EPR of Cu2+ Prion Protein Constructs at 2 GHz Using the g Region to Characterize Nitrogen Ligation

James S. Hyde, Medical College of Wisconsin
Brian Bennett, Marquette University
Eric D. Walter, University of California - Santa Cruz
Glenn L. Millhauser, University of California - Santa Cruz
Jason Walter Sidabras, Medical College of Wisconsin
William E. Antholine, Medical College of Wisconsin

Published version. Biophysical Journal, Vol. 96, No. 8 (April 22, 2009): 3354-3362. DOI. © Elsevier (Cell Press) 2009. Used with permission.

Brian Bennett was affiliated with the Medical College of Wisconsin at the time of publication.


A double octarepeat prion protein construct, which has two histidines, mixed with copper sulfate in a 3:2 molar ratio provides at most three imidazole ligands to each copper ion to form a square-planar Cu2+ complex. This work is concerned with identification of the fourth ligand. A new (to our knowledge) electron paramagnetic resonance method based on analysis of the intense features of the electron paramagnetic resonance spectrum in the g region at 2 GHz is introduced to distinguish between three and four nitrogen ligands. The methodology was established by studies of a model system consisting of histidine imidazole ligation to Cu2+. In this spectral region at 2 GHz (S-band), g-strain and broadening from the possible rhombic character of the Zeeman interaction are small. The most intense line is identified with the MI = +1/2 extra absorption peak. Spectral simulation demonstrated that this peak is insensitive to cupric Ax and Ay hyperfine interaction. The spectral region to the high-field side of this peak is uncluttered and suitable for analysis of nitrogen superhyperfine couplings to determine the number of nitrogens. The spectral region to the low-field side of the intense extra absorption peak in the g part of the spectrum is sensitive to the rhombic distortion parameters Ax and Ay. Application of the method to the prion protein system indicates that two species are present and that the dominant species contains four nitrogen ligands. A new loop-gap microwave resonator is described that contains ∼1 mL of frozen sample.