{'fraction_non_alphanumeric': 0.038551401869158876, 'fraction_numerical': 0.0397196261682243, 'mean_word_length': 5.096085409252669, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Cytoplasmic Loop Connecting Helices IV and V of the Melibiose Permease from Escherichia coli Is Involved in the Process of Na+-coupled Sugar Translocation*

Previous photolabeling and limited proteolysis studies suggested that one of the four basic residues (Arg-141) of the N-terminal cytoplasmic loop connecting helices IV and V (loop 4–5) of the melibiose permease (MelB) from Escherichia coli has a potential role in its symport function (Ambroise, Y., Leblanc, G., and Rousseau, B. (2000) Biochemistry 39, 1338–1345). A mutagenesis study of Arg-141 and of the other three basic residues of loop 4–5 was undertaken to further examine this hypothesis. Cys replacement analysis indicated that Arg-141 and Arg-149, but not Lys-138 and Arg-139, are essential for MelB transport activity. Replacement of Arg-141 by neutral residues (Cys or Gln) inactivated transport and energy-independent carrier-mediated flows of substrates (counterflow, efflux), whereas it had a limited effect on co-substrate binding. R141C sugar transport was partially rescued on reintroducing a positive charge with a charged and permeant thiol reagent. Whereas R149C was completely inactive, R149K and R149Q remained functional. Strikingly, introduction of an additional mutation in the C-terminal helix X (Gly for Val-343) of R149C restored sugar transport. Impermeant thiol reagents inhibited R149C/V343G transport activity in right-side-out membrane vesicles and prevented sugar binding in a sugar-protected manner. All these data suggest that MelB loop 4–5 is close to the sugar binding site and that the charged residue Arg-141 is involved in the reaction of co-substrate translocation or substrate release in the inner compartment.