Rapid Determination of Fast Protein Dynamics from NMR Chemical Exchange Saturation Transfer Data

Functional motions of ¹⁵N‐labeled proteins can be monitored by solution NMR spin relaxation experiments over a broad range of timescales. These experiments however typically take of the order of several days to a week per protein. Recently, NMR chemical exchange saturation transfer (CEST) experiments have emerged to probe slow millisecond motions complementing R_1ρ and CPMG‐type experiments. CEST also simultaneously reports on site‐specific R₁ and R₂ parameters. It is shown here how CEST‐derived R₁ and R₂ relaxation parameters can be measured within a few hours at an accuracy comparable to traditional relaxation experiments. Using a “lean” version of the model‐free approach S² order parameters can be determined that match those from the standard model‐free approach applied to ¹⁵N R₁, R₂, and {¹H}‐¹⁵N NOE data. The new methodology, which is demonstrated for ubiquitin and arginine kinase (42 kDa), should serve as an effective screening tool of protein dynamics from picosecond‐to‐millisecond timescales.