Solution structure of the carboxyl terminus of a human class Mu glutathione S-transferase: NMR assignment strategies in large proteins

Strategies to obtain the NMR assignments for the H-N, N, CO, C-alpha and C- beta resonance frequencies for the human class mu glutathione-S-transferase GSTM2-2 are reported. These assignments were obtained with deuterated prot ein using a combination of scalar and dipolar connectivities and various sp ecific labeling schemes. The large size of this protein (55 kDa, homodimer) necessitated the development of a novel pulse sequence and specific labeli ng strategies. These aided in the identification of residue type and were e ssential components in determining sequence specific assignments. These ass ignments were utilized in this study to characterize the structure and dyna mics of the carboxy-terminal residues in the unliganded protein. Previous c rystallographic studies of this enzyme in complex with glutathione suggeste d that this region may be disordered, and that this disorder may be essenti al for catalysis. Furthermore, in the related class alpha protein extensive changes in conformation of the C terminus are observed upon ligand binding . On the basis of the results presented here, the time-averaged conformatio n of the carboxyl terminus of unliganded GSTM2-2 is similar to that seen in the crystal structure. NOE patterns and H-1-N-15 heteronuclear nuclear Ove rhauser enhancements suggest that this region of the enzyme does not underg o motion on a rapid time scale. (C) 1999 Academic Press.