Recent developments in isotope-aided NMR methods for supramolecular protein complexes –SAIL aromatic TROSY

• Published in: Biochimica et biophysica acta. General subjects Vol. 1864; no. 2; p. 129439
• Main Authors: Miyanoiri, Yohei, Takeda, Mitsuhiro, Terauchi, Tsutomu, Kainosho, Masatsune
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.02.2020
• Subjects: amino acid substitution; amino acids; aromatic compounds; Aromatic ring CH TROSY; carbon; Identification of aromatic ring clusters; isotope labeling; lipid bilayers; malate synthase; Malate synthase G (MSG); mutants; nuclear magnetic resonance spectroscopy; proteins; protons; Sequence-specific assignment by single-amino acid substitution; stable isotopes; Stereo-array isotope labeled (SAIL) aromatic amino acids; structural biology; structure-activity relationships; Identification of aromatic ring clusters; Sequence-specific assignment by single-amino acid substitution; Aromatic ring CH TROSY; Stereo-array isotope labeled (SAIL) aromatic amino acids; Malate synthase G (MSG)
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2019.129439

The structure-function relationships for large protein complexes at the atomic level would be comprehensively understood, if hitherto unexplored aromatic ring NMR signals became accessible in addition to the currently used backbone amide and side-chain methyl signals. The 82 kDa malate synthase G (MSG) proteins, selectively labeled with Trp and Phe bearing relaxation optimized isotope-labeled rings, were prepared to investigate the optimal conditions for obtaining the aromatic TROSY spectra. The MSG proteins, selectively labeled with either [δ1,ε1,ε3,η2]-SAIL Trp or ζ-SAIL Phe, provided well-separated, narrow TROSY signals for the 12 Trp and 19 Phe residues in MSG. The signals were assigned sequence-specifically, using the set of single amino acid substitution mutants. The site-specific substitution of each Phe with Tyr or Leu induced substantial chemical shifts for the other aromatic ring signals, allowing us to identify the aromatic clusters in MSG, which were comparable to the structural domains proposed previously. We demonstrated that the aromatic ring 13CH pairs without directly bonded 13C and adjacent 1H spins provide surprisingly narrow TROSY signals, if the rings are surrounded by fully deuterated amino acids. The observed signals can be readily assigned by either the single amino acid substitution or the NOEs between the aromatic and methyl protons, if the methyl assignments are available. The method described here should be generally applicable for difficult targets, such as proteins in lipid bilayers or possibly in living cells, thus providing unprecedented opportunities to use these new probes in structural biology. •Innovative NMR strategy for investigating the aromatic residues in large proteins (81).•Sequence-specific aromatic ring NMR signal assignment by mutation (65)•Aromatic ring cluster identification for the 82 kDa malate synthase G protein (77).