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Magnetochemistry 2018, 4(2), 25; https://doi.org/10.3390/magnetochemistry4020025

Efficient Stereospecific Hβ2/3 NMR Assignment Strategy for Mid-Size Proteins

1
Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, 12801 East 17th Avenue, Aurora, CO 80045, USA
2
Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
3
Department of Pharmacology, University of Colorado Anschutz Medical Campus, 12801 East 17th Avenue, Aurora, CO 80045, USA
*
Author to whom correspondence should be addressed.
Received: 2 May 2018 / Revised: 25 May 2018 / Accepted: 25 May 2018 / Published: 1 June 2018
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Abstract

We present a strategy for stereospecific NMR assignment of Hβ2 and Hβ3 protons in mid-size proteins (~150 residues). For such proteins, resonance overlap in standard experiments is severe, thereby preventing unambiguous assignment of a large fraction of β-methylenes. To alleviate this limitation, assignment experiments may be run in high static fields, where higher decoupling power is required. Three-bond Hα–Hβ J-couplings (3JHα–Hβ) are critical for stereospecific assignments of β-methylene protons, and for determining rotameric χ1 states. Therefore, we modified a pulse sequence designed to measure accurate 3JHα–Hβ couplings such that probe heating was reduced, while the decoupling performance was improved. To further increase the resolution, we applied non-uniform sampling (NUS) schemes in the indirect 1H and 13C dimensions. The approach was applied to two medium-sized proteins, odorant binding protein 22 (OBP22; 14.4 kDa) and Pin1 (18.2 kDa), at 900 MHz polarizing fields. The coupling values obtained from NUS and linear sampling were extremely well correlated. However, NUS decreased the overlap of Hβ2/3 protons, thus supplying a higher yield of extracted 3JHα-Hβ coupling values when compared with linear sampling. A similar effect could be achieved with linear prediction applied to the linearly sampled data prior to the Fourier transformation. Finally, we used 3JHα–Hβ couplings from Pin1 in combination with either conventional or exact nuclear Overhauser enhancement (eNOE) restraints to determine the stereospecific assignments of β-methylene protons. The use of eNOEs further increased the fraction of unambiguously assigned resonances when compared with procedures using conventional NOEs. View Full-Text
Keywords: NMR spectroscopy; biological macromolecules; structure calculation; stereospecific assignment; J-coupling; proteins NMR spectroscopy; biological macromolecules; structure calculation; stereospecific assignment; J-coupling; proteins
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Born, A.; Henen, M.A.; Nichols, P.; Wang, J.; Jones, D.N.; Vögeli, B. Efficient Stereospecific Hβ2/3 NMR Assignment Strategy for Mid-Size Proteins. Magnetochemistry 2018, 4, 25.

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