Structural and Functional Characterization of a His-Tagged PhoE Pore Protein ofEscherichia coli

Gelder, Patrick Van; Steiert, Matthias; Khattabi, Mohamed El; Rosenbusch, Jürg P.; Tommassen, Jan

doi:10.1006/bbrc.1996.1894

Cited by 18 publications

(12 citation statements)

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“…S1). This construct was engineered by the GenScript Corporation (GenScript USA Inc.) and is similar to that for PhoE (Gelder et al, 1996). Mutants were constructed using Quikchange site-directed mutagenesis (Agilent), Bacteria were grown at 37°C in Luria-Bertani (LB) broth at 37 °C, supplemented with kanamycin (30 g/ml).…”

Section: Methodsmentioning

confidence: 99%

The Binding of Antibiotics in OmpF Porin

2013

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The structure of OmpF porin in complex with three common antibiotics (zwitterionic ampicillin, anionic ertapenem, and di-anionic carbenicillin) was determined using X-ray crystallography. The three antibiotics are found to bind within the extracellular and periplasmic pore vestibules, away from the narrow OmpF constriction zone. Using the X-ray structures as a starting point, non-equilibrium MD simulations with an applied membrane voltage show that ionic current through the OmpF channel is blocked with bound ampicillin, but not with bound carbenicillin. The susceptibility of E. coli expressing OmpF mutants to ampicillin and carbenicillin was also experimentally characterized using microbiological assays. These results show that general diffusion by OmpF porins allows for transfer of molecules with varied charged states and give new insights into the design of more efficient antibiotics. A better understanding of this mechanism will shed light on nature's way of devising channels able to enhance the transport of molecules through membranes.

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Section: Methodsmentioning

confidence: 99%

The Binding of Antibiotics in OmpF Porin

2013

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“…2A). As has been done to investigate various types of OM proteins, including other iron transporters (22,23,50,89,106,122,127,130), we introduced a His 6 -tagged version of LbtU into 130b and then used anti-His antibodies and immunoblot analysis to track the location of LbtU in cellular fractions. LbtU-His 6 did not diminish the growth of the wild type and complemented an iron-related growth defect of an lbtU mutant (see below, Fig.…”

Section: Vol 193 2011mentioning

confidence: 99%

Legionella pneumophila LbtU Acts as a Novel, TonB-Independent Receptor for the Legiobactin Siderophore

et al. 2011

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Gram-negative Legionella pneumophila produces a siderophore (legiobactin) that promotes lung infection. We previously determined that lbtA and lbtB are required for the synthesis and secretion of legiobactin. DNA sequence and reverse transcription-PCR (RT-PCR) analyses now reveal the presence of an iron-repressed gene (lbtU) directly upstream of the lbtAB-containing operon. In silico analysis predicted that LbtU is an outer membrane protein consisting of a 16-stranded transmembrane ␤-barrel, multiple extracellular domains, and short periplasmic tails. Immunoblot analysis of cell fractions confirmed an outer membrane location for LbtU. Although replicating normally in standard media, lbtU mutants, like lbtA mutants, were impaired for growth on iron-depleted agar media. While producing typical levels of legiobactin, lbtU mutants were unable to use supplied legiobactin to stimulate growth on iron-depleted media and displayed an inability to take up iron. Complemented lbtU mutants behaved as the wild type did. The lbtU mutants were also impaired for infection in a legiobactin-dependent manner. Together, these data indicate that LbtU is involved in the uptake of legiobactin and, based upon its location, is most likely the Legionella siderophore receptor. The sequence and predicted two-dimensional (2D) and 3D structures of LbtU were distinct from those of all known siderophore receptors, which generally contain a 22-stranded ␤-barrel and an extended N terminus that binds TonB in order to transduce energy from the inner membrane. This observation coupled with the fact that L. pneumophila does not encode TonB suggests that LbtU is a new type of receptor that participates in a form of iron uptake that is mechanistically distinct from the existing paradigm.

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“…For example, the addition of several hisitidine residues allows an efficient purification of the modified protein by metal chelate affinity chromatography (7)(8)(9)(10). In recent years, this technology has been widely applied for the purification of different types of proteins from several host systems: bacteria, yeast, insect, and mammalian cells (11)(12)(13)(14). The addition of a small His-tag has advantages with respect to the other fusion protein systems.…”

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confidence: 99%

“…Only 0.84 kDa is added to the mass of the protein by the cloning of a 6-histidine tag to the N-or C-terminus of the protein, whereas other fusion protein systems utilize larger affinity groups that must often be removed to allow normal protein function. His-tag fusion proteins typically retain their normal biological function (11)(12)(13)(14).…”

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confidence: 99%