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Jiajun successfully defended his PhD at Jacobs University (Bremen, Germany) on May the 18th, 2017!
Here is a summary.
A facilitated Method to Characterize Rapid Substrate Binding to Membrane Channels
The bacterial outer membrane porins are regarded as one of the main pathways for hydrophilic antibiotic molecules to penetrate through the bacterial outer membrane. To understand the interaction mechanisms of antimicrobial molecules with the porins, the latter are extracted from their natural environment, reconstituted into free standing artificial lipid bilayer and characterized by electrophysiology. A broad range of general diffusion porins, i.e. OmpF and OmpC from E. coli, and their orthologs Omp35, Omp36 from E. aerogenes; OmpK35, OmpK36 from K.pneumoniae; OmpE35, OmpE36 from E. cloacae were characterized to study the biophysical properties of porins in different bacterial species. Electrophysiology together with MIC (Minimum Inhibitory Concentration) determination, high-resolution protein crystal structure and all atom MD (Molecular Dynamic) simulation were used to study the interaction mechanisms of ten ß-lactam molecules. A further research question was about the effect of naturally abundant divalent ions like calcium and magnesium on fluoroquinolone molecules. Although these molecules easily chelate in presence of divalent ions, the chelation appears to be unstable at the porin lumen and the single fluoroquinolone follow the electrostatic properties at the porin lumen.
Within this thesis we have in particular applied a relatively rapid screening approach by means of membranes-on-chip. This provides the potential to study the molecule-porin interaction at single molecule level in a higher throughput manner. β-lactamase inhibitors, though have little intrinsic antibacterial activity, inhibit the activity of massive plasmid-mediated β-lactamases. These agents are normally dosed in combination with β-lactams to tackle with MDR (Multi-Drug Resistant) bacteria. We found that the combinations of β-lactam and β-lactamase inhibitor combination gave a lower interaction rate than pure ß-lactam substrates via electrophysiology characterization.
With respect to data analysis of our approach we need to break the data acquisition limitation (10 kHz). We introduced a physical model dedicated to extract the signal out of noise up to 2 MHz for the interaction between OmpF and meropenem at 40 oC. A software analysis package with user interface has been developed for the easy proceeding of the bilayer electrophysiology recordings.
Silvia successfully defended her PhD at the University of Cagliari (Physics Department) on March the 1st, 2017!
Here is a summary.
Permeability in Gram-negative bacteria: A microscopic journey
Bacteria multi-drug resistance is a challenging problem of contemporary medicine and a new molecular framework for antibiotics is needed. General bacterial porins are recognized as the main pathway for polar antibiotics, but the permeability rules are still under debate. Recent works in literature pointed the electrostatics of the channel to be responsible for its filtering mechanism, and some theoretical investigations are already reported in the literature aimed at characterizing the electrostatics inside water-filled channels.
Using Molecular dynamics simulations we revealed the electrostatic filtering mechanism for porins, using water as sensing tool. We further quantify from water polarization density inside the channel the macroscopic internal electric field inside porins. This method allowed us to put forward an ultra-coarse-grained model in which the channel is described by its cross-section area, internal electric field and electrostatic potential along the axis of diffusion. Once these three descriptors are defined, it is possible to estimate the whole free energy along the channel axis of diffusion for a molecule represented by its size, charge and electric dipole moment in a few seconds.
This model would allow to virtually screening libraries of molecules searching for hits with enhanced permeability. These results may have important implications for the formulation of a general model for antibiotics translocation, and can be taken into account for rational drug design.
Venkata successfully defended his PhD at the University of Cagliari (Physics Department) on March the 1st, 2017!
Here is a summary.
Molecular rationale behind the differential substrate specificity of homologous RND transporters in E. coli and P. aeruginosa
The discovery of medicinal antibiotics was a crucial breakthrough in the treatment of infectious diseases. Unfortunately, bacteria proved to be highly competitive by expeditiously developing various survival mechanisms to deal with most (if not all) antibiotics available today. One such highly efficient mechanism is the over expression of specific and general transporters that recognize a wide spectrum of substrates (including many chemically different antibiotics) and actively expel them out of the bacterial cell, thereby contributing to multidrug resistance. Resistance-Nodulation-Division (RND) transporters like AcrB and AcrD in Escherichia coli, and MexB and MexY in Pseudomonas aeruginosa are the most prominent multi-component drug efflux pumps exporting a wide range of substrates ranging from lipophilic to amphiphilic molecules. Despite a comparable overall sequence homology among these RND transporters of E. coli and P. aeruginosa, they exhibit varied substrate specificity, the underlying basis of which still remains elusive. In an attempt to provide better insights into the substrate-transporter complementarity underlying the recognition and transport events in both the Acr pumps of E. coli and the Mex pumps of P. aeruginosa, I performed a comparative analysis of multi-copy microsecond-long molecular dynamics simulations of the apo-forms of AcrB, AcrD, MexB and MexY transporters. To this effect, I chose a set of important physicochemical descriptors like pocket volume, molecular lipophilic potential, electrostatic potential and hydration to characterize the two putative binding pockets (Access and Deep Pockets) in these transporters. Owing to the absence of experimentally resolved structures of AcrD and MexY, I also built their atomistic models based on the high-resolution crystal structure of their closest homologues.
The results suggested that the interactions of ligands with and their affinity to these transporters arise from an interplay between physicochemical properties, such as volume, lipophilicity, electrostatic potential, and certain specific features like changes in the loop conformations, altogether tuned by the dynamics of the systems. My doctoral thesis discusses in detail the important findings from the microsecond-long MD simulations of AcrB, AcrD, MexB and MexY proteins in the absence of a bound substrate, emphasizing the molecular determinants governing the partially different substrate specificity of the two couples of proteins in E. coli and P. aeruginosa. In addition, certain key interaction types needed for a substrate to bind to its transporter and/or for a transporter to recognize its substrate are also discussed for Acr transporters in E. coli.