Abstract
Paramagnetic relaxation enhancements (PREs) are routinely used to provide long-range distance restraints for the determination of protein structures, to resolve protein dynamics, ligand–protein binding sites, and lowly populated species, using Nuclear Magnetic Resonance Spectroscopy (NMR). Here, we propose a simultaneous 1H-15 N, 1H-13C SESAME based pulse scheme for the rapid acquisition of 1HC/N-R2 relaxation rates for the determination of backbone and sidechain PREs of proteins. The 1HN-R2 rates from the traditional and our approach on Ubiquitin (UBQ) are well correlated (R2 = 0.99), revealing their potential to be used quantitatively. Comparison of the S57C UBQ calculated and experimental PREs provided backbone and side chain Q factors of 0.23 and 0.24, respectively, well-fitted to the UBQ NMR structure, showing that our approach can be used to acquire accurate PRE rates from the functionally important sites of proteins but in at least half the time as traditional methods.
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Acknowledgements
We would like to thank Dr. Duck-Yeon Lee of the Biochemistry Core at NHLBI for his assistance in the LC-MS data acquisition and analysis. We also would like to thank Dr. Marie-Paule Strub for her assistance in producing the mutant plasmids of Ubiquitin.
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This work was supported by the Intramural Research Program of the NHLBI, NIH.
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Conceived and designed the experiments: PM, NT. Performed the experiments: AB, MS and PM. Analyzed the data: PM, AB, NT, MS. Contributed reagents/materials/analysis tools: AB, NT. Wrote the paper: PM, AB.
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Barnes, C.A., Starich, M.R., Tjandra, N. et al. Simultaneous measurement of 1HC/N-R2′s for rapid acquisition of backbone and sidechain paramagnetic relaxation enhancements (PREs) in proteins. J Biomol NMR 75, 109–118 (2021). https://doi.org/10.1007/s10858-021-00359-9
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DOI: https://doi.org/10.1007/s10858-021-00359-9