Stereospecific assignments of glycine in proteins by stereospecific deuteration and 15N labeling

Summary A method is described for stereospecifically assigning the -protons of glycine residues in proteins. The approach involves the stereospecific deuteration and ¹⁵N labeling of glycine and subsequent selective incorporation of this residue into the protein. The stereospecific assignments of the glycine -protons are obtained from a comparison of a 3D ¹⁵N-resolved TOCSY spectrum of the uniformly ¹⁵N-labeled protein with a 2D/3D ¹⁵N-edited TOCSY spectrum of the protein, containing the stereospecifically deuterated and ¹⁵N-labeled glycine. The approach is demonstrated by stereospecifically assigning the glycine -protons of the FK506 binding protein when bound to the immunosuppressant ascomycin.