Nadja Hellmann

Johannes Gutenberg-Universität Mainz, Mayence, Rheinland-Pfalz, Germany

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Publications (45)136.27 Total impact

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    ABSTRACT: A novel homogenous core-shell formulation ‘AmbiShell’ of the hydrophobic antifungal polyene drug Amphotericin B was prepared from the pure drug, lecithin, and gelatin. The established ‘Hydrophilic Solubilisation Technology’ (HST) was used for embedding the lipophilic drug with lecithin and gelatin, yielding core-shell microparticles of 1 µm size. The particle size of the drug, the formulation, and the thickness of the coating were investigated by using dynamic light scattering (DLS), microscopy, UV–Visible spectroscopy and small angle neutron scattering (SANS). The drug in the formulation was not in the oligomer form but in a competent uptake form, as shown by the UV–Visible spectroscopy. The DLS and microscopy depicted the product to consist of spheres (∼1 µm). The SANS experiment using deuterium contrast matching of the drug cores revealed a thin layer span of 5.6 nm, which is similar, but slightly larger as compared to the biological lipid membranes. The finding offers opportunities for further improvements of the formulation, e.g., by surface modification.
    European Journal of Lipid Science and Technology 07/2014; · 2.27 Impact Factor
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    ABSTRACT: The characteristics of intestinal model fluids were investigated at conditions, which simulate the passage from the middle to the end of the duodenum. The formation and decay of liposomes and micelles in model bile fluids were studied, because of their role as an intermediate host for the resolution and uptake of hydrophobic drugs (BCS classes II, IV). The conditions, which may influence the formation of these nanoparticulate intermediates were studied, i.e. the lipid composition of the bile, the preparation method, the time of the passage through the modelled duodenum segment and the concentration, which results from the variable dilution of the bile by mixing with the transfer medium representing the fluid arriving from the stomach. The variation of the lecithin entity revealed an equivalence of Egg-lecithin of a high purity (99%) with its main component 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPC, while 1,2-di-oleoyl-sn-glycero-3-phosphocholine DOPC resulted in a slight delay of the micelle-liposome conversion. The FeSSIF preparation method was best with the sequential-film method, while the bile-film method yielded comparable results; the shake method showed a slightly different kinetics of the nanoparticle conversion. The time and concentration dependence of the formation and decay of lipidic nanoparticles indicates that these strongly depend on the passage time (speed) and bile dilution rate. The corresponding physiological conditions at healthy persons may vary in vivo individually and due to diseases. The studied conditions cover typical physiological conditions, which should be taken into consideration in the exploration of in vitro tests of formulations of hydrophobic drugs.
    European Journal of Lipid Science and Technology 07/2014; · 2.27 Impact Factor
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    ABSTRACT: Human papillomaviruses (HPV) induce warts and cancers on skin and mucosa. The HPV16 capsid is composed of the proteins L1 and L2. After cell entry and virus disassembly, the L2 protein accompanies the viral DNA to promyelocytic leukemia nuclear bodies (PML-NBs) within the host nuclei enabling viral transcription and replication. Multiple components of PML-NBs are regulated by small ubiquitin-like modifiers (SUMOs) either based on covalent SUMO modification (SUMOylation), or noncovalent SUMO interaction via SUMO interacting motifs (SIMs). We show here that the HPV16 L2 comprises at least one SIM, which is crucial for the L2 interaction with SUMO2 in immunoprecipitation and colocalization with SUMO2 in PML-NBs. Biophysical analysis confirmed a direct L2 interaction with SUMO substantiated by identification of potential L2-SUMO interaction structures in molecular dynamics simulations. Mutation of the SIM resulted in absence of the L2-DNA complex at PML-NB and in a loss of infectivity of mutant HPV16 pseudoviruses. In contrast, we found that L2 SUMOylation has no effect on L2 localization in PML-NBs and SUMO interaction. Our data suggest that the L2 SIM is important for L2 interaction with SUMO and/or SUMOylated proteins, which is indispensable for the delivery of viral DNA to PML-NBs and efficient HPV infection.
