Partial NMR assignments and secondary structure mapping of the isolated α subunit of Escherichia coli tryptophan synthase, a 29‐kD TIM barrel protein

• Published in: Protein science Vol. 12; no. 1; pp. 185 - 191
• Main Authors: Vadrevu, Ramakrishna, Falzone, Christopher J., Matthews, C. Robert
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01-01-2003
• Subjects: 15N‐ selective labeling; 3D NMR; Amino Acid Sequence; CSI, Chemical shift index; DTE, dithioerythritol; Escherichia coli - enzymology; folding intermediates; For the Record; HSQC, heteronuclear single quantum coherence; H‐D exchange; H‐D, hydrogen‐deuterium; I1, equilibrium intermediate of αTS maximally populated at ∼3 M urea; I2, equilibrium intermediate of αTS maximally populated at ∼5 M urea; K2EDTA, ethylenediaminetetraacetic acid, dipotassium salt; Molecular Sequence Data; NOE, nuclear Overhauser enhancement; NOESY, nuclear Overhauser enhancement spectroscopy; nuclear magnetic resonance; Nuclear Magnetic Resonance, Biomolecular; ppm, parts per million; Protein Folding; Protein Structure, Secondary; Protein Subunits; TIM, triosephosphate isomerase; Tryptophan Synthase - chemistry; αTS, the α subunit of tryptophan synthase from Escherichia coli
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.0221103

The α subunit of tryptophan synthase (αTS) from S. typhimurium belongs to the triosephosphate isomerase (TIM) or the (β/α)8 barrel fold, one of the most common structures in biology. To test the conservation of the global fold in the isolated Escherichia coli homolog, we have obtained a majority of the backbone assignments for the 29‐kD αTS by using standard heteronuclear multidimensional NMR methods on uniformly 15N‐ and 15N/13C‐labeled protein and on protein selectively 15N‐labeled at key hydrophobic residues. The secondary structure mapped by chemical shift index, nuclear Overhauser enhancements (NOEs), and hydrogen‐deuterium (H‐D) exchange, and several abnormal chemical shifts are consistent with the conservation of the global TIM barrel fold of the isolated E. coli αTS. Because most of the amide protons that are slow to exchange with solvent correspond to the β‐sheet residues, the β‐barrel is likely to play an important role in stabilizing the previously detected folding intermediates for E. coli αTS. A similar combination of uniform and selective labeling can be extended to other TIM barrel proteins to obtain insight into the role of the motif in stabilizing what appear to be common partially folded forms.