Simulating the Distance Distribution between Spin-Labels Attached to Proteins

By Shahidul M Islam and Benoît Roux.

Published in J. Phys. Chem. B, 2015 Feb 2;119(10). PMID: 25645890.

Core Facility: Computational Modeling.


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 Cα 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 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 RXi,i+3. Simplified dummy spin-label models 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 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.


Figure 1. Spin-label side chains, R1 and RX, resulting from linking MTSSL to cysteine through a disulfide bond, and dummy spin-labels, OND and ONDX, which mimics the dynamics of the R1 and RX, respectively.

Figure 2. Polyalanine α-helix with (A) RXi,i+3 and (B) RXi,i+4 (left panel) and the corresponding dynamics of the nitroxide oxygen of the respective RX with respect to their Cα atoms obtained from MD simulation (right panel).

Figure 4. Comparison of distribution and the potential of mean force of the distances, angle, and dihedral angle obtained from MD simulations with RX and dummy nitroxide atoms at positions (A) i, i + 3 and (B) i, i + 4 of the polyalanine α-helix.

Figure 5. (A) Cartoon representation of T4 lysozyme with the OND at positions 109 and 131 and the OND4 at positions 109–113 and 127–131 and corresponding distance distributions obtained from MDDS and EPR/DEER. (B) Cartoon representation of KCNE1 with the OND at positions 47 and 66 and the OND4 at positions 46–50 and 66–70 and corresponding distance distributions obtained from MDDS and EPR/DEER.