Heiko Heerklotz

University of Toronto, Toronto, Ontario, Canada

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Publications (91)385.23 Total impact

  • Source
    Sebastian Fiedler · Heiko Heerklotz ·
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    ABSTRACT: Cyclic lipopeptides act against a variety of plant pathogens and are thus highly efficient crop-protection agents. Some pesticides contain Bacillus subtilis strains that produce lipopeptide families, such as surfactins (SF), iturins (IT), and fengycins (FE). The antimicrobial activity of these peptides is mainly mediated by permeabilizing cellular membranes. We used a fluorescence-lifetime based leakage assay to examine the effect of individual lipid components in model membranes on lipopeptide activity. Leakage induction by FE was strongly inhibited by cholesterol (CHOL) as well as by phosphatidylethanolamine (PE) and -glycerol (PG) lipids. Already moderate amounts of CHOL increased the tolerable FE content in membranes by an order of magnitude to 0.5 FE per PC + CHOL. This indicates reduced FE-lipid demixing and aggregation, which is known to be required for membrane permeabilization and explains the strong inhibition by CHOL. Ergosterol (ERG) had a weak antagonistic effect. This confirms results of microbiological tests and agrees with the fungicidal activity and selectivity of FE. SF is known to be much less selective in its antimicrobial action. In line with this, liposome leakage by SF was little affected by sterols and PE. Interestingly, PG increased SF activity and changed its leakage mechanism toward all-or-none, suggesting more specific, larger, and/or longer-lived defect structures. This may be because of the reduced energetic cost of locally accumulating anionic SF in an anionic lipid matrix. IT was found largely inactive in our assays. B. subtilis QST713 produces the lipopeptides in a ratio of 6 mol SF: 37 mol FE: 57 mol IT. Leakage induced by this native mixture was inhibited by CHOL and PE, but unaffected by ERG and by PG in the absence of PE. Note that fungi contain anionic lipids, but little PE. Hence, our data explain the strong, fungicidal activity and selectivity of B. subtilis QST713 lipopeptides.
    Biophysical Journal 11/2015; 109(10):2079. DOI:10.1016/j.bpj.2015.09.021 · 3.97 Impact Factor
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    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, was developed by incorporating histidine-mediated pH-sensitivity, amphipathic helix and amino acid pairing self-assembly design. The design strategy renders C8 as pH-responsive nanostructure that is switched on and off by cytotoxicity: Non-cytotoxic 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, we propose its physical mechanisms of pore-forming action and selectivity, 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 non-selective 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 multi-functional self-assembling peptides for therapeutic and drug delivery applications.
    Advanced Healthcare Materials 09/2015; DOI:10.1002/adhm.201500636 · 5.80 Impact Factor
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    Sara G Hovakeemian · Runhui Liu · Samuel H Gellman · Heiko Heerklotz ·
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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; 11(34). DOI:10.1039/c5sm01521a · 4.03 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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    Sebastian Fiedler · Quang Huynh · Hiren Patel · Heiko Heerklotz ·

    Biophysical Journal; 01/2015
  • 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
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    Alekos D Tsamaloukas · Sandro Keller · Heiko Heerklotz ·

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    Helen Y. Fan · Mozhgan Nazari · Gaurav Raval · Zubeir Khan · Hiren Patel · 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(9). 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 · 2.39 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
  • Sara G. Hovakeemian · Runhui Liu · Samuel H. Gellman · Heiko Heerklotz ·

    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 · 3.02 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
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    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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    Biophysical Journal 01/2013; 104(2):26-. DOI:10.1016/j.bpj.2012.11.181 · 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 · 3.38 Impact Factor

Publication Stats

3k Citations
385.23 Total Impact Points


  • 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