HELIX PACKING IN THE LACTOSE PERMEASE DETERMINED BY METAL-NITROXIDE INTERACTION

The magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances within proteins [Voss, J. , Salwinski, L., Kaback, H. R., and Hubbell, W. L. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 12295-12299; Voss, J., Hubbell, W. L., and Kabac k, H. R. (1995) PI oc. Natl. Acad. Sci. U.S.A. 92, 12300-12303], and t he approach is utilized here to measure helix proximities in the lacto se permease of Escherichia coli. A high-affinity divalent metal bindin g site was created by replacing Arg302 (helix IX) and Glu325 (helix X) with His residues in permease mutants containing single Cys residues in helices II, V, or VII and a biotin acceptor domain to facilitate pu rification. Mutant proteins were purified by avidin affinity chromatog raphy, labeled specifically with a nitroxide free radical and investig ated by electron paramagnetic resonance spectroscopy in the absence or presence of Cu(II). Spectral broadening due to bound Cu(II) was used to estimate distances between the metal center and the spin-labeled si de chains. For each of the transmembrane domains probed, the variation in interspin distance with sequence position is consistent with an a- helical structure. The measured distances were also used to construct a model that is in good agreement with packing data obtained from othe r approaches.