Parallels transporter download1/13/2024 ![]() For details see "Experimental Procedures." C, the protein content of the eluted fractions was analyzed using SDS-PAGE and visualized by Coomassie stain. B, elution profile of o-PDM-treated EmrE T108C on a 5-ml Q FF-Sepharose column. A, EmrE T108C (left lane) radiolabeled with methionine (15) was solubilized and cross-linked with o-PDM (right lane) as described under "Experimental Procedures." The proteins were analyzed using SDS-PAGE, visualized with a Fujifilm LAS-1000 imaging system, and digitally analyzed with Image Gauge 3.46 Fujifilm software. Purification of EmrE cross-linked at position T108C with o-PDM. Functionality ofĮmrE with the suggested antiparallel orientation of the monomers remains to be characterized. That the antiparallel orientation observed is a result of the arrangement of the monomers in the crystal. We show that the detergents used in crystallization increase the fraction of monomers in solution. The results support the contention that EmrE with parallel topology isįully functional. The cross-linked dimers do not interact with non-cross-linked dimers as judged from the fact that inactive mutantsĭo not affect their activity (negative dominance). A purifiedĬross-linked dimer binds substrate and transports it in proteoliposomes with kinetic constants similar to those of the non-cross-linkedĭimer. ![]() To examine this apparent contradiction we chemically cross-linked dimers withĪ rigid bifunctional maleimide using Cys replacements at positions not permissible by an antiparallel topology. However, it is at odds with biochemical data that demonstrated the same topology for all protomers in the intactĬell and with extensive cross-linking studies. The recently suggested antiparallel topology of EmrE has intriguing implications for many aspects of the biology of ioncoupled
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