Mathias Winterhalter

Designed to penetrate: Time-resolved interaction of single antibiotic molecules with bacterial pores

Membrane permeability barriers are among the factors contributing to the intrinsic resistance of bacteria to antibiotics. We have been able to resolve single ampicillin molecules moving through a channel of the general bacterial porin, OmpF (outer membrane protein F), believed to be the principal pathway for the β-lactam antibiotics. With ion channel reconstitution and high-resolution conductance recording, we find that ampicillin and several other efficient penicillins and cephalosporins strongly interact with the residues of the constriction zone of the OmpF channel. Therefore, we hypothesize that, in analogy to substrate-specific channels that evolved to bind certain metabolite molecules, antibiotics have “evolved” to be channel-specific. Molecular modeling suggests that the charge distribution of the ampicillin molecule complements the charge distribution at the narrowest part of the bacterial porin. Interaction of these charges creates a region of attraction inside the channel that facilitates drug translocation through the constriction zone and results in higher permeability rates.

Protein encapsulation in liposomes: efficiency depends on interactions between protein and phospholipid bilayer.

BackgroundWe investigated the encapsulation mechanism of enzymes into liposomes. The existing protocols to achieve high encapsulation efficiencies are basically optimized for chemically stable molecules. Enzymes, however, are fragile and encapsulation requires in addition the preservation of their functionality. Using acetylcholinesterase as a model, we found that most protocols lead to a rapid denaturation of the enzyme with loss in the functionality and therefore inappropriate for such an application. The most appropriate method is based on lipid film hydration but had a very low efficiency.ResultsTo improve it and to propose a standard procedure for enzyme encapsulation, we separate each step and we studied the effect of each parameter on encapsulation: lipid and buffer composition and effect of the different physical treatment as freeze-thaw cycle or liposomes extrusion. We found that by increasing the lipid concentration, increasing the number of freeze-thaw cycles and enhancing the interactions of the enzyme with the liposome lipid surface more than 40% of the initial total activity can be encapsulated.ConclusionWe propose here an optimized procedure to encapsulate fragile enzymes into liposomes. Optimal encapsulation is achieved by induction of a specific interaction between the enzyme and the lipid surface.

Stealth® liposomes: from theory to product

Abstract The development of an effective anti-cancer liposomal formulation — doxorubicin in sterically stabilized liposomes — will be discussed. We shall argue that for many tumors the necessary condition for an effective anti-cancer activity of systemically administered liposomal doxorubicin formulation is the long circulation life of liposomes in blood and stable drug encapsulation. Theoretical basis for stabilization of liposomes in biological environments and for the stabilization of drug encapsulation will be shown. When a formulation with acceptable stability was obtained it was tested in pre-clinical models and simultaneously scaled-up and it entered into clinical studies. After successfully passing all these tests, doxorubicin in sterically stabilized liposomes (Doxil ™ by Sequus Pharmaceuticals, Inc., Menlo Park, CA) was approved by Food and Drug Administration and is commercially available since late 1995.

Nanoreactors based on (polymerized) ABA-triblock copolymer vesicles

A new kind of nanoreactor has been prepared by the incorporation of a channel protein into the shell of (polymerized) vesicles formed from an amphiphilic ABA-triblock copolymer.

Kinetics of pore size during irreversible electrical breakdown of lipid bilayer membranes

The kinetics of pore formation followed by mechanical rupture of lipid bilayer membranes were investigated in detail by using the charge-pulse method. Membranes of various compositions were charged to a sufficiently high voltage to induce mechanical breakdown. The subsequent decrease of membrane voltage was used to calculate the conductance. During mechanical breakdown, which was probably caused by the widening of one single pore, the membrane conductance was a linear and not exponential function of time after the initial starting process. In a large number of experiments using various lipids and electrolytes, the characteristic opening process of the pore turned out to be independent of the actual membrane potential and electrolyte concentration. Our theoretical analysis of the pore formation suggested that the voltage-induced irreversible breakdown is due to a decrease in edge energy when the pore had formed. After initiation of the pore, the electrical contribution to surface tension is negligible. The time course of the increase of pore size shows that our model of the irreversible breakdown is in good agreement with mechanical properties of membranes reported elsewhere.

Black lipid membranes

Abstract New techniques have been introduced or pushed forward to render this model system more powerful. Dynamical light scattering allows us to quantify, in non-pertubative way, viscoelastic properties. Ion current fluctuation analysis gives information on channels on a single molecular level. Highly promising is the current development in fluorescence detection.

Understanding the function of bacterial outer membrane channels by reconstitution into black lipid membranes

Structural and functional information is obtained by the reconstitution of membrane channel forming proteins into black lipid membranes. Due to this outstanding sensitivity only little material is needed and single molecule detection can be easily achieved. An overview on different types of detection will be given.

Deformation of spherical vesicles by electric fields

Abstract We calculate the strength of the ellipsoidal deformation of spherical vesicles in water by AC and DC electric fields.

Molecular origin of the cation selectivity in OmpF porin: single channel conductances vs. free energy calculation

Ion current through single outer membrane protein F (OmpF) trimers was recorded and compared to molecular dynamics simulation. Unidirectional insertion was revealed from the asymmetry in channel conductance. Single trimer conductance showed particularly high values at low symmetrical salt solution. The conductance values of various alkali metal ion solutions were proportional to the monovalent cation mobility values in the bulk phase, LiCl<NaCl<KCl<RbCl approximately CsCl, but the conductance differences were quantitatively larger than conductivity differences in bulk solutions. Selectivity measurements at low concentration showed that OmpF channels favored permeation of alkali metal ions over chloride and suggested size preference for smaller cations. These results suggest that there are specific interactions between the permeating cation and charged residues lining the channel walls. This hypothesis was supported by computational study which predicted that monovalent cations bind to Asp113 at low concentration. Here, free energy calculations revealed that the affinity of the alkali metal ions to its binding site increased with their atomic radii, Li(+) approximately Na(+)<K(+) approximately Rb(+) approximately Cs(+). A detailed inspection of both experimental and computational results suggested that stronger binding at the central constriction of the channel increases the translocation rate of cations under applied voltage by increasing their local concentration relative to the bulk solution.

Molecular basis of enrofloxacin translocation through OmpF, an outer membrane channel of Escherichia coli--when binding does not imply translocation.

The molecular pathway of enrofloxacin, a fluoroquinolone antibiotic, through the outer membrane channel OmpF of Escherichia coli is investigated. High-resolution ion current fluctuation analysis reveals a strong affinity for enrofloxacin to OmpF, the highest value ever recorded for an antibiotic-channel interaction. A single point mutation in the constriction zone of OmpF, replacing aspartic acid at the 113 position with asparagine (D113N), lowers the affinity to a level comparable to other antibiotics. All-atom molecular dynamics simulations allow rationalizing the translocation pathways: wild-type OmpF has two symmetric binding sites for enrofloxacin located at each channel entry separated by a large energy barrier in the center, which inhibits antibiotic translocation. In this particular case, our simulations suggest that the ion current blockages are caused by molecules occupying either one of these peripheral binding sites. Removal of the negative charge on position 113 removes the central barrier and shifts the two peripheral binding sites to a unique central site, which facilitates translocation. Fluorescence steady-state measurements agree with the different location of binding sites for wild-type OmpF and the mutant. Our results demonstrate how a single-point mutation of the porin, and the resulting intrachannel shift of the affinity site, may substantially modify translocation.