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 13 C-dispersed NOESY-HSQC spectrum. Combination of these experiments with H(CC)NH-TOCSY and (H)C(C)NH-TOCSY may provide complete backbone and side-chain assignments for large proteins using only one or two samples.

[1]  B. Farmer,et al.  ASSIGNMENT OF SIDE-CHAIN 13C RESONANCES IN PERDEUTERATED PROTEINS , 1995 .

[2]  L. Kay,et al.  Assignment of 15N, 13Cα, 13Cβ, and HN Resonances in an 15N,13C,2H Labeled 64 kDa Trp Repressor−Operator Complex Using Triple-Resonance NMR Spectroscopy and 2H-Decoupling , 1996 .

[3]  S. Grzesiek,et al.  Carbon-13 line narrowing by deuterium decoupling in deuterium/carbon-13/nitrogen-15 enriched proteins. Application to triple resonance 4D J connectivity of sequential amides , 1993 .

[4]  P. Domaille,et al.  An Approach to the Structure Determination of Larger Proteins Using Triple Resonance NMR Experiments in Conjunction with Random Fractional Deuteration , 1996 .

[5]  Ad Bax,et al.  Rapid recording of 2D NMR spectra without phase cycling. Application to the study of hydrogen exchange in proteins , 1989 .

[6]  C. Allis,et al.  Solution structure of the catalytic domain of GCN5 histone acetyltransferase bound to coenzyme A , 1999, Nature.

[7]  Gareth A. Morris,et al.  Enhancement of nuclear magnetic resonance signals by polarization transfer , 1979 .

[8]  F. Richards,et al.  NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation. , 1988, Biochemistry.

[9]  E. Zuiderweg,et al.  Use of13C-13C NOE for the assignment of NMR lines of larger labeled proteins at larger magnetic fields , 1996 .

[10]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

[11]  S. Fesik,et al.  Heteronuclear three-dimensional NMR spectroscopy of isotopically labelled biological macromolecules , 1990, Quarterly Reviews of Biophysics.

[12]  Ad Bax,et al.  Three-dimensional heteronuclear NMR of nitrogen-15 labeled proteins , 1989 .

[13]  Weontae Lee,et al.  A Suite of Triple Resonance NMR Experiments for the Backbone Assignment of 15N, 13C, 2H Labeled Proteins with High Sensitivity , 1994 .

[14]  A. J. Shaka.  Composite pulses for ultra-broadband spin inversion , 1985 .

[15]  R. Freeman,et al.  An adaptable NMR broadband decoupling scheme , 1996 .

[16]  L. Mueller,et al.  Selective shaped pulse decoupling in NMR: homonuclear [carbon-13]carbonyl decoupling , 1992 .

[17]  Ad Bax,et al.  Methodological advances in protein NMR , 1993 .

[18]  Paul A. Keifer,et al.  Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity , 1992 .

[19]  Ad Bax,et al.  Proton-proton correlation via carbon-carbon couplings: a three-dimensional NMR approach for the assignment of aliphatic resonances in proteins labeled with carbon-13 , 1990 .