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An extracellular domain of the EsaA membrane component of the type VIIb secretion system: expression, purification and crystallization

The membrane protein EsaA is a conserved component of the type VIIb secretion system. Limited proteolysis of purified EsaA from Staphylococcus aureus USA300 identified a stable 48 kDa fragment, which was mapped by fingerprint mass spectrometry to an uncharacterized extracellular segment of EsaA. Ana...

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Detalles Bibliográficos
Autores principales: Mietrach, Nicole, Schlosser, Andreas, Geibel, Sebastian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: International Union of Crystallography 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891578/
https://www.ncbi.nlm.nih.gov/pubmed/31797813
http://dx.doi.org/10.1107/S2053230X1901495X
Descripción
Sumario:The membrane protein EsaA is a conserved component of the type VIIb secretion system. Limited proteolysis of purified EsaA from Staphylococcus aureus USA300 identified a stable 48 kDa fragment, which was mapped by fingerprint mass spectrometry to an uncharacterized extracellular segment of EsaA. Analysis by circular dichroism spectroscopy showed that this fragment folds into a single stable domain made of mostly α-helices with a melting point of 34.5°C. Size-exclusion chromatography combined with multi-angle light scattering indicated the formation of a dimer of the purified extracellular domain. Octahedral crystals were grown in 0.2 M ammonium citrate tribasic pH 7.0, 16% PEG 3350 using the hanging-drop vapor-diffusion method. Diffraction data were analyzed to 4.0 Å resolution, showing that the crystals belonged to the enantiomorphic tetragonal space groups P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 197.5, b = 197.5, c = 368.3 Å, α = β = γ = 90°.