A method for distance determination in proteins using a designed metal ion binding site and site-directed spin labeling: evaluation with T4 lysozyme.
The use of molecular genetics to introduce both a metal ion binding site and a nitroxide spin label into the same protein opens the use of paramagnetic metalnitroxyl interactions to estimate intramolecular distances in a wide variety of proteins. In this report, a His-Xaa3-His metal ion binding motif was introduced at the N terminus of the long interdomain helix of T4 lysozyme (Lys-65 --> His/Gln-69 --> His) of three mutants, each containing a single nitroxide-labeled cysteine residue at position 71, 76, or 80. The results show that Cu(II)-induced relaxation effects on the nitroxide can be quantitatively analyzed in terms of interspin distance in the range of 10-25 A using Redfield theory, as first suggested by Leigh [Leigh, J.S. (1970) J. Chem. Phys. 52, 2608-2612]. Of particular interest is the observation that distances can be determined both under rigid lattice conditions in frozen solution and in the presence of motion of the spins at room temperature under physiological conditions. The method should be particularly attractive for investigating structure in membrane proteins that are difficult to crystallize. In the accompanying paper, the technique is applied to a polytopic membrane protein, lactose permease.
Voss
J
J
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90095, USA.
Salwiński
L
L
Kaback
H R
HR
Hubbell
W L
WL
D017122:Bacteriophage T4 / Q000201:enzymology*; D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D008961:Models, Structural; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D013113:Spin Labels
D008670:Metals; D011994:Recombinant Proteins; D013113:Spin Labels; D003300:Copper; D009113:Muramidase