Functional lactose permease mutants containing single-Cys residues at positions 387-402 [He, M. M., Sun, J., & Kaback, H. R. (1996) Biochemistry 35, 12909-12914] and a biotin acceptor domain in the middle cytoplasmic loop were solubilized in n-dodecyl-beta-D-maltopyranoside and purified by avidin affinity chromatography. Each mutant protein was derivatized with a thiol-selective nitroxide reagent and examined by conventional and power saturation electron paramagnetic resonance spectroscopy. Analysis of the electron paramagnetic resonance spectral line shapes and the influence of O2 on the saturation behavior of the spin-labeled proteins were measured in order to obtain information on the mobility of the spin-labeled side chains and their accessibility to O2, respectively. The data show a periodic dependence of both mobility and accessibility on sequence position consistent with an alpha-helical structure. These results provide direct support for the contention that transmembrane domain XII is in an alpha-helical conformation and on the periphery of the 12-helix bundle that comprises the lactose permease molecule.

Engineering divalent metal-binding sites into the lactose permease of Escherichia coli by introducing bis-His residues has been utilized to confirm the proximity of helices VIII (Glu269 --> His) and X (His322) [Jung, K., Voss, J., He, M., Hubbell, W. L., & Kaback, H. R. (1995) Biochemistry 34, 6272] and helices VII (Asp237 --> His) and XI (Lys358 --> His) [He, M. M., Voss, J., Hubbell, W. L., & Kaback, H.R. (1995) Biochemistry 34, 00000--00000]. In this paper, the approach is used to confirm and extend the relationship between helices IX (Arg302) and X (His322 and Glu325) [Jung, K., Jung, H., Wu, J., Prive, G. G., l& Kaback, H. R. (1993) Biochemistry 32, 12273]. Thus, mutants Arg302 --> His, Glu325 --> His, and Arg302 --> His/Glu325 --> His were constructed, and Mn2+ binding was assayed by electron paramagnetic resonance. Mutant Arg302 --> His binds Mn2+ with a KD of about 24 microM and a stoichiometry approximating unity in all likelihood because the His residue at position 302 forms a metal-binding site in conjunction with the native His residue at position 322. Mutant Arg302 --> His/Glu325 --> His also binds Mn2+ with a 1:1 stoichiometry, but the KD is decreased to about 13 microM. The results suggest that Arg302 is sufficiently close to both Glu325 and His322 to form a tridentate metal-binding site in mutant Arg302 --> His/Glu325 --> His. In contrast, replacement of Glu325 with His in permease with a native His residue at position 322 does not lead to Mn2+ binding. The results provide strong support for the helix packing model proposed.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain XII and the periplasmic loop between putative helices XI and XII (loop XI/XII) was replaced individually with Cys. Out of 34 mutants, 31 exhibit 60-100% or more of C-less activity, mutants Gly377-->Cys and Leu385-->Cys exhibit lower rates of transport but accumulate lactose about 60-70% as well as C-less, and mutant Leu400-->Cys exhibits < 20% of C-less activity. Immunoblots reveal that all of the mutant proteins are present in the membrane in amounts comparable to that of C-less with the exception of mutants Gly377-->Cys and Leu385-->Cys which are expressed about 40% as well as C-less and mutant Leu400-->Cys which is hardly detectable. When transferred to the wild-type background, however, mutant Leu400-->Cys is expressed normally and exhibits highly significant transport activity. Finally, each active Cys-replacement mutant was assayed for sensitivity to N-ethylmaleimide, and with three exceptions, the mutants are essentially unaffected by the alkylating agent. Mutants Val367-->Cys, Gly370-->Cys, and Tyr373-->Cys which are predicted to be immediately distal to helix XI in loop XI/XII are significantly inactivated. The periodicity observed suggests that the periplasmic end of transmembrane domain XI may extend to position 373. In the following paper [Voss, J., He, M. M., Hubbell, W. L., & Kaback, H. R. (1996) Biochemistry 35, 12915-12918], site-directed spin labeling of single-Cys mutants at positions 387-402 is used to demonstrate that transmembrane domain XII is in an alpha-helical conformation.