Multi-state Unfolding of the Alpha Subunit of Tryptophan Synthase, a TIM Barrel Protein: Insights into the Secondary Structure of the Stable Equilibrium Intermediates by Hydrogen Exchange Mass Spectrometry

The urea-induced unfolding of the α subunit of tryptophan synthase (αTS) from Escherichia coli, an eight-stranded (β/α)8 TIM barrel protein, has been shown to involve two stable equilibrium intermediates, I1 and I2, well populated at ∼3 M and 5 M urea, respectively. The characterization of the I1 intermediate by circular dichroism (CD) spectroscopy has shown that I1 retains a significant fraction of the native ellipticity; the far-UV CD signal for the I2 species closely resembles that of the fully unfolded form. To obtain detailed insight into the disruption of secondary structure in the urea-induced unfolding process, a hydrogen exchange-mass spectrometry study was performed on αTS. The full-length protein was destabilized in increasing concentration of urea, the amide hydrogen atoms were pulse-labeled with deuterium, the labeled samples were quenched in acid and the products were analyzed by electrospray ionization mass spectrometry. Consistent with the CD results, the I1 intermediate protects up to ∼129 amide hydrogen atoms against exchange while the I2 intermediate offers no protection. Electrospray ionization mass spectrometry analysis of the peptic fragments derived from αTS labeled at 3 M urea indicates that most of the region between residues 12–130, which constitutes the first four β strands and three α helices, (β/α)1–3β4, is structured. The (β/α)1–3β4 module appears to represent the minimum sub-core of stability of the I1 intermediate. A 4+2+2 folding model is proposed as a likely alternative to the earlier 6+2 folding mechanism for αTS.