NMR analysis of partially folded states and persistent structure in the alpha subunit of tryptophan synthase: implications for the equilibrium folding mechanism of a 29-kDa TIM barrel protein
Department of Biochemistry and Molecular Pharmacology
Amides; Amino Acids; Deuterium; Escherichia coli; Hydrogen; Hydrogen Bonding; Isotope Labeling; Magnetic Resonance Spectroscopy; Molecular Weight; Nitrogen Isotopes; *Protein Folding; Protein Structure, Secondary; Protein Subunits; Thermodynamics; Time Factors; Tryptophan Synthase; Urea
Life Sciences | Medicine and Health Sciences
Structural insights into the equilibrium folding mechanism of the alpha subunit of tryptophan synthase (alpha TS) from Escherichia coli, a (beta alpha)(8) TIM barrel protein, were obtained with a pair of complementary nuclear magnetic resonance (NMR) spectroscopic techniques. The secondary structures of rare high-energy partially folded states were probed by native-state hydrogen-exchange NMR analysis of main-chain amide hydrogens. 2D heteronuclear single quantum coherence NMR analysis of several (15)N-labeled nonpolar amino acids was used to probe the side chains involved in stabilizing a highly denatured intermediate that is devoid of secondary structure. The dynamic broadening of a subset of isoleucine and leucine side chains and the absence of protection against exchange showed that the highest energy folded state on the free-energy landscape is stabilized by a hydrophobic cluster lacking stable secondary structure. The core of this cluster, centered near the N-terminus of alpha TS, serves as a nucleus for the stabilization of what appears to be nonnative secondary structure in a marginally stable intermediate. The progressive decrease in protection against exchange from this nucleus toward both termini and from the N-termini to the C-termini of several beta-strands is best described by an ensemble of weakly coupled conformers. Comparison with previous data strongly suggests that this ensemble corresponds to a marginally stable off-pathway intermediate that arises in the first few milliseconds of folding and persists under equilibrium conditions. A second, more stable intermediate, which has an intact beta-barrel and a frayed alpha-helical shell, coexists with this marginally stable species. The conversion of the more stable intermediate to the native state of alpha TS entails the formation of a stable helical shell and completes the acquisition of the tertiary structure.
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Citation: J Mol Biol. 2008 Mar 14;377(1):294-306. Epub 2007 Nov 13. Link to article on publisher's site
DOI of Published Version
Journal of molecular biology
Vadrevu, Ramakrishna; Wu, Ying; and Matthews, C. Robert, "NMR analysis of partially folded states and persistent structure in the alpha subunit of tryptophan synthase: implications for the equilibrium folding mechanism of a 29-kDa TIM barrel protein" (2008). Open Access Articles. 2051.