    Cellular Microbiology 01/2014; · 4.81 Impact Factor
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    ABSTRACT: Mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) are responsible for a subset of amyotrophic lateral sclerosis cases presumably by the acquisition of as yet unknown toxic properties. Additional overexpression of wild-type SOD1 in mutant SOD1 transgenic mice did not improve but rather accelerated the disease course. Recently, it was documented that the presence of wild-type SOD1 (SOD(WT)) reduced the aggregation propensity of mutant SOD1 by the formation of heterodimers between mutant and SOD1(WT) and that these heterodimers displayed at least a similar toxicity in cellular and animal models. In this study we investigated the biochemical and biophysical properties of obligate SOD1 dimers that were connected by a peptide linker. Circular dichroism spectra indicate an increased number of unstructured residues in SOD1 mutants. However, SOD1(WT) stabilized the folding of heterodimers compared to mutant homodimers as evidenced by an increase in resistance against proteolytic degradation. Heterodimerization also reduced the affinity of mutant SOD1 to antibodies detecting misfolded SOD1. In addition, the formation of obligate dimers resulted in a detection of substantial dismutase activity even of the relatively labile SOD1(G85R) mutant. These data indicate that soluble, dismutase-active SOD1 dimers might contribute at least partially to mutant SOD1 toxicity.
    Neurobiology of Disease 11/2013; · 5.62 Impact Factor
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  • M Schwiering, A Brack, R Stork, N Hellmann
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    ABSTRACT: The pore forming toxin Hla (α-toxin) from S. aureus is an important pathogenic factor of the bacterium S. aureus and also a model system for the process of membrane-induced protein oligomerisation and pore formation. It has been shown that binding to lipid membranes at neutral or basic pH requires the presence of a phosphocholine-headgroup. Thus, sphingomyelin and phosphatidylcholine may serve as interaction partners in cellular membranes. Based on earlier studies it has been suggested that rafts of sphingomyelin are particularly efficient in toxin binding. In this study we compared the oligomerisation of Hla on liposomes of various lipid compositions in order to identify the preferred interaction partners and conditions. Hla seems to have an intrinsic preference for sphingomyelin compared to phosphatidylcholine due to a higher probability of oligomerisation of membrane bound monomer. We also can show that increasing the surface density of Hla-binding sites enhances the oligomerisation efficiency. Thus, preferential binding to lipid rafts can be expected also in the cellular context. On the other hand, sphingomyelin in the liquid disordered phase is a more favourable binding partner for Hla than SM in the liquid ordered phase, which makes the membrane outside of lipid rafts the preferred region of interaction. Thus, the partitioning of Hla is expected to strongly depend on the exact composition of raft and non-raft domains in the membrane.
