An improved strategy for determining resonance assignments for isotopically enriched proteins and its application to an engineered domain of staphylococcal protein A.
Sequence-specific resonance assignments provide the basis for interpreting multidimensional NMR spectra and for determining 3D structures of proteins from these data. We have developed an improved strategy for determining these sequence-specific NMR assignments in small proteins and applied this method in determining proton and nitrogen resonance assignments for an 8.2-kDa engineered domain (the Z-domain) of the cell wall protein A of Staphylococcus aureus. First, HCCNH-TOCSY [Lyons, B. A. & Montelione, G.T. (1993) J. Magn. Reson. 101B, 206] data were used together with 2D 2QF-COSY, TOCSY, and 15N-HSQC data to identify amino acid spin systems. Most asparagine and glutamine spin systems were also identified uniquely from these triple-resonance data. Next, complementary HCC(CO)-NH-TOCSY [Montelione, G. T., et al. (1992) J. Am. Chem. Soc. 114, 10975] data were used to identify sequential connections from the aliphatic H alpha, H beta, H gamma, H delta, and H epsilon resonances of residue i to the amide and nitrogen resonances of residue i + 1. By combined analysis of HCCNH-TOCSY and HCC(CO)NH-TOCSY spectra we have determined most of the proton and nitrogen resonance assignments for the Z-domain. This represents the first example of the use of this triple-resonance technique to determine extensive resonance assignments in a small protein.
Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08854-5638.
B A
BA
Lyons
M
M
Tashiro
L
L
Cedergren
B
B
Nilsson
G T
GT
Montelione
D000595:Amino Acid Sequence; D001666:Binding Sites, Antibody; D002247:Carbon Isotopes; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000302:isolation & purification
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011522:Protons; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A