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[摘要]：AEI, which exists in the erythrocyte plasma membrane as a noncovalent dimer, facilitates transmembrane Cl-/HCO3- exchange. Here a concatamer of AEI (two AEI monomers fused via a two-residue linker to form an intramolecular dimer) was designed to facilitate fluorescence resonance energy transfer (FRET) studies. The concatameric protein (AEI.AEI) was expressed at the plasma membrane at levels similar to that of wild-type AEI and had Cl-/HCO3- exchange activity indistinguishable from that of wild-type AEI. Nondenaturing gel electrophoresis revealed that AEI. AEI does not associate into higher-order oligomers when expressed in HEK293 cells and Xenopus laevis oocytes. The cysteine-less concatamer (AEI.AEI-C-) enabled introduction of unique cysteine residues into the whole intramolecular dimer. AEI (Q434C).AEI(Q434C)-C-, with a single cysteine residue in each AEI subunit, was labeled with the donor Alexa Fluor 488 C-5-maleimide (AF) and the acceptor tetramethylrhodamine methanethiosulfonate (TMR-MTS). Energy transfer efficiency revealed that the distance between these residues in the AEI dimer is 49 +/- 5 angstrom. The 72% FRET efficiency observed between AEI(Q434C).AEl-C- labeled with AF and the lipid bilayer labeled with 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate indicates that Q434 is less than 33 angstrom from the lipid bilayer. We thus provide two distance constraints for the position of Q434, which is located in extracellular loop 1, connecting the first two transmembrane segments of AEl. |
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