Analysis of nitroxide spin label motion in a protein-protein complex using multiple frequency EPR spectroscopy.
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.
White
G F
GF
School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK.
Ottignon
L
L
Georgiou
T
T
Kleanthous
C
C
Moore
G R
GR
Thomson
A J
AJ
Oganesyan
V S
VS
D001665:Binding Sites; D003087:Colicins / Q000737:chemistry*; D003198:Computer Simulation; D003851:Deoxyribonucleases / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D009589:Nitrogen Oxides / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D013113:Spin Labels
D003087:Colicins; D029968:Escherichia coli Proteins; D009589:Nitrogen Oxides; D013113:Spin Labels; C488858:immE8 protein, E coli; C488859:immE9 protein, E coli; D003851:Deoxyribonucleases; C039900:nitroxyl