Simulating the Distance Distribution between Spin-Labels Attached to Proteins

E'PR/DEER spectroscopy-is playing an increasingly important role in the characterizatiOn Of the conformational states of proteins: In this study, forte 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-alpha atom of the backbone to the -nitroxide ring moiety of the RX spin-label attached to i and + 4 positions in a poIyalanine alpha-helix agree very well with those observed in the X=ray crystallography. Both RXi,i+4 and RXi,i+3 are more rigid than the monofunctional spin-label (R1) commonly used in EPR/DEER, while RXi,i+4 is more rigid and causeS less distortion in a protein backbone than Simplified dummy spin-label ihodels with a single effective particle representing the RXi,i+3 and RXi,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 IVID (re-MD) simulations to carry out structural refinement of 3D models. Applications of this methodology to T4 lysozyme, KCNE1, and LeuT ate shown to provide important insights about their conformational dynamics.