Efficient Stereospecific H(β2/3) NMR Assignment Strategy for Mid-Size Proteins

We present a strategy for stereospecific NMR assignment of H(β2) and H(β3) 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 H(α)–H(β) J-couplings ((3)J(Hα–Hβ)) are critical for stereospecific assignments of β-methylene protons, and for determining rotameric χ(1) states. Therefore, we modified a pulse sequence designed to measure accurate (3)J(Hα–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 (1)H and (13)C 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 H(β2/3) protons, thus supplying a higher yield of extracted (3)J(Hα-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 (3)J(Hα–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.