A Method for Distance Determination in Proteins Using a Designed Metal Ion Binding Site and Site-Directed Spin Labeling: Evaluation with T4 Lysozyme

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 92; no. 26; pp. 12295 - 12299
• Main Authors: Voss, J, Salwiński, L, Kaback, H R, Hubbell, W L
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 19-12-1995; National Acad Sciences; National Academy of Sciences
• Subjects: Amplitude; Bacteriophage T4 - enzymology; Binding Sites; Biochemistry; Copper - metabolism; Crystal lattices; Electron Spin Resonance Spectroscopy; Ions; Line spectra; Magnetic fields; Metal ions; Metals; Metals - metabolism; Models, Molecular; Models, Structural; Muramidase - chemistry; Muramidase - metabolism; Mutagenesis, Site-Directed; phage T4; Point Mutation; Protein Structure, Secondary; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Reproducibility of Results; Room temperature; Spin Labels
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.92.26.12295

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 metal-nitroxyl 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 Å 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.