Selective 15N labeling and direct observation by NMR of the active-site glutamine of Fe-containing superoxide dismutase.
The glutamine in position 69 is one of only three conserved active-site amino acid differences between Fe- and Mn-containing superoxide dismutases (SODs). We have refined the conditions for extremely selective labeling of the side chains of glutamine with 15N, and thus obtained dramatically simplified spectra, despite the large size of Fe-SOD. The improved resolution afforded by such highly specific labeling permits the use of direct 15N detection to observe and assign Gln 69, even though its distance to the paramagnetic Fe2+ is only 5 A. Selective glutamine side-chain labeling is inexpensive and has general utility for large (and paramagnet-containing) proteins.
Vance
C K
CK
Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA.
Kang
Y M
YM
Miller
A F
AF
D001665:Binding Sites; D003001:Cloning, Molecular; D005973:Glutamine; D007501:Iron; D007527:Isoenzymes / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008345:Manganese; D009587:Nitrogen Isotopes; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D013482:Superoxide Dismutase / Q000737:chemistry*
D007527:Isoenzymes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D005973:Glutamine; D008345:Manganese; D007501:Iron; D013482:Superoxide Dismutase