Motional clustering in supra-τ; c; conformational exchange influences NOE cross-relaxation rate

Biomolecular spin relaxation processes, such as the NOE, are commonly modeled by rotational τ; c; -tumbling combined with fast motions on the sub-τ; c; timescale. Motions on the supra-τ; c; timescale, in contrast, are considered to be completely decorrelated to the molecular tumbling and therefore invisible. Here, we show how supra-τ; c; dynamics can nonetheless influence the NOE build-up between methyl groups. This effect arises because supra-τ; c; motions can cluster the fast-motion ensembles into discrete states, affecting distance averaging as well as the fast-motion order parameter and hence the cross-relaxation rate. We present a computational approach to estimate methyl-methyl cross-relaxation rates from extensive (>100×τ; c; ) all-atom molecular dynamics (MD) trajectories on the example of the 723-residue protein Malate Synthase G. The approach uses Markov state models (MSMs) to resolve transitions between metastable states and thus to discriminate between sub-τ; c; and supra-τ; c; conformational exchange. We find that supra-τ; c; exchange typically increases NOESY cross-peak intensities. The methods described in this work extend the theory of modeling sub-μs dynamics in spin relaxation and thus contribute to a quantitative estimation of NOE cross-relaxation rates from MD simulations, eventually leading to increased precision in structural and functional studies of large proteins.