    Biochimica et Biophysica Acta 04/2013; · 4.66 Impact Factor
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    ABSTRACT: The oxygen binding properties of hemocyanins are regulated on a short time scale by effectors such as L-lactate, urate and protons, and on longer time scales by expression of different types of subunits. For A. leptodactylus it was shown previously that acclimation to higher temperatures leads to increased levels of a 6-meric hemocyanin species, whereas at lower temperatures the 12-meric form prevails. Here we show that the temperature dependence of the two forms supports the idea, that maintenance of high affinity towards oxygen is the driving force for the differential expression of these hemocyanins. Furthermore, the two different types of hemocyanin differ not only in the affinity to oxygen, but also with respect to their interaction with L-lactate: while the 12-meric form displays a normal shift in oxygen affinity upon addition of L-lactate this allosteric regulation is absent in the 6-meric form. Exclusive binding of L-lactate to the 12-meric form was supported by isothermal titration calorimetry. These results indicate that L-lactate binds either at the interface between the two hexamers or at subunit α' which is responsible for formation of the 12-mers and is not present in the 6-meric form. Urate has a comparable effect on the oxygen affinity of 6-meric and 12-meric forms and also binds to a similar extent to the oxygenated state as determined by isothermal titration calorimetry. Thus, urate and L-lactate does not seem to share the same binding sites. Interestingly, urate binding sites with no allosteric effect seem to exist, which is unusual. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
    Biochimica et Biophysica Acta 03/2013; · 4.66 Impact Factor
  • Biophysical Journal 01/2013; 104(2):97-. · 3.67 Impact Factor
  • Markus Schwiering, Nadja Hellmann
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    ABSTRACT: Liposomes of different sizes are frequently used model systems for cellular membranes. To mimic the cellular environment, these liposomes are often prepared from a mixture of different lipids in organic solution. The preparation involves, at some point, the transfer into aqueous solution. Thus, both the total amount of lipid and the relative amount of each lipid species might deviate from the original composition in the organic solvent. We used thin-layer chromatography combined with a lipid extraction step to check whether the liposomes in the final aqueous solution have the intended composition. This allows determination of the lipid composition not only for large unilamellar vesicles, but was also applied, for the first time, to giant lamellar vesicles (GUVs), which typically are available only with a low amount of lipid per preparation. For different ternary and quaternary mixtures of cholesterol, phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and sphingomyelin, the final composition agreed within 15% with the intended composition in most cases, but in certain cases, such as GUVs prepared with a large fraction of phosphatidylethanolamine, the deviation can be significant. This shows that in those cases where the composition plays an important role, it is advisable to check the final composition of these model membranes.
    Journal of Liposome Research 07/2012; · 1.91 Impact Factor
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    N Hellmann
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    ABSTRACT: Cooperative effects in ligand binding and dissociation kinetics are much less investigated than steady state kinetics or equilibrium binding. Nevertheless, cooperativity in ligand binding leads necessarily to characteristic properties with respect to kinetic properties of the system. In case of positive cooperativity as found in oxygen binding proteins, a typical property is an autocatalytic ligand dissociation behavior leading to a time dependent, apparent ligand dissociation rate. To follow systematically the influence of the various potentially involved parameters on this characteristic property, simulations based on the simple MWC model were performed which should be relevant for all types of models based on the concept of an allosteric unit. In cases where the initial conformational distribution is very much dominated by the R-state, the intrinsic kinetic properties of the T-state are of minor influence for the observed ligand dissociation rate. Even for fast conformational transition rates, the R-state properties together with the size of the allosteric unit and the allosteric equilibrium constant define the shape of the curve. In such a case, a simplified model of the MWC-scheme (the irreversible n-chain model) is a good approximation of the full scheme. However, if in the starting conformational distribution some liganded T-molecules are present (a few percent is enough), the average off-rates can be significantly altered. Thus, the assignment of the initial rates to R-state properties has to be done with great care. However, if the R-state strongly dominates initially it is even possible to get an estimation of the lower limit for the number of interacting subunits from kinetic data: similar to the Hill-coefficient for equilibrium conditions, a measure for "kinetic cooperativity" can be derived by comparing initial and final ligand dissociation rates.
    International Union of Biochemistry and Molecular Biology Life 05/2011; 63(5):329-36. · 2.79 Impact Factor
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    ABSTRACT: The extracellular hemoglobin multimer of the planorbid snail Biomphalaria glabrata, intermediate host of the human parasite Schistosoma mansoni, is presumed to be a 1.44 MDa complex of six 240 kDa polypeptide subunits, arranged as three disulfide-bridged dimers. The complete amino acid sequence of two subunit types (BgHb1 and BgHb2), and the partial sequence of a third type (BgHb3) are known. Each subunit encompasses 13 paralogus heme domains, and N-terminally a smaller plug domain responsible for subunit dimerization. We report here the recombinant expression of different functional fragments of BgHb2 in Escherichia coli, and of the complete functional subunits BgHb1 and BgHb2 in insect cells; BgHb1 was also expressed as disulfide-bridged dimer (480 kDa). Oxygen-binding measurements of the recombinant products show a P(50) of about 7 mmHg and the absence of a significant cooperativity or Bohr effect. The covalently linked dimer of BgHb1, but not the monomer, is capable to form aggregates closely resembling native BgHb molecules in the electron microscope.
