Simulating the Distance Distribution between Spin-Labels Attached to Proteins

[Image: see text] EPR/DEER spectroscopy is playing an increasingly important role in the characterization of the conformational states of proteins. In this study, force field parameters for the bifunctional spin-label (RX) used in EPR/DEER are parametrized and tested with molecular dynamics (MD) simulations. The dihedral angles connecting the C(α) atom of the backbone to the nitroxide ring moiety of the RX spin-label attached to i and i + 4 positions in a polyalanine α-helix agree very well with those observed in the X-ray crystallography. Both RX(i,i+4) and RX(i,i+3) are more rigid than the monofunctional spin-label (R1) commonly used in EPR/DEER, while RX(i,i+4) is more rigid and causes less distortion in a protein backbone than RX(i,i+3). Simplified dummy spin-label models with a single effective particle representing the RX(i,i+3) and RX(i,i+4) are also developed and parametrized from the all-atom simulations. MD simulations with dummy spin-labels (MDDS) provide distance distributions that can be directly compared to distance distributions obtained from EPR/DEER to rapidly assess if a hypothetical three-dimensional (3D) structural model is consistent with experiment. The dummy spin-labels can also be used in the restrained-ensemble MD (re-MD) simulations to carry out structural refinement of 3D models. Applications of this methodology to T4 lysozyme, KCNE1, and LeuT are shown to provide important insights about their conformational dynamics.