Efficient Stereospecific H?2/3 NMR Assignment Strategy for Mid-Size Proteins.

ABSTRACT: We present a strategy for stereospecific NMR assignment of H?2 and H?3 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 ?-methylenes. To alleviate this limitation, assignment experiments may be run in high static fields, where higher decoupling power is required. Three-bond H?-H? J-couplings (3 J H?-H?) are critical for stereospecific assignments of ?-methylene protons, and for determining rotameric ?1 states. Therefore, we modified a pulse sequence designed to measure accurate 3 J H?-H? 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?2/3 protons, thus supplying a higher yield of extracted 3 J H?-H? 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 H?-H? couplings from Pin1 in combination with either conventional or exact nuclear Overhauser enhancement (eNOE) restraints to determine the stereospecific assignments of ?-methylene protons. The use of eNOEs further increased the fraction of unambiguously assigned resonances when compared with procedures using conventional NOEs.

SUBMITTER: Born A 

PROVIDER: S-EPMC6513325 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature