recorded for an array of different lipid and peptide concentrations. It turns that the effective charge of a membranebound peptide is not straightforward to be guessed, because it may depend sensitively on membrane-induced (de)protonation and counterion-specific neutralization effects. The importance of the effective charge for trans-membrane flip-flop and interactions with other membrane components underlines the value of its direct measurement as explained here. Another interesting feature of the zeta potential is that it specifically reflects the charge density in the outer leaflet of the liposome. This allows for addressing the asymmetric binding of a peptide and detecting its threshold for transmembrane equilibration due to bilayer asymmetry stress or pore formation. Finally, composition-dependent changes of the apparent charge already at low membrane content may indicate the formation of peptide-rich domains. These approaches are demonstrated for the Bacillus lipopeptides surfactin and fengycin, as well as for SDS in different buffers.
    Biophysical Journal 01/2013; 104(2):44-. DOI:10.1016/j.bpj.2012.11.280 · 3.97 Impact Factor
  • Biophysical Journal 01/2013; 104(2):26-. DOI:10.1016/j.bpj.2012.11.181 · 3.97 Impact Factor
  • Sara Hovakeemian · Hiren Patel · Heiko Heerklotz
    Biophysical Journal 01/2013; 104(2):80-. DOI:10.1016/j.bpj.2012.11.486 · 3.97 Impact Factor
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    Dew Das · Arpit Shah · Mozhgan Nazari · Hiren Patel · Heiko Heerklotz
    Biophysical Journal 01/2013; 104(2):81-. DOI:10.1016/j.bpj.2012.11.487 · 3.97 Impact Factor
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    ABSTRACT: This work investigated the capability of a new nanoparticulate system, based on terpolymer of starch, polymethacrylic acid and polysorbate 80, to load and release doxorubicin (Dox) as a function of pH and to evaluate the anticancer activity of Dox-loaded nanoparticles (Dox-NPs) to overcome multidrug resistance (MDR) in human breast cancer cells in vitro. The Dox-NPs were characterized by Fourier transform infrared spectroscopy (FTIR), isothermal titration calorimetry (ITC), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The cellular uptake and cytotoxicity of the Dox-loaded nanoparticles were investigated using fluorescence microscopy, flow cytometry, and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. The nanoparticles were able to load up to 49.7±0.3% of Dox with a high loading efficiency of 99.9±0.1%, while maintaining good colloidal stability. The nanoparticles released Dox at a higher rate at acidic pH attributable to weaker Dox-polymer molecular interactions evidenced by ITC. The Dox-NPs were taken up by the cancer cells in vitro and significantly enhanced the cytotoxicity of Dox against human MDR1 cells with up to a 20-fold decrease in the IC50 values. The results suggest that the new terpolymeric nanoparticles are a promising vehicle for the controlled delivery of Dox for treatment of drug resistant breast cancer.
    European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 09/2012; 82(3):587–597. DOI:10.1016/j.ejpb.2012.09.001 · 4.25 Impact Factor
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    Mozhgan Nazari · Helen Y Fan · Heiko Heerklotz
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    ABSTRACT: Volumetric parameters have long been used to elucidate the phenomena governing the stability of protein structures, ligand binding, or transitions in macromolecular or colloidal systems. In spite of much success, many problems remain controversial. For example, hydrophobic groups have been discussed to condense adjacent water to a volume lower than that of bulk water, causing a negative contribution to the volume change of unfolding. However, expansivity data were interpreted in terms of a structure-making effect that expands the water interacting with the solute. We have studied volume and expansivity effects of transfer of alkyl chains into micelles by pressure perturbation calorimetry and isothermal titration calorimetry. For a series of alkyl maltosides and glucosides, the methylene group contribution to expansivity was obtained as 5 uL/(mol K) in a micelle (mimicking bulk hydrocarbon) but 27 uL/(mol K) in water (20 °C). The latter value is virtually independent of temperature and similar to that obtained from hydrophobic amino acids. Methylene contributions of micellization are about -60 J/(mol K) to heat capacity and 2.7 mL/mol to volume. Our data oppose the widely accepted assumption that water-exposed hydrophobic groups yield a negative contribution to expansivity at low temperature that would imply a structure-making, water-expanding effect.
    Langmuir 09/2012; 28(40):14129-36. DOI:10.1021/la302276n · 4.46 Impact Factor
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    ABSTRACT: This work focused on the design of new pH-responsive nanoparticles for controlled delivery of anticancer drug doxorubicin (Dox). Nanoparticles of poly(methacrylic acid)-polysorbate 80-grafted starch (PMAA-PS 80-g-St) were synthesized by using a one-pot method that enabled simultaneous grafting of PMAA and PS 80 onto starch and nanoparticle formation in an aqueous medium. The particles were characterized by FTIR, (1)H NMR, TEM, DLS, and potentiometric titration. Dox loading and in vitro release from the nanoparticles were investigated. The FTIR and (1)H NMR confirmed the chemical composition of the graft terpolymer. The nanoparticles were relatively spherical with narrow size distribution and porous morphology. They exhibited pH-dependent swelling in a physiological pH range. The particle size and magnitude of phase transition were dependent on polymer composition and formulation parameters such as concentrations of surfactant and cross-linking agent and total monomer concentration. The nanoparticles with optimized compositions showed high loading capacity for Dox and sustained Dox release. The results suggest that the new pH-responsive terpolymer nanoparticles are useful in controlled drug delivery.
    Colloids and surfaces B: Biointerfaces 07/2012; 101:405-413. DOI:10.1016/j.colsurfb.2012.07.015 · 4.15 Impact Factor

Publication Stats

3k Citations
379.38 Total Impact Points

Institutions

  • 2007–2014
    • University of Toronto
      • Leslie L. Dan Faculty of Pharmacy
      Toronto, Ontario, Canada
  • 1999–2007
    • Universität Basel
      • Department of Biophysical Chemistry
      Bâle, Basel-City, Switzerland
  • 1998–2001
    • McMaster University
      • Health Sciences Centre
      Hamilton, Ontario, Canada
  • 1994–2000
    • University of Leipzig
      • Institute of Experimental Physics
      Leipzig, Saxony, Germany