an economical method for production of 2h,13ch3-threonine for solution nmr studies of large protein complexes application to the 670 kda proteasome一种经济的方法生产2 h,13大蛋白复合物应用程序的解决方案nmr研究ch3-threonine 670 kda蛋白酶体.pdfVIP

An Economical Method for Production of 2H,13CH - 3 Threonine for Solution NMR Studies of Large Protein Complexes: Application to the 670 kDa Proteasome 1 1 1,2 Algirdas Velyvis *, Amy M. Ruschak , Lewis E. Kay * 1 Departments of Molecular Genetics, Biochemistry, and Chemistry, University of Toronto, Toronto, Ontario, Canada, 2 Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, Canada Abstract NMR studies of very high molecular weight protein complexes have been greatly facilitated through the development of labeling strategies whereby 13CH3 methyl groups are introduced into highly deuterated proteins. Robust and cost-effective labeling methods are well established for all methyl containing amino acids with the exception of Thr. Here we describe an inexpensive biosynthetic strategy for the production of L-[a-2 2 13 H; b2 H;c- C]-Thr that can then be directly added during protein expression to produce highly deuterated proteins with Thr methyl group probes of structure and dynamics. These reporters are particularly valuable, because unlike other methyl containing amino acids, Thr residues are localized predominantly to the surfaces of proteins, have unique hydrogen bonding capabilities, have a higher propensity to be found at protein nucleic acid interfaces and can play important roles in signaling pathways through phosphorylation. The utility of the labeling methodology is demonstrated with an application to the 670 kDa