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. 2019 May 2;10:2032. doi: 10.1038/s41467-019-10001-w

Fig. 5.

Fig. 5

Generation and functional characterization of SLC26Dg-IL. a Surface representation of MD-simulated SLC26Dg clipped through the funnel toward the putative substrate-binding site. Cytoplasmic water molecules in a ~ 10 Å slab at the clipping plane are shown. Ile-45 and Ala-142 indicate the relative position of the cysteine mutants in the core (orange) and gate (gray) domain, respectively. b SDS-PAGE analysis of purified and cross-linked SLC26Dg-IL monomers in the absence and presence of DTT. Single and double stars indicate not-cross-linked and cross-linked protein, respectively. c Functional characterization of membrane-reconstituted and cross-linked SLC26Dg-IL (dark blue), wildtype SLC26Dg (orange), and both proteins mixed in equal ratio’s (pink). Closed and open symbols indicate the absence and presence of a pre-incubation step with DTT. d Initial transport rates of membrane-reconstituted and cross-linked samples containing wildtype and SLC26Dg-IL mixed in different ratio’s. Dark blue, pink, and orange dashed curves indicate the anticipated curves assuming an activity of the heterodimers corresponding to 0, 50, and 100% of the wildtype homodimers. These models were calculated assuming stochastic dimer formation (e.g., mixing WT:IL protomers in a 50:50 ratio results in 25% WT–WT, 50% WT–IL, and 25% IL–IL dimers) and specific transport activities of 32.3 or 6.8 nmol fumarate per mg WT or IL homodimer per min, respectively, and heterodimer activities corresponding to 0, 50, or 100% of WT homodimer. Data points represent mean and standard deviations of three technical replicates. Source data are provided as a Source Data file