Non-ergodic internal dynamics of a globular protein observed over fourteen orders in time*

Protein internal motions have been assumed ergodic, i.e., dynamics of a single molecule over time resembles that of an ensemble. Here, by performing 254 single-molecule fluorescence resonance energy transfer (smFRET) experiments and 100 molecular dynamics (MD) simulations of a multi-domain globular protein, cytoplasmic protein-tyrosine phosphatase, we demonstrate by examining large ensembles that the interdomain motion varies widely between  individual biomolecules and is non-ergodic over the time span ~10^-12 to 10² seconds. Analysis of MD trajectories reveals that observed heterogeneity derives from the high-dimensional complex energy landscape. As the characteristic time for the protein to conduct its dephosphorylation function is ~10s seconds, these findings suggest that, due to non-ergodicity  individual, otherwise seemingly identical protein molecules can be dynamically and functionally different.