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X- and W-band EPR spectra, at room and low temperatures, are reported for nitroxide spin labels attached to cysteine residues selectively introduced into two proteins, the DNase domain of colicin-E9 and its immunity protein, Im9. The dynamics of each site of attachment on the individual proteins and in the tight DNase-Im9 complex have been analysed by computer simulations of the spectra using a model of Brownian dynamics trajectories for the spin label and protein. Ordering potentials have been introduced to describe mobility of labels restricted by the protein domain. Label mobility varies with position from completely immobilised, to motionally restricted and to freely rotating. Bi-modal dynamics of the spin label have been observed for several sites. We show that W-band spectra are particularly useful for detection of anisotropy of spin label motion. On complex formation significant changes are observed in the dynamics of labels at the binding interface region. This work reveals multi-frequency EPR as a sensitive and valuable tool for detecting conformational changes in protein structure and dynamics especially in protein-protein complexes.

Original publication

DOI

10.1016/j.jmr.2006.12.009

Type

Journal article

Journal

J Magn Reson

Publication Date

04/2007

Volume

185

Pages

191 - 203

Keywords

Binding Sites, Colicins, Computer Simulation, Deoxyribonucleases, Electron Spin Resonance Spectroscopy, Escherichia coli Proteins, Models, Chemical, Models, Molecular, Nitrogen Oxides, Protein Binding, Protein Conformation, Spin Labels