Purification and Activity Testing of the Full-Length YycFGHI Proteins of Staphylococcus aureus

Background: The YycFG two-component regulatory system (TCS) of Staphylococcus aureus represents the only essential TCS that is almost ubiquitously distributed in Gram-positive bacteria with a low G+C-content. YycG (WalK/VicK) is a sensor histidine-kinase and YycF (WalR/VicR) is the cognate response regulator. Both proteins play an important role in the biosynthesis of the cell envelope and mutations in these proteins have been involved in development of vancomycin and daptomycin resistance. Methodology/Principal Findings: Here we present high yield expression and purification of the full-length YycG and YycF proteins as well as of the auxiliary proteins YycH and YycI of Staphylococcus aureus. Activity tests of the YycG kinase and a mutated version, that harbours an Y306N exchange in its cytoplasmic PAS domain, in a detergent-micelle-model and a phosholipid-liposome-model showed kinase activity (autophosphorylation and phosphoryl group transfer to YycF) only in the presence of elevated concentrations of alkali salts. A direct comparison of the activity of the kinases in the liposomemodel indicated a higher activity of the mutated YycG kinase. Further experiments indicated that YycG responds to fluidity changes in its microenvironment. Conclusions/Significance: The combination of high yield expression, purification and activity testing of membrane and membrane-associated proteins provides an excellent experimental basis for further protein-protein interaction studies an

La Atalaya : diario de la mañana: Año XIV Número 5340 - 1906 septiembre 7

Copia digital. Madrid : Ministerio de Educación, Cultura y Deporte. Subdirección General de Coordinación Bibliotecaria, 201

Characterization of Mixed Polypeptide Colloidal Particles by Light Scattering

Temperature-dependent polymer surfactants have been developed by connecting three elastin-like polypeptide (ELP) chains to a charged protein domain (foldon), forming a three-armed star polymer. At low temperatures the polymer is soluble, while at higher temperatures it forms micelles. The behavior of mixtures of the three-armed star ELP (E20-Foldon) and H40-Linear ELP chains was analyzed under different salt and protein concentrations and various foldon to linear ELP ratio using Depolarized Dynamic Light Scattering. It was expected that under certain conditions the pure E20-Foldon would form spherical micelles, which upon adding the linear ELP would change in size and possibly shape. The pure E20-Foldon indeed formed largely spherical micelles with Rh of 10-20nm in solutions with 15-100mM salt and protein concentration between 10μM and 100μM. For the mixtures of 50μM E20-Foldon and varying concentrations of H40-Linear in 25mM of salt, it was discovered that low and high H40-Linear concentration (4μM and 50μM) had only one transition. For the mixtures with of 10 and 25μM of H40-Linear the two distinct transition temperatures were observed by spectrophotometry. The first transition corresponded to significantly elongated diffusive particles of apparent Rh of 30-50nm, while the second transition corresponded to slightly anisotropic diffusive particles with apparent Rh of about 20nm. At all H40-Linear concentrations studied, diffusive particles were seen above the second transition. Their radius and ability to depolarize light increased with the increase of H40-Linear concentration

Redox-Induced Polymerisation/Depolymerisation of Metallo-Supramolecular Polymers

Redox-responsive metallo-supramolecular polymers (MSPs), consisting of a ditopic ligand end-capped poly(tetrahydrofuran) (pTHF) macromonomer self-assembled with copper ions, have been developed where the degree of polymerization of the MSP, and therefore the viscosity of its solutions, depends on the oxidation state of the copper ion. We use a combination of UV-Vis, viscosity, light scattering, cyclic voltammetry and SAXS to show that the ligands, 2,6-bis(N-methylbenzimidazolyl)pyridine (MeBip), at the ends of the pTHF, bind copper(II) in a 2:1 ratio and copper(I) in a 2:2 ratio in solution. Thus at fixed 1:1 or 1:2 stoichiometry of macromonomer to copper ions the viscosity of their solutions shows dramatic changes in response to addition of a chemical oxidant (nitroso tetrafluoroborate) or reductant (aqueous ascorbic acid)

Characterization of Mixed Polypeptide Colloidal Particles by Light Scattering

Temperature-dependent polymer surfactants have been developed by connecting three elastin-like polypeptide (ELP) chains to a charged protein domain (foldon), forming a three-armed star polymer. At low temperatures the polymer is soluble, while at higher temperatures it forms micelles. The behavior of mixtures of the three-armed star ELP (E20-Foldon) and H40-Linear ELP chains was analyzed under different salt and protein concentrations and various foldon to linear ELP ratio using Depolarized Dynamic Light Scattering. It was expected that under certain conditions the pure E20-Foldon would form spherical micelles, which upon adding the linear ELP would change in size and possibly shape. The pure E20-Foldon indeed formed largely spherical micelles with Rh of 10-20nm in solutions with 15-100mM salt and protein concentration between 10μM and 100μM. For the mixtures of 50μM E20-Foldon and varying concentrations of H40-Linear in 25mM of salt, it was discovered that low and high H40-Linear concentration (4μM and 50μM) had only one transition. For the mixtures with of 10 and 25μM of H40-Linear the two distinct transition temperatures were observed by spectrophotometry. The first transition corresponded to significantly elongated diffusive particles of apparent Rh of 30-50nm, while the second transition corresponded to slightly anisotropic diffusive particles with apparent Rh of about 20nm. At all H40-Linear concentrations studied, diffusive particles were seen above the second transition. Their radius and ability to depolarize light increased with the increase of H40-Linear concentration

Three-phase separation in nonionic surfactant/hydrophobically modified polymer aqueous mixtures

Langmuir23209967-9973LANG