Full Atom Simulations of Spin Label Conformations

Abstract

Interpretation of EPR 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; (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 Annealing, 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). Simulated Scaling method, avoids above flaws by modulating the forcefields between 0 (allowing crossing energy barriers) and full potential (sampling minima). Spin label diffuses on this surface while remaining in thermodynamic equilibrium. Simulations show that: (a) single conformation is rare, often there are 2–4 populated rotamers; (b) position of the NO varies up to 16Å. These results illustrate necessity for caution when interpreting EPR signals in terms of molecular structure. For example the 10–16Å 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, which motion is restricted 10,000-fold and the NO position is restricted to 2–5Å.

The contribution of modern EPR to structural biology

“…All these approaches end up with similar accuracy of ∼3 Å for predicting the mean distance 〈r ee 〉 from a known high-resolution structure [29]. Computationally more elaborate procedures sample the energy hypersurface of the labelled protein in a Monte Carlo approach [30] or by enhanced sampling MD [31], but do not improve strongly on that accuracy. Hence, at present, an accuracy of ∼2-3 Å must be accepted, which precludes building atomic-resolution models from only EPR restraints.…”

DEER-PREdict: Software for efficient calculation of spin-labeling EPR and NMR data from conformational ensembles

“…Since spin labels are conformationally dynamic, both protein and paramagnetic probes need to be described by conformational ensembles to obtain accurate predictions of DEER and PRE observables from molecular models [ 12 – 14 ]. Molecular dynamics (MD) simulations are one approach to obtain conformational ensembles that model the structure and dynamics of spin-labels for the calculation of EPR and NMR data [ 15 – 18 ].…”

High-resolution models of actin-bound myosin from EPR of a bifunctional spin label

“…The comparatively superior intrinsic resolution of EPR has proven especially powerful in the study of intra-myosin distances by double electron-electron resonance (DEER) (7,8), and of the orientation of myosin domains within oriented muscle fibers (9,10). Until recently, however, the effective resolving power of site-directed EPR has been limited by nanosecond mobility of the spectroscopic probe relative to its associated labeling site, contributing significant static and dynamic disorder to the resulting spectral waveform (11,12).…”