GLOBAL FOLDS OF HIGHLY DEUTERATED, METHYL-PROTONATED PROTEINS BY MULTIDIMENSIONAL NMR

The development of N-15, C-13, H-2 multidimensional NMR spectroscopy h as facilitated the assignment of backbone and side chain resonances of proteins and protein complexes with molecular masses of over 30 kDa. The success of these methods has been achieved through the production of highly deuterated proteins; replacing carbon-bound protons with deu terons significantly improves the sensitivity of many of the experimen ts used in chemical shift assignment. Unfortunately, uniform deuterati on also radically depletes the number of interproton distance restrain ts available for structure determination, degrading the quality of the resulting structures. Here we describe an approach for improving the precision and accuracy of global folds determined from highly deuterat ed proteins through the use of deuterated, selectively methyl protonat ed samples. This labeling profile maintains the efficiency of triple-r esonance NMR experiments while retaining a sufficient number of proton s at locations where they can be used to establish NOE-based contacts between different elements of secondary structure. We evaluate how thi s deuteration scheme affects the sensitivity and resolution of experim ents used to assign N-15, C-13, and H-1 chemical shifts and interproto n NOEs. This approach is tested experimentally on a 14 kDa SH2/phospho peptide complex, and a global protein fold is obtained from a set of m ethyl-methyl, methyl-NH, and NH-NH distance restraints. We demonstrate that the inclusion of methyl-NH and methyl-methyl distance restraints greatly improves the precision and accuracy of structures relative to those generated with only NH-NH distance restraints. Finally, we exam ine the general applicability of this approach by determining the stru ctures of several proteins with molecular masses of up to 40 kDa from simulated distance and dihedral angle restraint tables.