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 χ₁ 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 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 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.