311 research outputs found
Insertion of membrane proteins in artificial polymer membranes
No full textThe last few decades have seen a huge growth in research on “soft materials”. A large part of the research in this field was dedicated to the preparation of new types of artificial membranes, which behave similar to lipid or cell membranes. A particular challenge is the preparation of stabilized, flexible, adaptable and responsive materials. Similar to nature such systems can only be realized using hierarchically self-assembled systems. In this context we have introduced a new way of stabilizing lipid-bilayers using hydrophobic polymer scaffold. In contrast to other approaches, presented by Ringsdorf et al., 1988, the hydrophobic polymer scaffold allowed us to insert membrane proteins into the polymer stabilized membranes. One representative example of the functional insertion of a membrane protein into such stabilized membranes will be described in the present work. In another approach we used the self assembling capacity of amphiphilic block copolymers to prepare stable biomimetic membranes. The last few years have seen considerable progress in the development of block copolymer chemistry. Particularly, a whole series of new amphiphilic block copolymers with low glass-transition temperatures have been introduced. The diversity of block copolymer chemistry allows to vary the chemical constitution, such as the nature and the sequence of the repeat unit (as mentioned in a later section), the length and the different structures of the different blocks and even the molecular architecture of the whole polymer, block, graft, star, etc. This may lead to the formation of new types of self-assembled superstructures that are not accessible to conventional low molar mass amphiphiles. Recently our group introduced a new type of amphiphilic block copolymer composed of two hydrophilic side blocks poly-methyloxazoline (PMOXA) and one hydrophobic middle block polydimethylsiloxane (PDMS), forming an ABA triblock copolymer. The physico-chemical characterization of the ABA block copolymer has been described by Nardin et al. Similar to conventional low molar mass amphiphiles (lipids, surfactants), this polymer selfassembles in aqueous media and forms well defined superstructures. Depending on its molecular composition and the experimental conditions various lyotropic mesophases, vesicles and nanotubes have been identified. Most interestingly it has been shown that membranes formed by such polymers could be used as a matrix for the incorporation of integral membrane proteins.
In the present work we developed new procedures for membrane proteins that are adapted and
optimized with respect to the artificial polymer membranes. For that purpose we performed a series of
experiments with different membrane proteins that have different structural properties and
functionality. In all systems investigated we could successfully proof the incorporation and the
functionality of the proteins.
For a first experiment we used well characterized and stable membrane proteins like bacterial
porins. Porins are well characterized integral membrane proteins possessing interesting structural and
physical properties, such as hydrophobic β-strands, which can interact and insert into the hydrophobic
part of the block copolymer. Additionally, the porins form β-stranded pores, which allow a passive and
selective transfer of small molecules across a membrane.
Then, more complex membrane proteins were used such as hemagglutinin or
NADH:oxydoreductase. Both proteins are composed of a large soluble part which contributes to their
structural and functional particularities. The globular part of hemagglutinin is involved in the fusion of
two membranes whereas the soluble part of NADH:oxydoreductase is responsible for proton and
electron transfer across the membrane.
