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

Abstract

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 R1ρ and CPMG-type experiments. CEST also simultaneously reports on site-specific R1 and R2 parameters. It is shown here how CEST-derived R1, R2 relaxation parameters can be measured within 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 ps to ms timescales.

Applications of NMR and computational methodologies to study protein dynamics

“…k ex = 50–150 s −1 ). The CEST experiment can also simultaneously take advantage of the CEST-derived R 1 and R 2 data to extract S 2 order parameters reporting on the ps-ns time scale 30 . In spin-relaxation NMR, the order parameter S 2 is typically extracted from the model-free approach using experimental R 1 , R 2 , and hNOE NMR data 31–33 .…”

15N CEST data and traditional model-free analysis capture fast internal dynamics of DJ-1

“…Recent advances employing saturation transfer, such as chemical exchange saturation transfer (CEST) and dark-state exchange saturation transfer (DEST), have enabled the detection of these previously invisible protein states [7, 8]. Several studies have already reported the use of CEST to study the invisible conformers of slowly exchanging proteins on the millisecond to second timescale [1, 9–12]. Additionally, the fitting of CEST profiles have been shown to reliably extract R 1 and R 2 parameters that can be used for modelfree analysis of fast timescale dynamics (ps to ns).…”

“…To date, however, no study has combined CEST-derived R 1 and R 2 parameters with 1 H- 15 N NOE data to establish the picosecond to nanosecond dynamics of a protein. Instead, leaner versions of modelfree have been applied without the NOE data [1].…”

“… Previous studies have shown that relaxation parameters and fast protein dynamics can be quickly elucidated from 15 N-CEST experiments [1]. Longitudinal R 1 and transverse R 2 values were reliably derived from fitting of CEST profiles.…”

Characterization of Internal Protein Dynamics and Conformational Entropy by NMR Relaxation

“…Sometimes, an additional local R ex parameter is also fit to obtain information about slower timescale dynamics arising from conformational exchange which increases the minimum number of necessary experimental observables to 3 n +1 for the case of isotropic tumbling. So-called “lean” approaches (Gu, Hansen, Peng, & Bruschweiler, 2016) can be used to reduce data collection and a recent publication has provided a systematic and quantitative evaluation of using a reduced number of 15 N relaxation observables in the quantification of internal motion (Jaremko, Jaremko, Ejchart, & Nowakowski, 2018). Alternative methods utilizing cross correlated relaxation have also been introduced but will not be discussed further here (Pelupessy, Espallargas, & Bodenhausen, 2003; Reif, Diener, Hennig, Maurer, & Griesinger, 2000; Weaver, & Zuiderweg, 2008; Weaver, & Zuiderweg, 2009).…”