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.
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.
Vadrevu
Ramakrishna
R
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Wu
Ying
Y
Matthews
C Robert
CR
D000577:Amides / Q000737:chemistry; D000596:Amino Acids; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology*; D006859:Hydrogen; D006860:Hydrogen Bonding / Q000187:drug effects; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D009587:Nitrogen Isotopes; D017510:Protein Folding; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D013997:Time Factors; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
D000577:Amides; D000596:Amino Acids; D009587:Nitrogen Isotopes; D021122:Protein Subunits; D006859:Hydrogen; D014508:Urea; D003903:Deuterium; D014367:Tryptophan Synthase