Abstract
Isotopically labeled methyl groups provide NMR probes in large, otherwise deuterated proteins. However, the resonance assignment constitutes a bottleneck for broader applicability of methyl-based NMR. Here, we present the automated MethylFLYA method for the assignment of methyl groups that is based on methyl-methyl nuclear Overhauser effect spectroscopy (NOESY) peak lists. MethylFLYA is applied to five proteins (28–358 kDa) comprising a total of 708 isotope-labeled methyl groups, of which 612 contribute NOESY cross peaks. MethylFLYA confidently assigns 488 methyl groups, i.e. 80% of those with NOESY data. Of these, 459 agree with the reference, 6 were different, and 23 were without reference assignment. MethylFLYA assigns significantly more methyl groups than alternative algorithms, has an average error rate of 1%, modest runtimes of 0.4–1.2 h, and can handle arbitrary isotope labeling patterns and data from other types of NMR spectra.
Highlights
Labeled methyl groups provide NMR probes in large, otherwise deuterated proteins
MethylFLYA was applied to the five largest proteins of a benchmark data set that was originally prepared for evaluating the MAGMA algorithm for automated methyl assignment, as described in the original publication[32]
Methyl NMR data for the 20 kDa N-terminal domain of Heat Shock Protein 9038, which has been used previously with MAGMA, were used for evaluating MethylFLYA in combination with automated peak picking with CYPICK37
Summary
Labeled methyl groups provide NMR probes in large, otherwise deuterated proteins. For optimal gains in the signal enhancement and resolution of methyl-TROSY spectra, selectively protonated, 13C-labelled methyl groups are introduced into an otherwise highly deuterated background[9]. To this end, cost-effective and robust biosynthetic strategies have been established for the selective or simultaneous labelling of all methyl-containing amino acids in Escherichia coli[10,11]. The major bottleneck for NMR studies with selective methyllabeled proteins is the resonance assignment, i.e. relating 1H/13C signals in the NMR spectra to specific methyl groups in the protein (Fig. 1)[16]. For large proteins, backbone resonances and triple-resonance spectra cannot be observed, and, unless the protein is modified, only nuclear
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