Oral Presentation 23rd International Society of Magnetic Resonance Conference 2023

Relaxation optimized stereo-array isotope labeling method for NMR studies of supramolecular proteins and protein complexes (#10)

Yohei Miyanoiri 1 , Tsutomu Terauchi 2 , Masatsune Kainosho 3
  1. Institute for Protein Research, Osaka University, Suita, Osaka, Japan
  2. SI Innovation Center, Taiyo Nippon Sanso Corporation, Tama, Tokyo, Japan
  3. Graduate School of Science, Tokyo Metropolitan University, Hachioji, Tokyo, Japan

For over a decade, we have been continuing to develop the stereo-array isotope labeling (SAIL) method [1] to elucidate the structures and dynamics of supramolecular proteins and protein complexes. So far, we have successfully observed and assigned 1H-13C NMR signals for many of the aromatic ring and aliphatic moieties in the 82 kDa malate synthase G (MSG), by using amino acids possessing various relaxation optimized isotope labeling patterns [2,3]. The concomitant development of cost-effective cellular expression systems using various amino acid auxotrophic E. coli strains now allows the preparation of proteins with residues selectively labeled with ro-SAIL amino acids, and otherwise fully deuterated [4]. These advances have enabled us to routinely investigate molecular interactions and dynamics for biologically interesting proteins associated with bacterial motor machinery, signal transduction, and antibody-antigen complexes, which are regarded as difficult targets for the conventional NMR methodologies. During the past few years, we have further improved the relaxation properties of the ro-SAIL amino acids by using “1H-12C” instead of “1H-13C”. By sacrificing the 13C-dimension with this labeling approach, the sparsely distributed remaining protons of the “1H-12C” ro-SAIL amino acid residues gave surprisingly well-resolved, highly sensitive 1H-NMR signals, even for the 468 kDa aminopeptidase, TET2 12-mer. Some of our future prospects for this new ro-SAIL approach will also be presented.

 

References

[1] Kainosho M, et al. (2006) Optimal isotope labelling for NMR protein structure determinations, Nature 440:52-57.

[2] Kainosho M, et al. (2018) Perspective: Next Generation Isotope-Aided Methods for Protein NMR Spectroscopy, J Biomol NMR 71: 119-127.

[3] Miyanoiri Y, et al. (2020) Recent developments in isotope-aided NMR methods for supramolecular protein complexes-SAIL aromatic TROSY, BBA-General Subjects 1864:12439.

[4] Miyanoiri Y, et al. (2016) Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E-coli strain, J Biomol NMR, 65, 109-119.