Journal: bioRxiv

Article Title: Biophysical characterization and analysis of a mesophilic chorismate mutase from B. pumilus

doi: 10.1101/2023.04.20.537678

Figure Lengend Snippet: Heavy atom distances between the substrate hydroxyl group on C 4 and amide protons of Cys75 (BpCM in redBsCM in cyan) throughout the course of the chorismate mutase reaction.

Article Snippet: The gene encoding the Bacillus pumilus AroH-type Chorismate mutase (uniprot A8FEK3, BpCM) with a dual C-terminal stop codon (TAATAA) was codon optimized for expression in E . coli , synthesized and subcloned into pET22b by GenScript Biotech (Netherlands) B.V.

Techniques:

Journal: Chemical Science

Article Title: Metal selectivity and translocation mechanism characterization in proteoliposomes of the transmembrane NiCoT transporter NixA from Helicobacter pylori †

doi: 10.1039/d3sc05135h

Figure Lengend Snippet: Purification of NixA and reconstitution in proteoliposomes. (A) Size exclusion chromatogram profile of NixA purified in CYMAL-7 micelles, and (B) SDS-PAGE of purified NixA after SEC, and corresponding and western blot analysis utilizing an Anti-His 6 tag monoclonal antibody. (C) Dynamic light scattering (DLS) analysis of control liposomes and NixA proteoliposomes showing the monodisperse size of SUVs and corresponding polydispersity index (PDI). (D) SDS-PAGE analysis of NixA reconstitution in the proteoliposome fraction isolated by ultracentrifugation (pellet) compared to non-incorporated soluble NixA (supernatant, SUP), and corresponding control liposomes.

Article Snippet: Synthetic DNA encoding codon-optimized wild-type NixA (wtNixA Uniprot: Q48262) from H. pylori was synthetized and cloned in a pET-52b(+) vector between the SmaI and SacI restriction sites (Genscript Inc.), generating a construct encoding a thrombin cleavage site followed by a His-10-tag at the protein C-terminus.

Techniques: Purification, SDS Page, Western Blot, Control, Liposomes, Isolation

Journal: Chemical Science

Article Title: Metal selectivity and translocation mechanism characterization in proteoliposomes of the transmembrane NiCoT transporter NixA from Helicobacter pylori †

doi: 10.1039/d3sc05135h

Figure Lengend Snippet: Transport activity of NixA monitored by FZ-3-Zn( ii ) complex. (A) Overall scheme for the characterization of NixA transport properties utilizing fluorescent probes responsive to diverse stimuli. (B) Fluorescence quenching transport traces indicative of Ni( ii ) transport by NixA in proteoliposomes, as monitored by FZ-3-Zn( ii ) encapsulated in the SUV lumen, as a function of increasing Ni( ii ) concentrations (1–100 μM; λ exc = 480 nm; λ em = 515 nm) ( n = 3). Traces are plotted as differential fluorescence at time t ( F t − F 0 ; Δ F ) normalized to the fluorescence prior to the addition of Ni( ii ) ( F 0 ), with traces corrected by subtracting the signals obtained with control liposomes. (C) Michaelis–Menten-type fitting of the maximum F change (Δ F / F 0 ) as a function of Ni( ii ) concentrations ( K M, Ni( ii ) = 26.6 ± 1.5 μM and (Δ F / F 0 ) MAX = −0.52 ± −0.01 s −1 ) inset: maximal initial Ni( ii ) transport rates in NixA proteoliposomes and corresponding fit with a Michaelis–Menten-like equation: (δ F /δ t ) = (δ F /δ t ) MAX × [Ni( ii )]/( K M + [Ni( ii )]); K M, Ni( ii ) = 31.0 ± 1.2 μM and (δ F /δ t ) MAX = −0.0089 ± −0.0004 s −1 . (D) Fluorescence quenching transport traces upon addition of Ni( ii ) and Co( ii ) (25 μM) to NixA proteoliposomes, monitored by FZ-3-Zn( ii ) probe complex, and Zn( ii ) transport traces monitored by metal-free FZ-3. Signals were corrected by subtracting the background signal of control liposomes ( n = 3). (E) Maximal initial transport rates (initial slope) obtained for control (−1.22 ± 0.05 × 10 −5 s −1 ), Ni( ii ) (−5.51 ± 0.20 × 10 −3 s −1 ), and Co( ii ) (−4.35 ± 0.24 × 10 −6 s −1 ) (25 μM) in NixA proteoliposomes monitored by FZ-3-Zn( ii ) probe, and Zn( ii ) (−2.73 ± 0.20 × 10 −5 ) (25 μM) in NixA proteoliposomes using FZ-3. Signals were corrected by subtracting the background signal of control liposomes ( n = 3).

Article Snippet: Synthetic DNA encoding codon-optimized wild-type NixA (wtNixA Uniprot: Q48262) from H. pylori was synthetized and cloned in a pET-52b(+) vector between the SmaI and SacI restriction sites (Genscript Inc.), generating a construct encoding a thrombin cleavage site followed by a His-10-tag at the protein C-terminus.

