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Title: Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization

Authors:
; ; ; ORCiD logo; ; ; ; ; ORCiD logo;
Publication Date:
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1416244
Resource Type:
Journal Article: Publisher's Accepted Manuscript
Journal Name:
Protein Expression and Purification
Additional Journal Information:
Journal Volume: 134; Journal Issue: C; Related Information: CHORUS Timestamp: 2018-01-09 17:44:09; Journal ID: ISSN 1046-5928
Publisher:
Elsevier
Country of Publication:
United States
Language:
English

Citation Formats

Niedzialkowska, Ewa, Mrugała, Beata, Rugor, Agnieszka, Czub, Mateusz P., Skotnicka, Anna, Cotelesage, Julien J. H., George, Graham N., Szaleniec, Maciej, Minor, Wladek, and Lewiński, Krzysztof. Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization. United States: N. p., 2017. Web. doi:10.1016/j.pep.2017.03.019.
Niedzialkowska, Ewa, Mrugała, Beata, Rugor, Agnieszka, Czub, Mateusz P., Skotnicka, Anna, Cotelesage, Julien J. H., George, Graham N., Szaleniec, Maciej, Minor, Wladek, & Lewiński, Krzysztof. Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization. United States. doi:10.1016/j.pep.2017.03.019.
Niedzialkowska, Ewa, Mrugała, Beata, Rugor, Agnieszka, Czub, Mateusz P., Skotnicka, Anna, Cotelesage, Julien J. H., George, Graham N., Szaleniec, Maciej, Minor, Wladek, and Lewiński, Krzysztof. Thu . "Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization". United States. doi:10.1016/j.pep.2017.03.019.
@article{osti_1416244,
title = {Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization},
author = {Niedzialkowska, Ewa and Mrugała, Beata and Rugor, Agnieszka and Czub, Mateusz P. and Skotnicka, Anna and Cotelesage, Julien J. H. and George, Graham N. and Szaleniec, Maciej and Minor, Wladek and Lewiński, Krzysztof},
abstractNote = {},
doi = {10.1016/j.pep.2017.03.019},
journal = {Protein Expression and Purification},
number = C,
volume = 134,
place = {United States},
year = {Thu Jun 01 00:00:00 EDT 2017},
month = {Thu Jun 01 00:00:00 EDT 2017}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1016/j.pep.2017.03.019

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  • Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the component responsible for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has an amino acid sequence that is 36% identical to that of Streptomyces coelicolor AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomyces genus. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Å,more » binding properties with neutral red and deoxyadenosine were surveyed, backbone dynamics were measured, and thermal stability was assayed by circular dichroism spectroscopy. The protein is composed of four approximate repeats with a βαβββ topology arranged radially in consecutive pairs to form two continuous eight-strand β-sheets capped on both ends with an α-helix. The two β-sheets intersect in the center at roughly a right angle and form two asymmetric deep “saddles” that may serve to bind ligands. Nuclear magnetic resonance chemical shift perturbation experiments show that neutral red and deoxyadenosine bind to Rv0577. Binding to deoxyadenosine is weaker with an estimated dissociation constants of 4.1 ± 0.3 mM for saddle 1. Heteronuclear steady-state { 1H}– 15N nuclear Overhauser effect, T 1, and T 2 values were generally uniform throughout the sequence with only a few modest pockets of differences. As a result, circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C.« less
  • Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretionmore » chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 {angstrom} revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.« less
  • The 261-residue Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the responsible component for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has 36% sequence identity to AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomycetes genus from which many commercial antibiotics are derived. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Åmore » (3OXH), binding properties with neutral red and deoxyadenosine (Ado) surveyed, backbone dynamics measured, and thermal stability assayed by CD spectroscopy. The protein is composed of four approximate repeats with an topology arranged radially in consecutive pairs to form two continuous eight-strand -sheets capped on both ends with an -helix. The two -sheets intersect in the center at roughly a right angle and form an asymmetric deep “saddle” on both sides of the protein, saddle one (P11 to A129) and saddle two (L143 to A258), that may serve to bind ligands. NMR chemical shift perturbation experiments show that neutral red binds to Rv0577, further cementing the role of Rv0577 in the neutral red staining of virulent strains of M. tuberculosis. Similar experiments show that adenosine also bind to Rv0577, although less tightly, with estimated dissociation constants of 4.1 ± 0.3 mM for saddle one and > 1 M for saddle two. Heteronuclear steady-state {1H}-15N NOE, T1, and T2 values were generally uniform through-out the sequence with only a few modest pockets of differences suggestive of slightly different motion in loops between -strands in saddle 1. Circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C. While it is not known if Rv0577 has a kinase regulatory role similar to its Streptomyces homolog KbpA, protein kinase and phosphatase signaling help M. tuberculosis adapt to the hostile host environment during infections. Consequently, new anti-tuberculosis drugs targeting Rv0577 may act by interfering with multiple mechanisms; a potential signaling machinery as well as toll-like receptor 2 activation and the methylglyoxal detoxification pathway.« less
  • Transmissible gastroenteritis coronavirus (TGEV) is one of the most destructive agents, responsible for the enteric infections that are lethal for suckling piglets, causing enormous economic loss to the porcine fostering industry every year. Although it has been known that TGEV spiker protein is essential for the viral entry for many years, the detail knowledge of the TGEV fusion protein core is still very limited. Here, we report that TGEV fusion core (HR1-SGGRGG-HR2), in vitro expressed in GST prokaryotic expression system, shares the typical properties of the trimer of coiled-coil heterodimer (six {alpha}-helix bundle), which has been confirmed by a combinedmore » series of biochemical and biophysical evidences including size exclusion chromatography (gel-filtration), chemical crossing, and circular diagram. The 3D homologous structure model presents its most likely structure, extremely similar to those of the coronaviruses documented. Taken together, TGEV spiker protein belongs to the class I fusion protein, characterized by the existence of two heptad-repeat (HR) regions, HR1 and HR2, and the present knowledge about the truncated TGEV fusion protein core may facilitate in the design of the small molecule or polypeptide drugs targeting the membrane fusion between TGEV and its host.« less
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