Elucidating factors important for monovalent cation selectivity in enzymes: E. coli β-galactosidase as a model

The [Zn3(CitH)2] (1) (CitH4= citric acid), was dispersed in sodium lauryl sulphate (SLS) to form the micelle of SLS@[Zn3(CitH)2] (2). This material 2 was incorporated in hydrogel made by hydroxyethyl-methacrylate (HEMA), an ingredient of contact lenses, toward the formation of pHEMA@(SLS@[Zn3(CitH)2]) (3). Samples of 1 and 2 were characterized by UV-Vis, 1H-NMR, FT-IR, FT-Raman, single crystal X-ray crystallography, X-ray fluorescence analysis, atomic absorption and TG/DTA/DSC. The antibacterial activity of 1–3 as well as of SLS against Gram-positive (Staphylococcus epidermidis (St. epidermidis) and Staphylococcus aureus (St. aureus)) and Gram-negative (Pseudomonas aeruginosa (PAO1), and Escherichia coli (E. coli)) bacteria was evaluated by the means of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and inhibitory zone (IZ). 2 showed 10 to 20-fold higher activity than 1 against the bacteria tested. Moreover the 3 decreases the abundance of Gram-positive microbes up to 30% (St. aureus) and up to 20% (PAO1) the Gram-negative ones. The noteworthy antimicrobial activity of the obtained composite 3 suggests an effective antimicrobial additive for infection-free contact lenses.

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Probing the active site of the sugar isomerase domain from E. coli arabinose-5-phosphate isomerase via X-ray crystallography

Protein Science ◽

10.1002/pro.525 ◽

2010 ◽

Vol 19 (12) ◽

pp. 2430-2439 ◽

Cited By ~ 13

Author(s):

Louise J. Gourlay ◽

Silvia Sommaruga ◽

Marco Nardini ◽

Paola Sperandeo ◽

Gianni Dehò ◽

...

Keyword(s):

Active Site ◽

X Ray ◽

E Coli ◽

X Ray Crystallography

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A practical method for efficient and optimal production of selenomethionine-labeled recombinant protein complexes in the insect cells

10.1101/491548 ◽

2018 ◽

Author(s):

Sabine Wenzel ◽

Tsuyoshi Imasaki ◽

Yuichiro Takagi

Keyword(s):

Recombinant Protein ◽

Recombinant Proteins ◽

Model Building ◽

Insect Cells ◽

Protein Complexes ◽

Practical Method ◽

Optimal Production ◽

X Ray ◽

E Coli ◽

X Ray Crystallography

AbstractThe use of Selenomethionine (SeMet) incorporated protein crystals for single or multiwavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X-ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in E. coli. In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet-incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allows for incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60-90% compared to native protein expression.

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Structure of theEscherichia coliRNA polymerase α subunit C-terminal domain

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444910018470 ◽

2010 ◽

Vol 66 (7) ◽

pp. 806-812 ◽

Cited By ~ 6

Author(s):

Samuel Lara-González ◽

Jens J. Birktoft ◽

Catherine L. Lawson

Keyword(s):

Transcription Activation ◽

Reductive Methylation ◽

Α Subunit ◽

Subunit C ◽

X Ray ◽

E Coli ◽

X Ray Crystallography ◽

Terminal Domain ◽

Dimerization Interface ◽

Coordinate Model

The α subunit C-terminal domain (αCTD) of RNA polymerase (RNAP) is a key element in transcription activation inEscherichia coli, possessing determinants responsible for the interaction of RNAP with DNA and with transcription factors. Here, the crystal structure ofE. coliαCTD (α subunit residues 245–329) determined to 2.0 Å resolution is reported. Crystals were obtained after reductive methylation of the recombinantly expressed domain. The crystals belonged to space groupP21and possessed both pseudo-translational symmetry and pseudo-merohedral twinning. The refined coordinate model (Rfactor = 0.193,Rfree= 0.236) has improved geometry compared with prior lower resolution determinations of the αCTD structure [Jeonet al.(1995),Science,270, 1495–1497; Benoffet al.(2002),Science,297, 1562–1566]. An extensive dimerization interface formed primarily by N- and C-terminal residues is also observed. The new coordinates will facilitate the improved modeling of αCTD-containing multi-component complexes visualized at lower resolution using X-ray crystallography and electron-microscopy reconstruction.

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The organomercurial lyase Merb possesses unique metal-binding properties

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314083193 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1680-C1680

Author(s):

Haytham Wahba ◽

Ahmed Mansour ◽

Julien Vanasse ◽

Laurent Cappadocia ◽

Jurgen Sygusch ◽

...