    International Union of Biochemistry and Molecular Biology Life 05/2011; 63(5):323-8. · 2.79 Impact Factor
  • Biophysical Journal 01/2011; 100(3). · 3.67 Impact Factor
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    ABSTRACT: Myelin basic protein (MBP) is predominantly found in the membranes of the myelin sheath of the central nervous system and is involved in important protein-protein and protein-lipid interactions in vivo and in vitro. Furthermore, divalent transition metal ions, especially Zn(2+) and Cu(2+), seem to directly affect the MBP-mediated formation and stabilization of the myelin sheath of the central nervous system. MBP belongs to the realm of intrinsically disordered proteins, and only fragmentary information is available regarding its partial structure(s) or supramolecular arrangements. Here, using standard continuous wave and modern pulse electron paramagnetic resonance methods, as well as dynamic light scattering, we demonstrate the uptake and specific coordination of two Cu(2+) atoms or one Zn(2+) atom per MBP molecule in solution. In the presence of phosphates, further addition of divalent metal ions above a characteristic threshold of four Cu(2+) atoms or two Zn(2+) atoms per MBP molecule leads to the formation of large MBP aggregates within the protein solution. In vivo, MBP-MBP interactions may thus be mediated by divalent cations.
    Biophysical Journal 11/2010; 99(9):3020-8. · 3.67 Impact Factor
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    N Hellmann, M Paoli, F Giomi, M Beltramini
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    ABSTRACT: Hemocyanins from Crustacea usually are found as 1x6 or 2x6-meric assemblies. An exception is the hemocyanin isolated from thalassinidean shrimps where the main component is a 24-meric structure. Our analysis of oxygen binding data of the thalassinidean shrimp Upogebia pusilla based on a three-state MWC-model revealed that despite the 24-meric structure the functional properties can be described very well based on the hexamer as allosteric unit. In contrast to the hemocyanins from other thalassinidean shrimps the oxygen affinity of hemocyanin from U. pusilla is increased upon addition of l-lactate. A particular feature of this hemocyanin seems to be that l-lactate already enhances oxygen affinity under resting conditions which possibly compensates the rather low intrinsic affinity observed in absence of l-lactate. The fast rate of oxygen dissociation might indicate that in this hemocyanin a higher cooperativity is less important than a fast response of saturation level to changes in oxygen concentration.
    Archives of Biochemistry and Biophysics 03/2010; 495(2):112-21. · 3.37 Impact Factor
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    ABSTRACT: The allosteric respiratory protein hemocyanin occurs in gastropods as tubular di-, tri- and multimers of a 35 x 18 nm, ring-like decamer with a collar complex at one opening. The decamer comprises five subunit dimers. The subunit, a 400 kDa polypeptide, is a concatenation of eight paralogous functional units. Their exact topology within the quaternary structure has recently been solved by 3D electron microscopy, providing a molecular model of an entire didecamer (two conjoined decamers). Here we study keyhole limpet hemocyanin (KLH2) tridecamers to unravel the exact association mode of the third decamer. Moreover, we introduce and describe a more complex type of hemocyanin tridecamer discovered in fresh/brackish-water cerithioid snails (Leptoxis, Melanoides, Terebralia). The "typical" KLH2 tridecamer is partially hollow, whereas the cerithioid tridecamer is almost completely filled with material; it was therefore termed "mega-hemocyanin". In both types, the staggering angle between adjoining decamers is 36 degrees . The cerithioid tridecamer comprises two typical decamers based on the canonical 400 kDa subunit, flanking a central "mega-decamer" composed of ten unique ~550 kDa subunits. The additional ~150 kDa per subunit substantially enlarge the internal collar complex. Preliminary oxygen binding measurements indicate a moderate hemocyanin oxygen affinity in Leptoxis (p50 ~9 mmHg), and a very high affinity in Melanoides (~3 mmHg) and Terebralia (~2 mmHg). Species-specific and individual variation in the proportions of the two subunit types was also observed, leading to differences in the oligomeric states found in the hemolymph. In cerithioid hemocyanin tridecamers ("mega-hemocyanin") the collar complex of the central decamer is substantially enlarged and modified. The preliminary O2 binding curves indicate that there are species-specific functional differences in the cerithioid mega-hemocyanins which might reflect different physiological tolerances of these gill-breathing animals. The observed differential expression of the two subunit types of mega-hemocyanin might allow individual respiratory acclimatization. We hypothesize that mega-hemocyanin is a key character supporting the adaptive radiation and invasive capacity of cerithioid snails.
