NMR assignment of protein side chains using residue-correlated labeling and NOE spectra.
A new approach for the isotopic labeling of proteins is proposed that aims to facilitate side chain resonance assignments. Residue-correlated (RC) labeling is achieved by the expression of a protein on a medium containing a mixture of labeled, e.g., [U-13C,15N]amino acids, and NMR silent, [U-2H]amino acids. De novo synthesis of amino acids was suppressed by feedback inhibition by the amino acids in the growth medium and by the addition of beta-chloro-L-alanine, a transaminase inhibitor. Incorporation of these amino acids into synthesized proteins results in a relative diminution of inter-residue NOE interactions and a relative enhancement of intra-residue NOEs. Comparison of the resulting NOE spectra with those obtained from a uniformly labeled sample allows identification of intra-residue NOE peaks. Thus, this approach provides direct information for sidechain assignments in the NOE spectra, which are subsequently used for structural analysis. We have demonstrated the feasibility of this strategy for the 143 amino acid nuclease inhibitor NuiA, both at 35 degrees C, corresponding to a rotational correlation time of 9.5 ns, and at 5 degrees C, corresponding to a rotational correlation time of 22 ns.
Mueller
Geoffrey A
GA
National Institute of Environmental Health Sciences, Laboratory of Structural Biology, 111 Alexander Drive, PO Box 12233 MD MR-01, Research Triangle Park, NC 27709, USA.
Kirby
Thomas W
TW
DeRose
Eugene F
EF
London
Robert E
RE
D000465:Algorithms; D000596:Amino Acids / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D002247:Carbon Isotopes; D005240:Feasibility Studies; D005947:Glucose / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes; D010649:Phenylalanine / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D013113:Spin Labels / Q000138:chemical synthesis*; D013223:Statistics as Topic; D014633:Valine / Q000737:chemistry; D015091:beta-Alanine / Q000031:analogs & derivatives* / Q000737:chemistry*
D000596:Amino Acids; D001426:Bacterial Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; C106206:NuiA protein, Anabaena; D011506:Proteins; D013113:Spin Labels; D015091:beta-Alanine; C006977:3-chloroalanine; D010649:Phenylalanine; D014633:Valine; D005947:Glucose