An NMR experiment for measuring methyl-methyl NOEs in 13C-labeled proteins with high resolution

被引:82
作者
Zwahlen, C
Gardner, KH
Sarma, SP
Horita, DA
Byrd, RA [1 ]
Kay, LE
机构
[1] NCI, Frederick Canc Res & Dev Ctr, ABL Basic Res Program, Macromol NMR Sect, Frederick, MD 21702 USA
[2] Univ Toronto, Prot Engn Network Ctr Excellence, Toronto, ON M5S 1A8, Canada
[3] Univ Toronto, Dept Med & Mol Genet, Toronto, ON M5S 1A8, Canada
[4] Univ Toronto, Dept Chem & Biochem, Toronto, ON M5S 1A8, Canada
[5] Hosp Sick Children, Toronto, ON M5G 1X8, Canada
关键词
D O I
10.1021/ja981205z
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
A three-dimensional NMR experiment is presented for correlating NOEs between methyl groups in protonated, N-15,C-13-labeled or methyl protonated, highly deuterated N-15,C-13-labeled proteins. The high resolution of this experiment facilitates the assignment of NOEs between methyls that can be poorly resolved in other; three- and four-dimensional experiments and extends the utility of solution-based NMR structural studies to proteins in the 40 kDa molecular weight regime. Applications to methyl protonated, highly deuterated samples of the 370 residue maltose binding protein (122 methyl groups) and the dimer of the 124 residue amino terminal domain of human STAT-4 (59 methyls) are presented. The method is also well suited for studies of fully protonated proteins, as demonstrated with an application involving the 160 residue phosphotyrosine binding domain from Drosophila Numb. Distance restraints obtained from the present experiment are particularly useful in the generation of global protein folds since many methyls are located in hydrophobic protein cores and inter-methyl restraints therefore link elements of secondary structure that are often distant in the primary sequence.
引用
收藏
页码:7617 / 7625
页数:9
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