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Gram-negative porins are the main entry for small hydrophilic molecules. We studied translocation of structurally related cephalosporins, ceftazidime (CAZ), cefotaxime (CTX) and cefepime (FEP). CAZ is highly active on E. coli producing OmpF (Outer membrane protein F) but less efficient on cells expressing OmpC (Outer membrane protein C), whereas FEP and CTX kill bacteria regardless of the porin expressed. This matches with the different capacity of CAZ and FEP to accumulate into bacterial cells as quantified by LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry). Furthermore, porin reconstitution into planar lipid bilayer and zero current assays suggest permeation of ≈1,000 molecules of CAZ per sec and per channel through OmpF versus ≈500 through OmpC. Here, the instant killing is directly correlated to internal drug concentration. We propose that the net negative charge of CAZ represents a key advantage for permeation through OmpF porins that are less cation-selective than OmpC. These data could explain the decreased susceptibility to some cephalosporins of enterobacteria that exclusively express OmpC porins. The translocation of cephalosporins across enterobacterial OmpF and OmpC channels is monitored in real-time, demonstrating differential permeation of some cephalosporins through OmpF and OmpC. [ABSTRACT FROM AUTHOR]

This review explores various aspects of the interaction between microtubule targeting agents and tubulin, including binding site, affinity, and drug resistance. Starting with the basics of tubulin polymerization and microtubule targeting agent binding, we then highlight how the three-dimensional structures of drug–tubulin complexes obtained on stabilized tubulin are seeded by precise biological and biophysical data. New avenues opened by thermodynamics analysis, high throughput screening, and proteomics for the molecular pharmacology of these drugs are presented. The amount of data generated by biophysical, proteomic and cellular techniques shed more light onto the microtubule–tubulin equilibrium and tubulin–drug interaction. Combining these approaches provides new insight into the mechanism of action of known microtubule interacting agents and rapid in-depth characterization of next generation molecules targeting the interaction between microtubules and associated modulators of their dynamics. This will facilitate the design of improved and/or alternative chemotherapies targeting the microtubule cytoskeleton. [ABSTRACT FROM AUTHOR]

Smooth muscle thin filaments are made up of actin, tropomyosin, the inhibitory protein caldesmon and a Ca2+-binding protein. Thin filament activation of myosin MgATPase is Ca2+-regulated but thin filaments assembled from smooth muscle actin, tropomyosin and caldesmon plus brain or aorta calmodulin are not Ca2+-regulated at 25°C/50 mM KCl. We isolated the Ca2+-binding protein (CaBP) from smooth muscle thin filaments by DEAE fast-flow chromatography in 6 M urea and phenyl sepharose chromatography using sheep aorta as our starting material. CaBP combines with smooth muscle actin, tropomyosin and caldesmon to reconstitute a normally regulated thin filament at 25°C/50 mM KCl. It reverses caldesmon inhibition at pCa5 under conditions where CaM is largely inactive, it binds to caldesmon when complexed with actin and tropomyosin rather than displacing it and it binds to caldesmon independently of [Ca2+]. Amino acid sequencing, and electrospray mass spectrometry show the CaBP is identical to CaM. Structural probes indicate it is different: calmodulin increases caldesmon tryptophan fluorescence but CaBP does not. The distribution of charged species in electrospray mass spectrometry and nozzle skimmer fragmentation patterns are different indicating a less stable N-terminal lobe for CaBP. Brief heating abolishes these special properties of the CaBP. Mass spectrometry in aqueous buffer showed no evidence for the presence of any covalent or non-covalently bound adduct. The only remaining conclusion is that CaBP is calmodulin locked in a metastable altered state. [ABSTRACT FROM AUTHOR]