Chapter Twenty-Three – Full Atom Simulations of Spin Label Conformations

Interpretation of EPR measurables from spin labels in terms of structure and dynamics requires knowledge of label behavior. General strategies were developed for simulation of labels used in EPR of proteins. The criteria for those simulations are (a) exhaustive sampling of rotamer space, (b) consensus of results independent of starting points, and (c) inclusion of entropy. These criteria are satisfied only when the number of transitions in any dihedral angle exceeds 100 and the simulation maintains thermodynamic equilibrium. Methods such as conventional MD do not efficiently cross energetic barriers, simulated anealing, Monte Carlo or popular Rotamer Library methodologies are potential energy based and ignore entropy (in addition to their specific shortcomings: environment fluctuations, fixed environment, or electrostatics). The Simulated scaling method avoids the above flaws by modulating the force fields between a reduced (allowing crossing energy barriers) and full potential (sampling minima). Spin label diffuses on this surface while remaining in thermodynamic equilibrium. Simulations show that (a) adopting a single conformation is rare, often there are two to four populated rotamers and (b) position of the NO varies up to 16 A. These results illustrate necessity for caution when interpreting EPR signals in terms of molecular structure. For example, the 10–16 A distance change in DEER should not be interpreted as a large conformational change, it can well be a flip about Cα–Cβ bond. Rigorous exploration of possible rotamer structures of a spin label is paramount in signal interpretation. We advocate use of bifunctional labels, motion of which is restricted 10,000-fold and the NO position is restricted to 2–5 A.