EPR spectra of spin-labeled lysozyme in tetragonal crystals oriented in the magnetic field

Tetragonal crystals of spin-labeled lysozyme were studied. The protein was modified at the histidine residue 15 (the only one in the protein structure ) with spin labels containing an imidazolidine-based nitroxide radical. The average number of spin-labeled histidine residues per lysozyme molecule in the crystal was shown to be close to 0.1. The dependence of the EPR spectr a on the crystal orientation with respect to the magnetic field was obtaine d and analyzed by theoretical modeling of the spectra using an original com puter program, which simulates the quasicrystal EPR spectrum of the nitroxi de radical. It was demonstrated that the directions of the Z axes of the ni troxides on lysozyme molecules in the unit cell of a tetragonal crystal are reduced to two mutually perpendicular orientations along the crystallograp hic axes a and b, Without dipole-dipole interaction between labels in a cry stal, the substantial difference of the distance between outer wide peaks i n the triplet EPR spectra from the doubled principal value of the hyperfine structure tensor directly proves that the magnetic tensors are partially a veraged because of the rapid angle-confined reorientations of the nitroxide at the His-15 end. The noticeable broadening of peaks in the experimental spectra, as compared with narrow peaks in the spectra of the magnetically d iluted radical in single crystal, immediately follows from the validity of the predicted distribution of nitroxides among clusters that correspond to only certain types of fast reorientations, The mobility of the label at His -15 of lysozyme in a tetragonal crystal is comparable to the label mobility in protein solution, since His-15 residues are exposed into wide intermole cular channels of the crystal.