e. coli extract polar  (Avanti Polar)

Journal: bioRxiv

Article Title: Mechanism of phospholipid transport to the bacterial outer membrane by TAM

doi: 10.64898/2026.03.22.713439

Figure Lengend Snippet: ( a ) Structure of TAM predicted using AlphaFold2 and depicted embedded into both the inner membrane (IM) and outer membrane (OM). POTRA and β-barrel domains of TamA (yellow), and the location of an N-terminal His-tag, are indicated. The conserved TamB domain of unknown function 490 (DUF490) is colored light-green and the remaining N-terminal region of TamB is grey. The location of a Twin-StrepII (TS)-tag introduced into TamB is indicated. ( b ) E. coli BL21 cultures expressing His TamAB, His TamAB TS and BamABCDE, His TamAB490 TS and BamABCDE, or possessing an empty pTrc99a plasmid were induced with 0.4 mM IPTG at 25 ℃ for 1h. Total cell protein was probed by western immunoblotting using αTamA, αTamB, or αStrepII and culture density (OD 600 ) was recorded that this time point (n = 3). TAM subunit expression in uninduced samples was also probed by immunoblot (Fig. S1). Asterisk, putative TamA prior to signal peptide cleavage. Statistical tests are in Table S2. ( c ) His TamAB490 TS purified in LMNG detergent (left) or reconstituted into E. coli phospholipid membrane nanodiscs with membrane scaffold protein 1D1 (MSP1D1) (right) was resolved by SDS-PAGE. ( d ) Cryo-EM map of His TamAB490 TS in LMNG detergent (3.5 Å average resolution). His TamA and TamB490 TS are colored orange and dark-green respectively. ( e ) Cryo-EM map of His TamAB490 TS in phospholipid nanodiscs (left, 3.7 Å average resolution) and model (right). ( f ) Comparison of His TamAB490 TS -nanodisc (orange, dark-green) to the crystal structure of TamA alone (white, PDB ID: 4C00 ). Models were aligned on TamA β-barrel α-carbons Y440-L577. The difference in the position of α-carbon Y274 (red sphere, TamA β1) between each model was measured. ( g ) As in f except His TamAB490 TS -nanodisc and TamAB His -amphipol (TamA, cream; TamB, teal-green; PDB ID: 9XDC ). Additional comparisons in Fig. S8 and S9.

Article Snippet: A chloroform solution of E. coli polar phospholipid extract (Avanti) was dried under a N 2 stream for 20 min followed by drying in a vacuum desiccator overnight.

Techniques: Membrane, Expressing, Plasmid Preparation, Western Blot, Purification, SDS Page, Cryo-EM Sample Prep, Comparison, Cream

Journal: bioRxiv

Article Title: Mechanism of phospholipid transport to the bacterial outer membrane by TAM

doi: 10.64898/2026.03.22.713439

Figure Lengend Snippet: ( a ) Magnified His TamAB490 TS -nanodisc structure showing hybridization interface between TamA β-strand 1 (β1) and TamB DUF490 C-terminal β-signal (βS) strand. Register-paired lumen-facing residues that are substituted for cysteine in experiments in b and f are indicated by matching colors, except for T270 (grey) which is membrane-facing. ( b ) E. coli expressing wild-type (WT) His TamAB TS , or derivatives with cysteine-pair substitutions at the indicated residues, were mock treated (Ox -) or treated with 200 µM 4-DPS (Ox +), n = 2. Empty pTrc99a as vector control. Intermolecular disulfide-bonds (•) in total cell protein extracts were detected by double-immunoblotting with antibodies against TamA (αTamA) and the TS-tag in TamB (αStrepII). See Fig. S10 and S11 for reduced sample and single-cysteine substitution controls, respectively. ( c ) Efficiency of plating assay. Serial dilutions of WT or mutant derivatives of E. coli K-12 W3110 were spotted onto plain LB agar plates or plates containing 5 µg/mL vancomycin, n = 3. See Fig. S12 for results with 10 µg/mL vancomycin or with 0.2% deoxycholate. ( d ) Phospholipidomic analysis of W3110 or W3110Δ tamAB Δ yhdP outer membranes, (n = 4). d i , Abundance of phospholipid classes phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL), and lysophosphatidylethanolamine (LPE) possessing species with significant differences as in d ii . d ii , Volcano plot showing significant fold-changes in abundance of specific phospholipid species between strains (above the red dashed line is significant with false discovery rate correction). Letters correspond to the same species in d ii and Fig 5c. d ii , Abundances of all significantly different phospholipid species. See Table S3 for all identified lipid species ( e ) Experiment as in c except with W3110 Δ tamAB Δ yhdP or Δ tamAB Δ yhdP Δ rcsF strains spotted onto plain LB agar plates or plates containing 10 µg/mL vancomycin, n = 3. ( f ) Experiment as in c except with W3110 Δ tamAB Δ yhdP complemented with empty pTrc99a or harboring genes for expression of His TamAB TS , or derivatives with cysteine-pair substitutions at positions as in a , and spotted onto plates containing 50 µM 4-DPS and the absence or presence of 20 µg/mL vancomycin, n = 3. See Fig. S16 for different treatment concentrations of vancomycin, or 0.2% deoxycholate, and no-4-DPS controls.

