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Accurate measurement of dipole/dipole transverse cross-correlated relaxation \(\varGamma _2\) in methylenes and primary amines of uniformly \({}^{13}\text {C}/{}^{15}\text {N}\)-labeled proteins

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Abstract

Side chains possess a broader conformational space (compared to the backbone) and are directly affected by intra- and intermolecular interactions, hence their dynamics and the corresponding NMR relaxation data are more sensitive and informative. Nevertheless, transverse relaxation in \(\text {XH}_2\) (\(\text {X}={}^{13}\text {C}\) or \({}^{15}\text {N}\)) spin systems is predominantly non-measurable in uniformly \({}^{13}\text {C}/{}^{15}\text {N}\)-labeled proteins due to cross-correlation effects. In the present publication, we propose a number of pulse sequences for accurate and precise measurement of the dipole-dipole transverse cross-correlated relaxation rate \(\varGamma _{\text {2XH,XH}'}\), which, similarly to \(R_2\) measurements, provides information about the amplitudes of intramolecular dynamics. The suggested approach has allowed us to circumvent a number of obstacles that were limiting earlier applications of \(\varGamma _{\text {2XH,XH}'}\): (1) impossibility of transmission of the central component of the triplet of \(\text {XH}_2\) group to \({}^{1}\text {H}\)-acquisition via INEPT has been solved by transmission of the averaged signal of “inner” and “outer” components of the triplet; (2) direct recording of the entire triplets resulting in substantial overlap of side chain signals has been replaced by recording of individual singlets with the use of \(^1\!J_\text {CH}\)-modulated approach and constant-time evolution; (3) low sensitivity has been enhanced via proton acquisition which required special attention to a zero-quantum coherence evolution. The proposed method expands the set of “dynamics sensors” covering protein side chains and substantially improves the quality and the level of detail of experimental data describing dynamic processes in proteins and protein complexes.

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Acknowledgements

The work was supported by the Russian Science Foundation grant #18-14-00375: pulse sequence development and data analysis. NMR experiments were carried out using the equipment provided by the IBCH core facility (CKP IBCH, Grant RFMEFI62117X0018 from Russian Ministry of Education and Science) and partially supported by the Russian Academy of Science program “Molecular and cellular biology” and the Russian Foundation for Basic Research grant #18-04-01289. The authors thank Dr. A.A. Shulga for the sample of uniformly \({}^{13}\text {C}/{}^{15}\text {N}\)-labeled Neurotoxin II.

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Correspondence to Eduard V. Bocharov.

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Lesovoy, D.M., Dubinnyi, M.A., Nolde, S.B. et al. Accurate measurement of dipole/dipole transverse cross-correlated relaxation \(\varGamma _2\) in methylenes and primary amines of uniformly \({}^{13}\text {C}/{}^{15}\text {N}\)-labeled proteins. J Biomol NMR 73, 245–260 (2019). https://doi.org/10.1007/s10858-019-00252-6

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