Efficient side-chain and backbone assignment in large proteins: application to tGCN5.
In determining the structure of large proteins by NMR, it would be desirable to obtain complete backbone, side-chain, and NOE assignments efficiently, with a minimum number of experiments and samples. Although new strategies have made backbone assignment highly efficient, side-chain assignment has remained more difficult. Faced with the task of assigning side-chains in a protein with poor relaxation properties, the Tetrahymena histone acetyltransferase tGCN5, we have developed an assignment strategy that would provide complete side-chain assignments in cases where fast 13C transverse relaxation causes HCCH-TOCSY experiments to fail. Using the strategy presented here, the majority of aliphatic side-chain proton and carbon resonances can be efficiently obtained using optimized H(CC-CO)NH-TOCSY and (H)C(C-CO)NH-TOCSY experiments on a partially deuterated protein sample. Assignments can be completed readily using additional information from a 13C-dispersed NOESY-HSQC spectrum. Combination of these experiments with H(CC)NH-TOCSY and (H)C(C)NH-TOSCY may provide complete backbone and side-chain assignments for large proteins using only one or two samples.
Lin
Y
Y
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Wagner
G
G
D000123:Acetyltransferases / Q000737:chemistry*; D000818:Animals; D003903:Deuterium; D051548:Histone Acetyltransferases; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017433:Protein Structure, Secondary; D029701:Saccharomyces cerevisiae Proteins; D012680:Sensitivity and Specificity; D013768:Tetrahymena / Q000737:chemistry*
D029701:Saccharomyces cerevisiae Proteins; D003903:Deuterium; D000123:Acetyltransferases; D051548:Histone Acetyltransferases