Article Snippet: A chloroform solution of E. coli polar phospholipid extract (Avanti) was dried under a N 2 stream for 20 min followed by drying in a vacuum desiccator overnight.

Techniques: Hybridization, Membrane, Expressing, Plasmid Preparation, Control, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Mechanism of phospholipid transport to the bacterial outer membrane by TAM

doi: 10.64898/2026.03.22.713439

Figure Lengend Snippet: ( a ) Far left, His TamAB490 TS -nanodisc structure with views aligned to the TamB lipophilic β-taco. Left, His TamAB490 TS -nanodisc cryo-EM map with vertical slice across the TamB lipophilic β-taco revealing potential lipid densities (grey). Additional obstructing densities are transparent. Map was sharpened with LocScale2. See Fig. S21 and Videos S16-18 for cryo-EM map comparisons of sharpening methods and 3DVA analysis of rivulet of additional dynamic densities in the β-taco. Middle, model showing TamB β -taco surface hydrophobicity (blue, hydrophilic; brown, hydrophobic). TamA is transparent. Right, model showing TamB β -taco surface electrostatics (red, negative; blue, positive; white, neutral). Dashed box indicates channel terminus and the position of TamB I1102 (purple outline). Far right, magnified view of channel terminus, αH1-3 locations (2 and 3 transparent), and orientation of I1102. ( b ) Efficiency of plating experiment as in except Δ tamAB Δ yhdP strain complemented with empty pTrc99a or harboring genes for expression of His TamAB TS , or His TamAB I1102R TS . See Fig. S22 for 0.2% deoxycholate treatment condition. ( c ) Phospholipidomic experiment as in except that outer membrane phospholipids from plasmid complemented strains in b were analyzed, (n = 4). c i , Volcano plot showing significant fold-changes in abundance of specific phospholipid species in the absence of TamAB (vector) relative to the presence of WT His TamAB TS . Letters correspond to the same species in c ii and . c ii , as in c i except comparing His TamAB I1102R TS to His TamAB TS . c iii , Abundances of all significantly different phospholipid species. See Table S4 for all identified lipid species. c vi , Sum of abundances of all significantly different phospholipid species into classes. ( d ) Molecular mechanism of TAM-mediated phospholipid transport to the bacterial OM. Phospholipids enter the N-terminus of the TamB β-taco, move towards the OM through DUF490, and are released into the outer membrane through a reaction that requires the dynamicity of the conserved amphipathic α-helices.

Article Snippet: A chloroform solution of E. coli polar phospholipid extract (Avanti) was dried under a N 2 stream for 20 min followed by drying in a vacuum desiccator overnight.

Techniques: Cryo-EM Sample Prep, Expressing, Membrane, Plasmid Preparation

Journal: bioRxiv

Article Title: Structural Basis of Membrane Potential Coupled Vectorial CO 2 Hydration by the DAB2 Complex in Chemolithoautotrophs

doi: 10.64898/2026.03.13.711513

Figure Lengend Snippet: a Construct designs for purification of the wild-type DabA2B2 complex and the fusion variant (Dab2; top). TS: twin-strep tag. Both DabA2B2 and Dab2 were able to complement CA deficient Escherichia coli under low CO 2 condition (0.04%) to a similar extend (bottom). Strain transformed with empty plasmid pET24d was used as a negative control. Data points and error bars represent means and standard deviations, respectively (n = 4 biological replicates). b SDS-PAGE of purified proteins after size exclusion chromatography. c Superposition of AlphaFold predicted model of DabA2B2 (gray) on the Dab2 fusion protein (colored as in ; RMSD = 1.17 Å). d Size-exclusion chromatograms of the purified proteins in 0.03% DDM and after nanodisc (MSP1D1) reconstitution. Fractions under the gray area were collected.

Article Snippet: Chloroform dissolved E. coli polar lipid extract (Avanti Research) was evaporated under a gentle stream of nitrogen to from a thin lipid film.

Techniques: Construct, Purification, Variant Assay, Strep-tag, Transformation Assay, Plasmid Preparation, Negative Control, SDS Page, Size-exclusion Chromatography

Journal: bioRxiv

Article Title: Structural Basis of Membrane Potential Coupled Vectorial CO 2 Hydration by the DAB2 Complex in Chemolithoautotrophs

doi: 10.64898/2026.03.13.711513

Figure Lengend Snippet: a Structural comparison between Dab2 and representative β-CAs, shown in top and side views. DabA2 catalytic domain was characterized by two β-CA-like Rossmann folds, colored by secondary structure (orange: α-helix; cyan: β-sheet). The first fold consisted of four parallel β-sheets in the order of 2-1-3-4 (β2-β1-β7-β8) and an antiparallel sheet β9. β3-β6 formed the scaffold for the “finger-like” motifs . The second fold had an additional anti-parallel β-sheets, arranged in the ordered of β11-β10-β14-β15-β16-β17. β12 and β13 formed part of the interacting interface with DabB2. b Structure-based sequence alignment of DabA2 and β-CAs. Rv1284: Mycobacterium tuberculosis β-CA (PDB 1YLK); CsoSCA: Halothiobacillus neapolitanus β-CA (PDB 2FGY); PSCA: Pisum sativum β-CA (PDB 1EKJ); PACA: Pseudomonas aeruginosa β-CA (PDB 5BQ1); ECCA: E. coli β-CA (PDB 2ESF) HICA: Haemophilus influenzae β-CA (PDB 2A8D). Alignment is visualized in Jalview, and colored by the Clustal coloring scheme. Strictly conserved residues are marked by red triangles. DabA2 residues number and secondary structure are shown above the alignment. Un-aligned regions are trimmed and represented by dotted lines.

