Folding Mechanism of Indole-3-glycerol Phosphate Synthase from Sulfolobus solfataricus: A Test of the Conservation of Folding Mechanisms Hypothesis in (βα)8 Barrels

Structural Analysis of Kinetic Folding Intermediates for a TIM Barrel Protein, Indole-3-glycerol Phosphate Synthase, by Hydrogen Exchange Mass Spectrometry and Gō Model Simulation

“…1,2 By contrast, larger proteins often acquire substantial far-UV ellipticity in the sub-millisecond time range, far faster than the appearance of the native state. [3][4][5] The significance of this secondary structure to the folding mechanism has been a subject of controversy for several years. One view holds that this "burst-phase" species is simply the contraction of the ensemble of unfolded states in response to a solvent that favors the native state.…”

“…Eight α-helices alternate in sequence with the β-strands and form an amphipathic shell surrounding the barrel; loops between the C-termini of β-strands and the N-termini of the following α-helices form active sites that are capable of catalyzing a host of chemical reactions. 18,19 The chemically-induced unfolding of TIM barrels invariably involves at least one stable intermediate that is rich in secondary structure, 5,20 and the kinetic folding mechanism displays additional complexities. 21 Notable among these is the appearance of 25-50% of the far-UV ellipticity within a few milliseconds, i.e., burst-phase species that are candidates for the HX test of their stabilities and, thereby, roles in folding.…”

“…Previous studies of the kinetic folding mechanisms of three TIM barrels, the alpha subunit of tryptophan synthase, 21 indole-3-glycerol phosphate synthase 5 and a protein of unknown function from yeast, IOLI 22 assumed that the burst-phase species were folding intermediates. These assumptions were based upon the sigmoidal loss of the CD amplitude of the burst-phase with increasing final denaturant concentration in refolding jumps and, in the case of αTS, on the observation of a subsequent refolding phase that accelerated at increasing denaturant concentration.…”

“…The near coincidence of the urea-melting profiles for the far-UV ellipticities of the equilibrium intermediate in sIPGS and its burst-phase species led to the conclusion that they were one and the same species, i.e., the burst-phase reaction led directly to the on-pathway equilibrium intermediate. 5 A third type of response is displayed by IOLI, whose unfolded form is thought to partition between off-and on-pathway intermediates in the millisecond time frame. 22 Previous HX analyses of the equilibrium folding intermediates in αTS 23 and sIGPS, 24 employing pepsin digestion at acidic pH where mass-labeled amides can be examined by MALDI mass spectrometry, provided insights into the structures of their respective partiallyfolded forms.…”

“…In the case of sIGPS, the HX-MS data were sufficiently rich to allow the determination of the structures of a pair of stable intermediates predicted by kinetic studies of the folding reaction. 5 Differences in the structures of these equilibrium intermediates were attributed to sequence-specific hydrophobic clusters formed by isoleucine, leucine and valine side chains. 24 Complications with the folding properties of αTS, i.e., four parallel folding channels, and IOLI, i.e., a weakly-folded and self-associating native state, led to the choice of sIGPS as the initial target of an HX-MS analysis of protection in its burst-phase species.…”