Cys-Scanning Mutagenesis of the Transmembrane Helix II and Cytoplasmic Loops in Melibiose Permease of Salmonella typhimurium

Melibiose permease of Salmonella typhimurium (MelB_St), a member of the galactosides-pentoses-hexuronide family of membrane transport proteins, catalyzes the symport of a galactopyranoside sugar and its coupling cation (H⁺, Na⁺, or Li⁺).  A 3-D model of MelB_St, generated by threading through the lactose permease of E. coli, shows that eight out of twelve transmembrane a-helices (I, II, IV, V, VII, VIII, X, and XI) form an internal cavity containing cosubstrate-binding sites facing the cytoplasm.  Two loops connecting helices VI and V (L_4-5), as well as X and XI (L_10-11), are found within the cavity of the protein.  The helix II bears important residues for Na⁺ binding, and L_4-5 and L_10-11 are important for melibiose transport. In order to probe substrate-induced conformational dynamics of the protein, a functional Cys-less MelB_St mutant has been constructed, in which all four endogenous Cys were mutated to Ala.  In this study, Cys displaces single amino acid residues, at each position of helix II, L_4-5, and L_10-11 via site-directed mutagenesis. A total of 60 single-Cys mutants have been generated, or are under construction. Further studies including melibiose fermentation assays, [³H] melibiose transport assays, and alkylation by thiol reagents are expected to provide important information for understanding the melibiose/Na⁺ symport mechanism.