Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of Escherichia coli.
As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure. To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins. Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV. In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification. After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II). The results demonstrate that positions 103, 111, and 121 are 8, 14, and > 23 A from the metal binding site. These data are consistent with an alpha-helical conformation of transmembrane domain IV of the permease. Application of the technique to determine helix packing in lactose permease is discussed.
Voss
J
J
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90095-1662, USA.
Hubbell
W L
WL
Kaback
H R
HR
D000595:Amino Acid Sequence; D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D003545:Cysteine; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D008961:Models, Structural; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013113:Spin Labels; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D008670:Metals; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; D013113:Spin Labels; D027981:Symporters; D003300:Copper; C021499:lactose permease; D003545:Cysteine