| Autor: |
Born, Alexandra, Henen, Morkos A., Nichols, Parker, Jing Wang, Jones, David N., Vögeli, Beat |
| Předmět: |
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| Zdroj: |
Magnetochemistry; 2018, Vol. 4 Issue 2, p1-15, 15p |
| Abstrakt: |
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 fi-methylenes. To alleviate this limitation, assignment experiments may be run in high static fields, where higher decoupling power is required. Three-bond Hα-β J-couplings (3JHα-β) are critical for stereospecific assignments of fi-methylene protons, and for determining rotameric -1 states. Therefore, we modified a pulse sequence designed to measure accurate 3JHα-β 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-β 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-β couplings from Pin1 in combination with either conventional or exact nuclear Overhauser enhancement (eNOE) restraints to determine the stereospecific assignments of fi-methylene protons. The use of eNOEs further increased the fraction of unambiguously assigned resonances when compared with procedures using conventional NOEs. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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