By exploiting substrate protection of Cys148 in lactose permease, a methanethiosulfonate nitroxide spin-label was directed specifically to one of two Cys residues in a double-Cys mutant, followed by labeling of Cys148 with a thiol-reactive chelator that binds Gd(III) quantitatively. Distances between bound Gd(III) and the nitroxide spin-label were then studied by electron paramagnetic resonance. The results demonstrate that the Gd(III)-induced relaxation effects on nitroxides at positions 228, 226 (helix VII), and 275 (helix VIII) agree qualitatively with results obtained by studying spin - spin interactions [Wu, J., Voss, J., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 10123-10127]. Thus, a nitroxide attached to position 228 (helix VII) is closest to the lanthanide at position 148 (helix V), a nitroxide at position 275 (helix VIII) is further away, and the distance between positions 226 (helix VII) and 148 is too long to measure. However, the Gd(III) - spin-label distances are significantly longer than those estimated from nitroxide - nitroxide interactions between the same pairs due to the nature of the chelator. Although the results provide strong confirmation for the contention that helix V lies close to both helices VII and VIII in the tertiary structure of lactose permease, other methods for binding rare earth metals are discussed which do not involve the use of bulky chelators with long linkers.
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