Search

The spin label method was used to observe the nature of the fast motions of side chains in protein monocrystals. The EPR spectra of spin-labeled lysozyme monocrystals (with different orientations of the tetragonal protein crystal in relation to the direction of the magnetic field) were interpreted using the method of molecular dynamics (MD). Within the proposed simple model, MD calculations of the spin label motion trajectories are performed in a reasonable real time. The model regards the protein molecule as frozen as a whole and the spin-labeled amino acid residue as unfrozen. To calculate the trajectories in vacuum, a model of spin-labeled lysozyme was assembled, and the parameters of the force fields were specified for atoms of the protein molecule, including the spin label. The calculations show that the protein environment sterically limits the area of the possible angular reorientations for the NO reporter group of the nitroxide (within the spin label), and this, in turn, affects the shape of the EPR spectrum. However, it turned out that the spread in the positions of the reporter group in the angle space strictly adheres to the Gaussian distribution. Using the coordinates of the spin label atoms obtained by the MD method within a selected time range and considering the distribution of the spin label states over the ensemble of spin-labeled macromolecules in a crystal, the EPR spectra of spin-labeled lysozyme monocrystals were simulated. The resultant theoretical EPR spectra appeared to be similar to experimental ones.

The EPR spectra of the preparations produced by spin labeling of the carbohydrate parts in monoclonal IgM and normal IgG with 2,2,6,6-tetramethyl-4-aminopiperidine-1-oxyl as the spin label indicate the existence of a rapid spin-spin exchange interaction between two spin labels. In the case of spin-labeled IgM, the carrier of such a spectrum is shown to be a glycopeptide noncovalently bound to IgM; it includes two spin labels and may be detached from the macromolecule by a combination of dialysis and gel filtration.