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Lactose permease is a paradigm for the major facilitator superfamily, the largest family of ion-coupled membrane transport proteins known at present. LacY carries out the coupled stoichiometric symport of a galactoside with an H⁺, using the free energy released from downhill translocation of H⁺to drive accumulation of galactosides against a concentration gradient. In neutrophilicEscherichia coli, internal pH is kept at ∼7.6 over the physiological range, but the apparent pK (pK^app) for galactoside binding is 10.5. Surface-enhanced infrared absorption spectroscopy (SEIRAS) demonstrates that the high pK_ais due to Glu325 (helix X), which must be protonated for LacY to bind galactoside effectively. Deprotonation is also obligatory for turnover, however. Here, we utilize SEIRAS to study the effect of mutating residues in the immediate vicinity of Glu325 on its pK_a. The results are consistent with the idea that Arg302 (helix IX) is important for deprotonation of Glu325.