The combination of natural proteins with artificial polymer membranes allows the formation of
a new type of hybrid material combining the mechanical, chemical, and biological stability of the
amphiphile block copolymer and the functional specificity of membrane proteins
Using Molecular Simulations to Screen for Antibiotics with Enhanced Permeation Properties through Bacterial Pores
No full textRole of Antibiotic Side Chains in Uptake Through OmpPst1 Channel from Providencia Stuartii
No full text
Sensing Single Molecule Penetration into Nanopores: Pushing the Time Resolution to the Diffusion Limit
No full textTo quantify small molecule penetration into and eventually permeation through nanopores, we applied an improved excess-noise analysis of the ion current fluctuation caused by entering molecules. The kinetic parameters of substrate entry and exit are derived from a two-state Markov model, analyzing the substrate concentration dependence of the average ion current and its variance. Including filter corrections allows one to detect the transition rates beyond the cutoff frequency, fc, of the instrumental ion-current filter. As an application of the method, we performed an analysis of the single-channel ion current of Meropenem, an antibiotic of the carbapenem family, interacting with OmpF, the major general outer membrane channel of Escherichia coli bacteria. At 40 °C we detected the residence time of Meropenem inside OmpF of about 500 ns - more than 2 orders of magnitude smaller than fc-1 and close to the diffusion limit of few hundred nanoseconds. We also have established theoretical limit conditions under which the substrate-induced channel blockages can be detected and suggest that submicrosecond-scale gating kinetic parameters are accessible with existing experimental equipment
Triggered release of liposome contents by the divalent anion B12H11SH(2-): Biochemical studies
No full textBSH is one of the two boron compounds in use in BNCT. Triggered release of liposome contents by BSH has been achieved. We used liposomes of different compositions as a model for studying the interaction of BSH with cell membranes. The interaction of BSH with cationic liposomes of different compositions was investigated. Zeta potential and size measurements of the liposomes in absence and presence of BSH indicated a firm binding of BSH with the headgroups of these liposomes. Beside exploiting the liposomes as model for cell membranes, there is also great interest in liposomes as drug carriers. To have a pronounced therapeutic effect, the release of the drug from the liposomes should be as complete as possible. Because of this, developing tools for triggering release of agents encapsulated inside the aqueous compartment of the liposomes is an important factor in the liposome drug delivery systems.We studied the interaction of BSH with neutral liposomes prepared either from DMPC or DPPC lipids and proved that they released their contents under the influence of BSH within minutes
How the physical properties of bacterial porins match environmental conditions
No full textTransmembrane beta-barrel proteins are key systems for transport phenomena in biology. Based on their broad substrate specificity, they represent good candidates for present and future technological applications, such as DNA/RNA and protein sequencing, sensing of biomedical analytes, and production of blue energy. For a better understanding of the process at the molecular level, we applied parallel tempering simulations in the WTE ensemble to compare two beta-barrel porins from Escherichia coli, OmpF and OmpC. Our analysis showed a different behavior of the two highly homologous porins, where subtle amino acid substitutions can modulate critical properties of mass transport. Interestingly, the differences can be mapped to the respective environmental conditions under which the two porins are expressed. Apart from reporting on the advantages of the enhanced sampling methods in assessing the molecular properties of nanopores, our comparative analysis provided new and key results to better understand biological function and technical applications. Eventually, we showed how results from molecular simulations align well with experimental single-channel measurements, thus demonstrating the mature evolution of numerical methodologies for predicting properties in this field crucial for future biomedical applications
Molecular Basis of Enrofloxacin Translocation through OmpF, an Outer Membrane Channel of Escherichia coli - When Binding Does Not Imply Translocation
No full textThe 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
Robotic voltammetry with carbon nanotube-based sensors: a superb blend for convenient high-quality antimicrobial trace analysis
No full textSomjai Theanponkrang,1,2 Wipa Suginta,2,3 Helge Weingart,4 Mathias Winterhalter,4 Albert Schulte1,2 1School of Chemistry, 2Biochemistry–Electrochemistry Research Unit, Institute of Science, 3School of Biochemistry, Suranaree University of Technology, Nakhon Ratchasima, Thailand; 4Life Sciences, School of Engineering and Science, Jacobs University Bremen, Bremen, Germany Abstract: A new automated pharmacoanalytical technique for convenient quantification of redox-active antibiotics has been established by combining the benefits of a carbon nanotube (CNT) sensor modification with electrocatalytic activity for analyte detection with the merits of a robotic electrochemical device that is capable of sequential nonmanual sample measurements in 24-well microtiter plates. Norfloxacin (NFX) and ciprofloxacin (CFX), two standard fluoroquinolone antibiotics, were used in automated calibration measurements by differential pulse voltammetry (DPV) and accomplished were linear ranges of 1–10 µM and 2–100 µM for NFX and CFX, respectively. The lowest detectable levels were estimated to be 0.3±0.1 µM (n=7) for NFX and 1.6±0.1 µM (n=7) for CFX. In standard solutions or tablet samples of known content, both analytes could be quantified with the robotic DPV microtiter plate assay, with recoveries within ±4% of 100%. And recoveries were as good when NFX was evaluated in human serum samples with added NFX. The use of simple instrumentation, convenience in execution, and high effectiveness in analyte quantitation suggest the merger between automated microtiter plate voltammetry and CNT-supported electrochemical drug detection as a novel methodology for antibiotic testing in pharmaceutical and clinical research and quality control laboratories. Keywords: antibiotics, electroanalysis, automation, microtiter plates, pharmaceutical screening, pharmacoanalytic
- …
