Multiquantum Chemical Exchange Saturation Transfer NMR to Quantify Symmetrical Exchange: Application to Rotational Dynamics of the Guanidinium Group in Arginine Side Chains.

Gogulan Karunanithy,Jochen Reinstein,D Flemming Hansen

doi:10.1021/acs.jpclett.0c01322

Abstract

Chemical exchange saturation transfer (CEST) NMR experiments have emerged as a powerful tool for characterizing dynamics in proteins. We show here that the CEST approach can be extended to systems with symmetrical exchange, where the NMR signals of all exchanging species are severely broadened. To achieve this, multiquantum CEST (MQ-CEST) is introduced, where the CEST pulse is applied to a longitudinal multispin order density element and the CEST profiles are encoded onto nonbroadened nuclei. The MQ-CEST approach is demonstrated on the restricted rotation of guanidinium groups in arginine residues within proteins. These groups and their dynamics are essential for many enzymes and for noncovalent interactions through the formation of hydrogen bonds, salt-bridges, and π-stacking interactions, and their rate of rotation is highly indicative of the extent of interactions formed. The MQ-CEST method is successfully applied to guanidinium groups in the 19 kDa L99A mutant of T4 lysozyme.

Highlights

Chemical exchange saturation transfer (CEST) NMR experiments have emerged as a powerful tool for characterizing dynamics in proteins
A number of NMRbased approaches for characterizing exchange on these time scales exist and provide important insights into conformations that are transiently populated, invisible to other high-resolution methods, and broadened beyond detection in traditional NMR experiments.[3−6] chemical exchange saturation transfer (CEST) methods have traditionally been used within the MRI field,[7−9] CEST approaches have recently emerged as powerful tools for studying biomolecular dynamics on a time scale from 20 to 200 s−1.10,11 In these experiments, first developed in the 1960s,12 saturation of magnetization by a weak pulse is transferred by exchange events within a network of exchanging conformers, and in particular, magnetization is transferred from invisible species to visible species in order to report on chemical shifts and rates of exchange
We present a multiquantum CEST (MQ-CEST) NMR experiment that is ideally suited to characterizing dynamics in symmetrically exchanging groups, and we apply this methodology to quantify the rotational dynamics of guanidinium groups in the side chains of arginine residues within proteins

Summary

■ EXPERIMENTAL METHODS

Sample preparations are described in the Supporting Information. All spectra were processed using NMRPipe[32] and visualized with NMRFAM-Sparky.[33]. Samples of arginine and T4L99A sample preparations; a detailed list of experimental conditions for all NMR experiments, Table S1; detailed description of the NMR and computational methods; supporting tables with parameters derived and pulse sequences for the experiments performed;. Supplementary Figures S1−S9 showing simulated MQ-CEST data; detailed NMR pulse sequence diagrams including for the 1H-detected sequence; a comparison of the sensitivity of 13C and 1H detected MQ-CEST spectra; all experimental data for T4L99A; and an error analysis of fitted parameters for T4L99A and analysis of the effects of relaxation rates on the results (PDF).

■ ACKNOWLEDGMENTS

■ REFERENCES