Progress toward automated methyl assignments for methyl-TROSY applications

Abstract

Highlights d Presenting an interface between the data analysis platform and MAGIC algorithm d MAGIC-Net methyl network analysis for selection of methyl labeling scheme d MAGIC-Act semi-automated preparation of experimental data d MAGIC-View for unbiased comparison of different NOEbased assignment protocols

Synthesis of 13C-methyl-labeled amino acids and their incorporation into proteins in mammalian cells

“…[14][15][16][17][18] Procedures have also been developed for the backbone-independent assignment of methyl signals, the most reliable of which is mutagenesis, 19 though for larger proteins with many methyl groups this may be impractical, and other approaches that leverage existing structural information may be used to attain assignments. [20][21][22][23][24][25][26][27][28] Methyl labeling, coupled with sensitivity enhancement by cross-correlation approaches, has had an enormous impact in terms of extending the applicability of NMR to study the structure and function of much larger and more complex systems than previously possible using uniform labeling with 15 N, 13 C, and 2 H isotopes. 14,16 Nonetheless, there are still many proteins, such as mammalian membrane receptors or secreted proteins that are heavily disulfide-bonded, that can be very difficult or impossible to express in bacteria, precluding methyl labeling using the aforementioned metabolic precursors.…”

“…14–18 Procedures have also been developed for the backbone-independent assignment of methyl signals, the most reliable of which is mutagenesis, 19 though for larger proteins with many methyl groups this may be impractical, and other approaches that leverage existing structural information may be used to attain assignments. 20–28…”