Stereospecific assignments of glycine in proteins by stereospecific deuteration and 15N labeling.
A method is described for stereospecifically assigning the alpha-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 alpha-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 alpha-protons of the FK506 binding protein when bound to the immunosuppressant ascomycin.
Curley
R W
RW
Ohio State University, Columbus 43210.
Panigot
M J
MJ
Hansen
A P
AP
Fesik
S W
SW
D002352:Carrier Proteins / Q000737:chemistry*; D002460:Cell Line; D003001:Cloning, Molecular; D003903:Deuterium; D005998:Glycine / Q000032:analysis*; D006360:Heat-Shock Proteins / Q000737:chemistry*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013601:T-Lymphocytes; D016559:Tacrolimus / Q000378:metabolism; D022021:Tacrolimus Binding Proteins
D002352:Carrier Proteins; D006360:Heat-Shock Proteins; D009587:Nitrogen Isotopes; D011506:Proteins; D011994:Recombinant Proteins; D003903:Deuterium; D022021:Tacrolimus Binding Proteins; D005998:Glycine; D016559:Tacrolimus