Supplementary Figure 4: Inward-stabilizing cross-links abolish the transport activity of VcINDY.
From: The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanism
(a) Cartoon representation of the dimeric VcINDY inward-facing crystal structure (left) and the outward-facing model (right) viewed from the membrane plane. Substrate and Na+ are shown as yellow and pink spheres. Cα-atoms of the inward-stabilizing residues are shown as black spheres (left-hand side protomers). (b) SDS-PAGE band-shift assay showing the number of free cysteines present in L60C S381C with (+) and without (-) prior treatment with HgCl2 (left panel) or CuPhen (right panel). The following protein species seen in the gels are indicated by colored arrows; unmodified VcINDY (red arrow), dimeric VcINDY (orange), singly PEGylated VcINDY (blue), and doubly PEGylated VcINDY (magenta). (c) Normalized initial rates of [3H]-succinate transport in the presence of proteoliposomes containing Cysless and L60C S381C mutant after treatment with (+) and without (-) HgCl2 and DTT. Results from triplicate datasets are shown and error bars represent S.E.M. These assays were repeated twice with the same outcome.