Keyword(s):

Heavy Metals ◽

Metal Binding ◽

Catalytic Triad ◽

Bacterial Strains ◽

Paramagnetic Resonance ◽

Mer Operon ◽

X Ray ◽

E Coli ◽

X Ray Crystallography ◽

Organomercurial Lyase

Select bacterial strains survive in mercury-contaminated environments due to acquisition of a transferable genetic element known as the mer operon. The mer operon typically encodes for a series of proteins that includes two enzymes, MerA and MerB. The organomercurial lyase (MerB) cleaves carbon-mercury bonds of organomercurial compounds yielding ionic mercury Hg (II) and a reduced-carbon compound. The Hg (II) ion product remains bounds until it is shuttled directly to the mercuric ion reductase (MerA) to be reduced. Based on NMR spectroscopy and X-ray crystallography studies1, we have determined that Cys96, Asp99 and Cys159 of E. Coli MerB form a catalytic triad required for cleavage of the carbon-Hg bond and binding of the Hg (II) ion product. The three catalytic residues are conserved in 61 of 65 known variants of MerB and the four remaining variants retain both cysteine residues, but contain a serine in place of Asp99. Given its unique activity, we have examined the role of serine as a catalytic residue and the ability of MerB to cleave other organometals such as organotin (known substrates or inhibitors) and organolead compounds. Soaking MerB crystals with either dimethyltindibromide or trimethylleadchloride compound indicates that MerB crystals have the capacity to cleave both carbon-Sn and carbon-Pb bonds, and we have determined crystal structures of a MerB-Sn and a MerB-Pb complex. Furthermore, substitution of Ser for Asp99 (MerB D99S) in E. coli MerB alters the metal-binding specificity, as MerB D99S chelated an unknown metal during its purification. X-ray crystallography, ICP-MS and electron paramagnetic resonance (EPR) studies were performed to identify the unknown metal and the results of these studies will be presented. Given that mercury contaminated sites are often contaminated with other heavy metals, these studies indicate that other heavy metals may have important implications when using MerA and MerB in bioremediation of organomercurial compounds.

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Structural details of the binding of guanosine diphosphate to elongation factor Tu from E. coli as studied by X-ray crystallography.

The EMBO Journal ◽

10.1002/j.1460-2075.1985.tb03943.x ◽

1985 ◽

Vol 4 (9) ◽

pp. 2385-2388 ◽

Cited By ~ 306

Author(s):

T.F. la Cour ◽

J. Nyborg ◽

S. Thirup ◽

B.F. Clark

Keyword(s):

Elongation Factor ◽

Elongation Factor Tu ◽

X Ray ◽

E Coli ◽

X Ray Crystallography ◽

Guanosine Diphosphate ◽

Structural Details

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Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Journal of Visualized Experiments ◽

10.3791/53845 ◽

2016 ◽

Author(s):

Mila M. Leuthold ◽

Anna D. Koromyslova ◽

Bishal K. Singh ◽

Grant S. Hansman

Keyword(s):

Human Norovirus ◽

X Ray ◽

E Coli ◽

X Ray Crystallography

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Self-Assembled Synthesis and Characterization of Novel [Co(1-vinylimidazole)6].NO3 Polymer: Highly Efficient Antimicrobial Agent

Journal of the chemical society of pakistan ◽

10.52568/000973/jcsp/43.06.2021 ◽

2021 ◽

Vol 43 (6) ◽

pp. 706-706

Author(s):

Fouzia Chang Fouzia Chang ◽

Najma Memon Najma Memon ◽

Shahabuddin Memon Shahabuddin Memon ◽

Ayaz Ali Memon Ayaz Ali Memon ◽

Saddam Hussain Bughio Saddam Hussain Bughio ◽

...

Keyword(s):

Cobalt Atom ◽

Diffusion Method ◽

Cobalt Metal ◽

Orthorhombic Unit Cell ◽

Disk Diffusion Method ◽

X Ray ◽

Cell Parameters ◽

E Coli ◽

X Ray Crystallography ◽

Vis Spectroscopy

Cobalt coordination polymer i.e. [Co(1-VI)6]and#183;NO3 was successfully synthesized with cobalt metal and (VI = vinylimidazole) and characterized by elemental analysis, FTIR, UV/Vis spectroscopy and X-ray crystallography. The structure of the compound was determined by single X-ray crystallography at temperature 296 K with a Bruker APEX II CCD diffractometer using Mo-Kα radiations (λ = 0.71073 and#197;), R = 0.0642 and 0.0989. Orthorhombic unit cell parameters are a = 16.1341 (6) and#197;, b = 16.5179(16) and#197;, c = 18.2664(16) and#197;, V= 4868.0 (8) and#197;, Dx = 4, Mr = 937.91. The X-ray crystallography studies showed that the compound is polymeric in nature, in which cobalt atom coordinated with six N atoms of the 1-vinylimidazole ligands in a distorted orthorhombic geometry. As-prepared compound was screened in vitro against a variety of microorganisms, such as Gram-positive (G +ve) Staphylococcusaureus (S. aureus) and Gram-negative (G -ve) Escherichia coli (E. coli) bacterial strains, and fungal species with Aspergillus niger (A. niger) and Rhizopus stolonifer(R.stolonifer)by applying disk diffusion method. MIC (minimum inhibitory concentration) value for prepared [Co(1-VI)6]and#183;NO3 compound is 125 μg.mL-1a this MIC value cobalt polymer showed higher activity against E. coli and S. aureus () as compared to fungi including; R. StoloniferandA. Niger at 125 μg.mL-1).This study helps to reduce the risk of infectious diseases caused by various microorganisms.

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