Rotamer Library of Spin Labeled Cysteines Attached to T4 Lysozyme Deduced from Molecular Dynamics Simulations Constrained by Double Electron-Electron Resonance (Deer) Experiments

Abstract

DEER (Double Electron Electron Resonance) is a powerful pulsed EPR technique allowing the determination of spin-spin distance histograms between side-directed nitroxide label sites on a protein in their native environment. In this study, a novel molecular dynamics simulation method, the restrained ensemble (RE) method, has been used for the refinement of spin labels inserted in T4 Lysozyme (T4L) structures. In the RE method, the spin-spin distance distribution histograms calculated from a multiple-copy molecular dynamics simulations are forced, via a special ensemble-based energy restraint, to match those extracted from 51 EPR/DEER experimental spin-spin pairs. To examine the effect of the restraint on the rotameric state of the spin label at the 37 different sites in T4L, conventional long molecular dynamics (MD) and locally enhanced sampling (LES) simulations were also performed. As expected, the distance histograms obtained from the RE simulations are very similar to those obtained from experiment; however, the distance histograms from conventional long MD and LES simulations deviate from experiment. From the analysis of these simulations, a general trend is found in rotamer population distribution along five dihedral angles connecting the nitroxide ring to the protein backbone. Overall, the rotamer population obtained from the RE simulation agrees with those obtained from x-ray structure. From the analysis of the dynamics of nitroxide atoms in spin label side chains, a force field for a pseudo nitroxide dummy atom has been parameterized. This pseudo nitroxide was used in an RE simulation coupled with the EPR/DEER experimental distance distribution data to refine distorted structures of T4L. Thus, experimental restraints from DEER experiments in conjunction with RE simulations can be used to refine structural propertied of biological systems.