Techniques: Activity Assay, Fluorescence, Control, Liposomes

Journal: Chemical Science

Article Title: Metal selectivity and translocation mechanism characterization in proteoliposomes of the transmembrane NiCoT transporter NixA from Helicobacter pylori †

doi: 10.1039/d3sc05135h

Figure Lengend Snippet: Determination of H + transport and electrogenicity in NixA proteoliposomes. (A) Determination of H + translocation in NixA proteoliposomes upon Ni( ii ) transport (Ni( ii ) = 25 μM) monitored by tracking changes in luminal pH as a function of time, utilizing the pH indicator pyranine ( n = 3). (B) Superimposition of kinetic traces of Ni( ii ) transport monitored by FZ-3-Zn( ii ) and transmembrane potential changes monitored by oxonol VI. The kinetic traces were normalized and corrected with corresponding the control background signals ( n = 3). The close correspondence of the two traces are consistent with a positive-inside transmembrane potential generated by NixA-mediated Ni( ii ) transport.

Article Snippet: Synthetic DNA encoding codon-optimized wild-type NixA (wtNixA Uniprot: Q48262) from H. pylori was synthetized and cloned in a pET-52b(+) vector between the SmaI and SacI restriction sites (Genscript Inc.), generating a construct encoding a thrombin cleavage site followed by a His-10-tag at the protein C-terminus.

Techniques: Translocation Assay, Control, Generated

Journal: Chemical Science

Article Title: Metal selectivity and translocation mechanism characterization in proteoliposomes of the transmembrane NiCoT transporter NixA from Helicobacter pylori †

doi: 10.1039/d3sc05135h

Figure Lengend Snippet: Structural model of NixA ( H. pylori ). (A) Schematic depicting the AlphaFold model for NixA and approach to study key transmembrane residues essential for transport activity. (B) Relative position of conserved NiCoT permeases recognition motifs and the putative translocation pathway based on the AlphaFold model.

Article Snippet: Synthetic DNA encoding codon-optimized wild-type NixA (wtNixA Uniprot: Q48262) from H. pylori was synthetized and cloned in a pET-52b(+) vector between the SmaI and SacI restriction sites (Genscript Inc.), generating a construct encoding a thrombin cleavage site followed by a His-10-tag at the protein C-terminus.

Techniques: Activity Assay, Translocation Assay

Journal: Chemical Science

Article Title: Metal selectivity and translocation mechanism characterization in proteoliposomes of the transmembrane NiCoT transporter NixA from Helicobacter pylori †

doi: 10.1039/d3sc05135h

Figure Lengend Snippet: NixA mutants and relative positions of the mutations based on the AlphaFold model

Article Snippet: Synthetic DNA encoding codon-optimized wild-type NixA (wtNixA Uniprot: Q48262) from H. pylori was synthetized and cloned in a pET-52b(+) vector between the SmaI and SacI restriction sites (Genscript Inc.), generating a construct encoding a thrombin cleavage site followed by a His-10-tag at the protein C-terminus.

Techniques: Translocation Assay

Journal: Chemical Science

Article Title: Metal selectivity and translocation mechanism characterization in proteoliposomes of the transmembrane NiCoT transporter NixA from Helicobacter pylori †

doi: 10.1039/d3sc05135h

Figure Lengend Snippet: NixA mutation studies. (A) SDS-PAGE analysis of NixA and NixA combination mutants reconstituted in proteoliposomes utilized for the Ni( ii ) transport assays. (B) Maximal initial Ni( ii ) transport rates by NixA and combination mutants monitored in proteoliposomes with FZ-3-Zn( ii ) encapsulated in the SUV lumen (25 μM; λ exc = 480 nm; λ em = 515 nm) ( n = 3). (C) NixA and NixA mutants Ni( ii ) binding stoichiometry measured by ICP-MS (Ni( ii ) concentration) and Bradford assays (protein concentration) ( n = 3). (D) Ni( ii ) titration of HpNixA followed by ITC. Heat response for injections of 400 μM Ni( ii ) into 20 μM HpNixA (top); integrated heat data of the titration as a function of Ni( ii )/NixA molar ratio. The continuous line represents the best fit (goodness of fit, GoF = 78.4%) obtained with a stoichiometric equilibria model which involves two sets of metal-binding sites (bottom). (E) Real-time traces of transmembrane potential generation by NixA, NX1, and NX2 mutants in proteoliposomes upon incubation with Ni( ii ) (25 μM), monitored by encapsulating oxonol VI in the proteoliposome lumen ( n = 3). (F) SDS-PAGE analysis of NixA and single-point NixA mutants reconstituted in proteoliposomes utilized for Ni( ii ) transport assays. (G) Maximal initial Ni( ii ) transport rates in NixA and NixA point-mutants monitored in proteoliposomes with FZ-3-Zn( ii ) encapsulated in the SUV lumen (Ni( ii ) = 25 μM; λ exc = 480 nm; λ em = 515 nm) ( n = 3).

Article Snippet: Synthetic DNA encoding codon-optimized wild-type NixA (wtNixA Uniprot: Q48262) from H. pylori was synthetized and cloned in a pET-52b(+) vector between the SmaI and SacI restriction sites (Genscript Inc.), generating a construct encoding a thrombin cleavage site followed by a His-10-tag at the protein C-terminus.

Techniques: Mutagenesis, SDS Page, Binding Assay, Concentration Assay, Protein Concentration, Titration, Incubation