PubMed · 4583786
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P Régnier, M N Thang. 1973-10-01. Membrane associated proteases in E. coli.. https://doi.org/10.1016/0014-5793(73)80330-8
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The time-dependent evolution of the phase-separated microstructure of a caseinate-stabilised emulsion containing xanthan gum added before emulsification has been investigated by confocal laser scanning microscopy, image analysis and rheology. Moderately low levels of xanthan addition lead to depletion flocculation and gravity-induced phase separation. Increasing the polysaccharide concentration causes immobilisation of the microstructure due to an increase in the local viscoelasticity: that is, the emulsion structure cannot easily rearrange to expel xanthan-enriched aqueous serum phase because a weak gel-like network is generated. The effect of xanthan on the evolving microstructure of phase-separated regions, which reflects indirectly the local emulsion micro-rheology, has been estimated from image analysis of time sequences of confocal micrographs. A comparison has been made between object shape analysis using four different shape descriptors. The roundness parameter has been found to be a convenient descriptor for reliably quantifying the structural change in terms of the relaxation rate of xanthan-rich aqueous drops. The Taylor parameter has been used to link the kinetics of drop relaxation to the time-dependent small-deformation rheological behaviour. The analysis of the combined experimental data reveals the difficulty of relating the evolving microstructure to bulk rheological measurements.
The HslVU complex is a bacterial two-component ATP-dependent protease, consisting of HslU chaperone and HslV peptidase. Investigation of protein-protein interactions using SPR in Escherichia coli HslVU and the protein substrates demonstrates that HslU and HslV have moderate affinity (Kd = 1 microM) for each other. However, the affinity of HslU for HslV fivefold increased (Kd approximately 0.2 microM) after binding with the MBP approximately SulA protein indicating the formation of a "ternary complex" of HslV-HslU-MBP approximately SulA. The molecular interaction studies also revealed that HslU strongly binds to MBP approximately SulA with 10(-9) M affinity but does not associate with nonstructured casein. Conversely, HslV does not interact with the MBP-SulA whereas it strongly binds with casein (Kd = 0.2 microM) requiring an intact active site of HslV. These findings provide evidence for "substrate-induced" stable HslVU complex formation. Presumably, the binding of HslU to MBP approximately SulA stimulates a conformational change in HslU to a high-affinity form for HslV.
Tissue transglutaminase (TGase) is a Ca(2+)-dependent enzyme that catalyzes cross-linking of intracellular proteins through a mechanism that involves isopeptide bond formation between Gln and Lys residues. In addition to its transamidation activity, TGase can bind guanosine 5'-triphosphate (GTP) and does so in a manner that is antagonized by calcium. Once bound, GTP undergoes hydrolysis to form guanosine 5'-diphosphate and inorganic phosphate. TGase is thought to play a pathogenic role in neurodegenerative diseases by promoting aggregation of disease-specific proteins that accumulate in these disorders. Thus, this enzyme represents a viable target for drug discovery. We now report the development of a mechanism-based assay for TGase and the results of a screen using this assay in which we tested 56,500 drug-like molecules for their ability to inhibit TGase. In this assay, the Gln- and Lys-donating substrates are N,N-dimethylated casein (NMC) and N-Boc-Lys-NH-CH(2)-CH(2)-NH-dansyl (KXD), respectively. Through a combination of steady state kinetic experiments and reaction progress curve simulations, we were able to calculate values for the initial concentrations of NMC, KXD, and Ca(2+) that would produce a steady state situation in which all thermodynamically significant forms of substrate-bound TGase exist in equal concentration. Under these conditions, the assay is sensitive to both competitive and mixed active-site inhibitors and to inhibitors that bind to the GTP site. The assay was optimized for automated screening in 384-well format and was then used to test our compound library. From among these compounds, 104 authentic hits that represent several mechanistic classes were identified.