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 Kaback, H. R. (1995) Proc. Natl. Acad. Sci. U.S. A. 92, 12300-12303], and the approach is utilized here to measure helix proximities in the lactose permease of Escherichia coli. A high-affinity divalent metal binding 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 purification. Mutant proteins were purified by avidin affinity chromatography, labeled specifically with a nitroxide free radical and investigated 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 side chains. For each of the transmembrane domains probed, the variation in interspin distance with sequence position is consistent with an alpha-helical structure. The measured distances were also used to construct a model that is in good agreement with packing data obtained from other approaches.
Voss
J
J
Howard Hughes Medical Institute, Department of Physiology, University of California at Los Angeles 90095-1662, USA.
Hubbell
W L
WL
Kaback
H R
HR
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002413:Cations, Divalent; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D009589:Nitrogen Oxides / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D013113:Spin Labels; D027981:Symporters
D001426:Bacterial Proteins; D002413:Cations, Divalent; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D009589:Nitrogen Oxides; D013113:Spin Labels; D027981:Symporters; D003300:Copper; C021499:lactose permease