Molecular modeling of nitroxide spin labels: Methods development and applications.

摘要

There are two major obstacles in the interpretation of EPR spectra: (a) most spin labels are not fully immobilized by the protein, hence it is difficult to distinguish the mobility of the label with respect to the protein from the reorientation of the protein itself and (b) even in cases where the label is fully immobilized its orientation with respect to the protein is not known, which prevents interpretation of probe reorientation in terms of protein reorientation.; A computational strategy was developed for determining (a) whether or not a spin label is immobilized and (b) if immobilized, predicting its conformation within the protein. A Monte Carlo minimization algorithm was developed to search the conformational space of labels within known atomic level structures of proteins. The method was validated using a series of spin labels of varying size and geometry docked to sites on the myosin head catalytic and regulatory domains. The predicted immobilization and conformation compared well with experimental values. Thus, probes can now be designed and targeted to report on various modes of molecular dynamics. Applications are presented in which the method has been used to predict (a) distance distributions between pairs of spin labels; (b) spin label solvent accessibility; (c) spin label mobility and (d) starting structures for molecular dynamics simulations.; In cases where immobilization of the spin label is not possible, the observed mobility is a convolution of protein dynamics and probe dynamics. In order to account for probe dynamics, a hybrid method was developed that combines molecular dynamics (MD) and the stochastic Liouville (SLE) approach to EPR lineshape simulation, MD-SLE. Molecular Dynamics was used to predict the motion of the spin probe in the local environment of the labeled site and to calculate an ordering potential, which was then used as input to the SLE simulations of EPR spectra. The method was validated against high-field EPR experiments, where the macromolecular motion is frozen out, and the spectrum is sensitive to the details of the probe diffusion within the local structure of the macromolecule.