    Frontiers in Zoology 01/2010; 7:14. · 3.87 Impact Factor
  • Biophysical Journal 01/2010; 98(3). · 3.67 Impact Factor
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    ABSTRACT: Anthranilate phosphoribosyltransferase from the hyperthermophilic archaeon Sulfolobus solfataricus (ssAnPRT) is encoded by the sstrpD gene and catalyzes the reaction of anthranilate (AA) with a complex of Mg(2+) and 5'-phosphoribosyl-alpha1-pyrophosphate (Mg.PRPP) to N-(5'-phosphoribosyl)-anthranilate (PRA) and pyrophosphate (PP(i)) within tryptophan biosynthesis. The ssAnPRT enzyme is highly thermostable (half-life at 85 degrees C = 35 min) but only marginally active at ambient temperatures (turnover number at 37 degrees C = 0.33 s(-1)). To understand the reason for the poor catalytic proficiency of ssAnPRT, we have isolated from an sstrpD library the activated ssAnPRT-D83G + F149S double mutant by metabolic complementation of an auxotrophic Escherichia coli strain. Whereas the activity of purified wild-type ssAnPRT is strongly reduced in the presence of high concentrations of Mg(2+) ions, this inhibition is no longer observed in the double mutant and the ssAnPRT-D83G single mutant. The comparison of the crystal structures of activated and wild-type ssAnPRT shows that the D83G mutation alters the binding mode of the substrate Mg.PRPP. Analysis of PRPP and Mg(2+)-dependent enzymatic activity indicates that this leads to a decreased affinity for a second Mg(2+) ion and thus reduces the concentration of enzymes with the inhibitory Mg(2).PRPP complex bound to the active site. Moreover, the turnover number of the double mutant ssAnPRT-D83G + F149S is elevated 40-fold compared to the wild-type enzyme, which can be attributed to an accelerated release of the product PRA. This effect appears to be mainly caused by an increased conformational flexibility induced by the F149S mutation, a hypothesis which is supported by the reduced thermal stability of the ssAnPRT-F149S single mutant.
    Biochemistry 05/2009; 48(23):5199-209. · 3.38 Impact Factor
  • Biophysical Journal 01/2009; 96(3). · 3.67 Impact Factor
  • Comparative Biochemistry and Physiology A-molecular & Integrative Physiology - COMP BIOCHEM PHYSIOL PT A. 01/2009; 154(1).

Publication Stats

254 Citations
136.27 Total Impact Points

Institutions

  • 1998–2014
    • Johannes Gutenberg-Universität Mainz
      • • Institute of Molecular Biophysics
      • • Institute of Zoology
      Mayence, Rheinland-Pfalz, Germany
    • Universität Basel
      • Department of Biophysical Chemistry
      Basel, BS, Switzerland
  • 2010
    • Max Planck Institute for Polymer Research
      Mayence, Rheinland-Pfalz, Germany
  • 2000–2007
    • University of Padova
      • Department of Biology
      Padova, Veneto, Italy