By using site-directed fluorescence spectroscopy, we have carried out structure/function studies on lactose permease purified from Escherichia coli in dodecyl beta,D-maltoside. Initially, permease containing a single native Cys at position 148 (helix V) was studied, since this residue is protected against alkylation by substrates of the permease. In the absence of ligand, Cys148 permease reacts rapidly with 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS), a fluorophore whose quantum yield increases dramatically upon reaction with a thiol, indicating that this residue is readily accessible to the probe. Various ligands of the permease block the reaction, and the concentration dependence is commensurate with the affinity of each ligand for the permease (i.e., beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside << lactose < galactose), but neither sucrose nor glucose has any effect whatsoever. Thus, the permease retains the ability to bind ligand specifically when the molecule is in dodecyl beta,D-maltoside. Permease containing single Cys substitutions in the vicinity of Cys 148 was also studied. Interestingly, labeling of Cys 145 which is presumed to be one helical turn removed from Cys148 exhibits properties similar to those observed with Cys148 permease, but the effects of ligand are far less dramatic. On the other hand, permease with a single Cys residue at position 146 or 147 behaves in a completely different manner. Studies with iodide indicate that MIANS at positions 145 or 148 is accessible to the collisional quencher, indicating that this face of helix V is solvent exposed, while MIANS at positions 146 or 147 is not quenched by iodide in the presence or absence of ligand. Finally, iodide quenching of MIANS at position 145 is clearly diminished in the presence of ligand. Taken together with the preceding paper in this issue [Jung, H., Jung, K., and Kaback, H. R. (1994) Biochemistry (preceding paper in this issue)], the results indicate that Cys148 is a likely component of a substrate binding site in lac permease, although it does not play an essential role in transport, and that residue 145 is in close proximity.
