Analyzing sub-millisecond timescale protein dynamics using eCPMG experiments.

Cellular functions require biomolecules to transition among various conformational sub-states in the energy landscape. A mechanistic understanding of cellular functions requires quantitative knowledge of the kinetics, thermodynamics, and structural features of the biomolecules experiencing exchange between several states. High-power Relaxation Dispersion (RD) NMR experiments have proven very effective for such measurements if the exchange occurs in timescales ranging from microseconds to milliseconds. However, scanning the significantly larger kinetic window within the time limit of instrumental availability and sample stability requires careful optimization of experiments. Understanding biomolecular functions at a mechanistic level depends on fitting such experimental data to theoretical models. However, the reliability of the fit parameters depends on the measurement schemes and is sensitive to experimental noise. Here, we benchmark different measurement schemes along with theoretical models for sub-millisecond timescale exchange and determine the robustness of these models in providing information when the measurements contain noise. Our results show that kinetics can be measured reliably from such experiments. The structural features of the exchanging sub-states, encoded in the chemical shift differences between the states, can be fitted, albeit with significant uncertainties. Information about the minor states is difficult to obtain exclusively from the RD data due to large uncertainties and sensitivity to noise.