Benchmarking all-atom simulations using hydrogen exchange

Molecular dynamics simulations have recently become capable of observing multiple protein unfolding and refolding events in a single-millisecond–long trajectory. This major advance produces atomic-level information with nanosecond resolution, a feat unmatched by experimental methods. Such simulations are being extensively analyzed to assess their description of protein folding, yet the results remain difficult to validate experimentally. We apply a combination of hydrogen exchange, NMR, and other techniques to test the simulations with a resolution of single H-bonds. Several significant discrepancies between the simulations and experimental data were uncovered for regions of the energy surface outside of the native basin. This comparison yields suggestions for improving the force fields and provides a general method for experimentally validating folding simulations.