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 |
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Authors: | Vadrevu Ramakrishna Wu Ying Matthews C Robert |
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Institution: | Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA |
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Abstract: | Structural insights into the equilibrium folding mechanism of the alpha subunit of tryptophan synthase (αTS) from Escherichia coli, a (βα)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 15N-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 αTS, 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 β-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 β-barrel and a frayed α-helical shell, coexists with this marginally stable species. The conversion of the more stable intermediate to the native state of αTS entails the formation of a stable helical shell and completes the acquisition of the tertiary structure. |
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Keywords: | αTS alpha subunit of tryptophan synthase CD circular dichroism EDTA ethylenediaminetetraacetic acid ΔGHXo free energy of protection against hydrogen exchange in the absence of denaturant HSQC heteronuclear single quantum coherence HX hydrogen-to-deuterium exchange of amide hydrogens I1 equilibrium unfolding intermediate of αTS highly populated at 3  M urea I2 equilibrium unfolding intermediate of αTS highly populated at 5  M urea IBP kinetic intermediate of αTS populated within the stopped-flow burst phase (< 5  ms) N state native state NMR nuclear magnetic resonance TIM triosephosphate isomerase U state unfolded state |
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