An NMR analysis of the reaction of ubiquitin with [acetyl-1-13C]aspirin.
The acetylation of ubiquitin by [acetyl-1-13C]aspirin has been studied using 2D NMR methods. Studies performed in a 50:50 H2O:D2O medium show doubling of the acetyl carbonyl resonances, indicating that all of the stable adducts formed involved amide linkages. Assignment of the heteronuclear multiple quantum coherence (HMQC) resonances was accomplished based on comparison of resonance intensities with the results of an Edman degradation analysis, pH titration studies of acetylated ubiquitin, and analysis of two ubiquitin mutants, K33R and K63R. The presence of a single tyrosine residue in close proximity to lysine-48 suggested another assignment strategy. Nitration of tyrosine-59 resulted in a small, pH-dependent shift of the resonance assigned to lysine-48, with a pK of 7.0, close to that expected for the nitrotyrosyl hydroxyl group. An additional adduct resonance with very low intensity also was observed and tentatively assigned to the acetylated N-terminal methionine residue. The relative rates of acetylation of the various lysine residues were obtained from time-dependent HMQC studies. Since no sample preparation artifacts were introduced, the levels of modification of the various residues could be determined with relatively high accuracy. Based on the time-dependent intensity data, the relative rate constants for modification of K6, K48, K63, K11, K33, and M1 were 1.0, 0.59, 0.43, 0.26, 0.23, and 0.03, respectively. These results were in much better agreement with amino accessibility predictions based on the crystal structure of the ubiquitin monomer than with predictions based on the ubiquitin structure in the crystallized dimeric and tetrameric forms. This approach provides a useful basis for understanding how local environmental factors can influence protein adduct formation, as well as for comparing the extent and specificity of various acetylation reagents.
Macdonald
J M
JM
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
LeBlanc
D A
DA
Haas
A L
AL
London
R E
RE
D000107:Acetylation; D001241:Aspirin / Q000138:chemical synthesis / Q000737:chemistry*; D002247:Carbon Isotopes; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D014452:Ubiquitins / Q000031:analogs & derivatives / Q000737:chemistry*
D002247:Carbon Isotopes; D014452:Ubiquitins; D001241:Aspirin