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