Structure-based methyl resonance assignment with MethylFLYA

Abstract

Isotope-labeled methyl groups provide NMR probes that can be observed in very large, otherwise deuterated systems and enable investigations of protein structure, dynamics and mechanisms.However, the assignment of resonances to specific methyls in the protein is expensive and timeconsuming, which limits the use of methyl-based NMR for large proteins. To resolve this bottleneck, methyl assignment methods have been developed. However, these remain limited regarding complete automation, computational feasibility, and/or the extent and accuracy of the assignments. 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 was applied to five proteins (28-358 kDa) comprising a total of 708 isotopelabeled methyl groups, of which 674 had manually determined 1 H/ 13 C reference assignments and 614 showed cross peaks in the available NOESY peak lists. MethylFLYA confidently assigned 488 methyl groups, i.e. 79% of those with NOESY data. Of these, 460 agreed with the reference, 5 were different, and 23 concerned methyls without reference assignment. For high-quality NOESY spectra, automatic NOESY peak picking followed by resonance assignment with MethylFLYA can yield results comparable to those obtained from manually prepared peak lists, indicating the feasibility of unbiased, fully automatic methyl resonance assignment starting directly from the NMR spectra. Overall, MethylFLYA assigns significantly more methyl groups than other algorithms, has an average error rate of 1%, modest runtimes of 0.4-1.2 h, and flexibility to handle arbitrary isotope labeling patterns and include data from other types of NMR spectra.

Assignment of IVL-Methyl side chain of the ligand-free monomeric human MALT1 paracaspase-IgL 3 domain in solution

“…We started with the conventional approach, where the methyl resonances were connected to the known backbone assignments using methyl out-and-back experiments (Tugarinov et al, 2014). Then, the methyl assignments were validated and further expanded using the second approach based on Nuclear Oberhausen Effect (NOE) cross-peak data, peak residue type classi cation and a known 3D structure or a reliable structural model (Rossi et al, 2016, Pritišanac et al, 2019, Nerli et al, 2021.…”

Spectral editing of intra- and inter-chain methyl–methyl NOEs in protein complexes

“…Such a sample can be used with the CT 2 -HMQC-NOESY-HMQC experiment (Fig. 1) to detect exclusively 'precious' intrachain NOEs, enabling assignment of methyl resonances based on a previously known structure of symmetric oligomers (Xu and Matthews 2013;Chao et al 2014;Pritišanac et al 2017;Monneau et al 2017;Pritišanac et al 2019).…”

“…These distance restraints are particularly useful to determine the structure of large proteins and protein complexes Gauto et al 2019). If the tertiary structure of a protein is previously known, the network of NOE connectivities can also be exploited for automated NMR assignment of methyl resonances (Xu and Matthews 2013;Chao et al 2014;Pritišanac et al 2017;Monneau et al 2017;Pritišanac et al 2019). In protein complexes, special care should be taken to separate the large number of intra-chain NOEs from the few inter-chain signals, as any erroneous interpretation could significantly impact NMR assignment or structure determination (Werner et al 1997).…”