3/15/2023 0 Comments Parallels transporter torrent![]() ![]() The projection map calculated from unstained two-dimensional crystals revealed the structure of AdiC-W293L at 6.5 Å resolution. Reconstitution experiments yielded first well ordered two-dimensional crystals of AdiC-W293L suitable for structure analysis by cryo-TEM. Negative-stain TEM and blue native-polyacrylamide gel electrophoresis (BN-PAGE) of both detergent-solubilized AdiC proteins revealed a homodimeric state. coli and purified both forms to homogeneity. To grow two-dimensional crystals, we overexpressed AdiC and the mutant AdiC-W293L in E. This assignment was further supported by the different projection structure of AdiC, as obtained by cryo-TEM of two-dimensional crystals, compared with that of the oxalate/formate antiporter from Oxalobacter formigenes, a typical member from the MFS. Phylogenetic analysis indicated that AdiC belongs to the APC superfamily and not to the MFS. Here we present for the first time structural data at subnanometer resolution of the l-arginine/agmatine antiporter AdiC. Thus, two-dimensional crystals of AdiC-W293L yielded the first detailed view of a transport protein from the APC superfamily at sub-nanometer resolution. Comparison of AdiC-W293L with the projection map of the oxalate/formate antiporter from Oxalobacter formigenes, a member from the major facilitator superfamily, indicated different structures. The projection map showed 10–12 density peaks per monomer and suggested mainly tilted helices with the exception of one distinct perpendicular membrane spanning α-helix. Reconstitution experiments yielded two-dimensional crystals of AdiC-W293L diffracting beyond 6 Å resolution from which we determined the projection structure at 6.5 Å resolution. Purified detergent-solubilized AdiC particles were dimeric. For functional and structural characterization, we cloned, overexpressed, and purified wild-type AdiC and the point mutant AdiC-W293L, which is unable to bind and consequently transport l-arginine. Phylogenetic analysis indicated that AdiC belongs to the amino acid/polyamine/organocation (APC) transporter superfamily having sequence identities of 15–17% to eukaryotic and human APC transporters. The l-arginine/agmatine antiporter AdiC is a key component of the arginine-dependent extreme acid resistance system of Escherichia coli. Glycobiology and Extracellular Matrices.
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