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Recent developments in isotope-aided NMR methods for supramolecular protein complexes -SAIL aromatic TROSY
Yohei Miyanoiri1, Mitsuhiro Takeda2, Tsutomu Terauchi3
1Research Center for State-of-the-Art Functional Protein Analysis, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan; Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
Biochimica Et Biophysica Acta. General Subjects
|October 9, 2019
Summary
This study introduces a new method to obtain atomic-level structural insights into large protein complexes by accessing previously unexplored aromatic ring NMR signals. This technique enhances structural biology by providing clearer data for complex protein structures.
Area of Science:
- Structural Biology
- Biophysics
- Nuclear Magnetic Resonance (NMR) Spectroscopy
Background:
- Understanding protein complex structure-function relationships at the atomic level is crucial.
- Current NMR methods primarily use backbone amide and side-chain methyl signals, limiting exploration of aromatic ring signals.
Purpose of the Study:
- To investigate optimal conditions for obtaining aromatic TROSY (Transverse Relaxation-Optimized Spectroscopy) spectra.
- To develop a method for accessing previously unexplored aromatic ring NMR signals in large protein complexes.
Main Methods:
- Prepared 82 kDa malate synthase G (MSG) proteins selectively labeled with Trp (Tryptophan) and Phe (Phenylalanine) bearing isotope-labeled rings.
- Utilized SAIL (Selectively Acquired Isotope Labeled) labeling for Trp and Phe residues.
- Employed single amino acid substitution mutants for sequence-specific signal assignment.
Main Results:
- Achieved well-separated, narrow TROSY signals for 12 Trp and 19 Phe residues in MSG.
- Identified aromatic clusters by observing chemical shifts induced by site-specific Phe substitutions.
- Demonstrated that aromatic ring 13CH pairs yield narrow TROSY signals when surrounded by deuterated amino acids.
Conclusions:
- Aromatic ring 13CH pairs without directly bonded 13C and adjacent 1H spins provide narrow TROSY signals under specific labeling conditions.
- Signal assignment is feasible using single amino acid substitution or NOEs (Nuclear Overhauser Effect) if methyl assignments are available.
- This method is generally applicable to challenging protein targets, including those in lipid bilayers or living cells.