Article Snippet: Chloroform dissolved E. coli polar lipid extract (Avanti Research) was evaporated under a gentle stream of nitrogen to from a thin lipid film.

Techniques: Comparison, Sequencing

Journal: bioRxiv

Article Title: Structural Basis of Membrane Potential Coupled Vectorial CO 2 Hydration by the DAB2 Complex in Chemolithoautotrophs

doi: 10.64898/2026.03.13.711513

Figure Lengend Snippet: Time-resolved “CO 2 -minus-N 2 ” ATR FTIR difference spectra for E. coli β-CA (ECCA, black), BSA (red), and Dab2 (blue). a After 25 s in the presence of 10% gaseous CO 2 , bands at 2341 and 2337 cm -1 indicate the presence of dissolved CO 2 or protein-bound CO 2 , respectively. b At lower frequencies, the increasingly more positive bands at 1614, 1360, and 1302 cm⁻¹ revealed HCO 3 − formation. All spectra run from light color (t = 5 s) to full color (t = 25 s). c Kinetics of CO 2 hydration with ECCA, BSA, and Dab2 . The arrow marks the switch from 100% N 2 to 90% N 2 and 10% CO 2 (t 0 ). Spectra in panel b relate to the time frame between 5–25 s highlighted in panel c .

Article Snippet: Chloroform dissolved E. coli polar lipid extract (Avanti Research) was evaporated under a gentle stream of nitrogen to from a thin lipid film.

Techniques:

Journal: bioRxiv

Article Title: Structural Basis of Membrane Potential Coupled Vectorial CO 2 Hydration by the DAB2 Complex in Chemolithoautotrophs

doi: 10.64898/2026.03.13.711513

Figure Lengend Snippet: Effect of L658 substitution on DAB2 activity . a Representative results of CO 2 hydration activity measured as a function of pH reduction, indicated by phenol red absorbance at 558 nm (n = 3 technical replicates). 5 nM Bovine carbonic anhydrase II (CA-II) was used as a positive control. Both Dab2 WT (500 nM) and L658Q variant (500 nM) showed similar background CO 2 hydration as the negative control (Buffer). b Substitutions of Leu658 did not impair DAB2 ability to complement CA deficient E. coli . Bar heights and error bars represent means and standard deviations, respectively (n = 4 biological replicates). “**” Indicates statistically significant difference compared to WT (P < 0.05) according to Holm-Bonferroni corrected two-tailed t-test.

Article Snippet: Chloroform dissolved E. coli polar lipid extract (Avanti Research) was evaporated under a gentle stream of nitrogen to from a thin lipid film.

Techniques: Activity Assay, Positive Control, Variant Assay, Negative Control, Two Tailed Test

Journal: bioRxiv

Article Title: Structural Basis of Membrane Potential Coupled Vectorial CO 2 Hydration by the DAB2 Complex in Chemolithoautotrophs

doi: 10.64898/2026.03.13.711513

Figure Lengend Snippet: MUSCLE alignment of DabB2 transmembrane helixes TM1 to TM11 on Complex I (-like) distal proton-pumping subunits from Homo sapiens , Escherichia coli , Thermus thermophilus and Thermosynechococcus vestitus . Residues are colored by the Clustal coloring scheme. Regulatory ion-pairs and residues likely involved in proton transfer are marked by black and red triangle respectively. DabA2 residues number and secondary structure are shown above the alignment.

Article Snippet: Chloroform dissolved E. coli polar lipid extract (Avanti Research) was evaporated under a gentle stream of nitrogen to from a thin lipid film.

Techniques:

Journal: bioRxiv

Article Title: Structural Basis of Membrane Potential Coupled Vectorial CO 2 Hydration by the DAB2 Complex in Chemolithoautotrophs

doi: 10.64898/2026.03.13.711513

Figure Lengend Snippet: a DAB2 did not restore growth of E. coli lacking both NhaA and NhaB sodium transporters under sodium stress (0.1 M). Wild-type E. coli MG1655 was used as a positive control. b Complementation of CA deficient E. coli by DAB2 in M9 medium prepared with potassium salts (M9-K) or sodium salts (M9-Na). The similar growth profile suggests the complex might not require sodium. Data points and error bars represent means and standard deviations, respectively (n = 4 biological replicates).

Article Snippet: Chloroform dissolved E. coli polar lipid extract (Avanti Research) was evaporated under a gentle stream of nitrogen to from a thin lipid film.

Techniques: Positive Control