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| - Role of the Extracellular Loop in the Folding of a CFTR Transmembrane HelicalHairpin
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| - The folding of membrane-spanning domains into their native functional forms depends oninteractions between transmembrane (TM) helices joined by covalent loops. However, the importance ofthese covalent linker regions in mediating the strength of helix−helix associations has not beensystematically addressed. Here we examine the potential structural impact of cystic fibrosis-phenotypicmutations in the extracellular loop 2 (ECL2) on interactions between the TM3 and TM4 helices of thecystic fibrosis transmembrane conductance regulator (CFTR) in constructs containing CFTR residues 194−241. When the effects of replacements in ECL2 (including the CF-phenotypic mutants E217G and Q220R)were evaluated in a library of wild-type and mutant TM3-ECL2-TM4 hairpin constructs, we found thatSDS−PAGE gel migration rates differed over a range of nearly 40% +/− the wild-type position and thatdecreased migration rates correlate with increasing hairpin α-helical content as measured by circulardichroism spectra in sodium dodecyl sulfate micelles. The decreased mobility of TM3/4 constructs byintroduction of non-native residues is interpreted in terms of an elongation or “opening” of the helicalhairpin and concomitant destabilization of membrane-based helix−helix interactions. Our results supporta role for short loop regions in dictating the stability of membrane protein folds and highlight the interplaybetween membrane-embedded helix−helix interactions and loop conformation in influencing the structureof membrane proteins.
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