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

Abstract

The structures of partially folded states appearing during the folding of a (betaalpha)(8) TIM barrel protein, the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS), was assessed by hydrogen exchange mass spectrometry (HX-MS) and Gō model simulations. HX-MS analysis of the peptic peptides derived from the pulse-labeled product of the sub-millisecond folding reaction from the urea-denatured state revealed strong protection in the (betaalpha)(4) region, modest protection in the neighboring (betaalpha)(1-3) and (betaalpha)(5)beta(6) segments and no significant protection in the remaining N and C-terminal segments. These results demonstrate that this species is not a collapsed form of the unfolded state under native-favoring conditions nor is it the native state formed via fast-track folding. However, the striking contrast of these results with the strong protection observed in the (betaalpha)(2-5)beta(6) region after 5 s of folding demonstrates that these species represent kinetically distinct folding intermediates that are not identical as previously thought. A re-examination of the kinetic folding mechanism by chevron analysis of fluorescence data confirmed distinct roles for these two species: the burst-phase intermediate is predicted to be a misfolded, off-pathway intermediate, while the subsequent 5 s intermediate corresponds to an on-pathway equilibrium intermediate. Comparison with the predictions using a C(alpha) Gō model simulation of the kinetic folding reaction for sIGPS shows good agreement with the core of the structure offering protection against exchange in the on-pathway intermediate(s). Because the native-centric Gō model simulations do not explicitly include sequence-specific information, the simulation results support the hypothesis that the topology of TIM barrel proteins is a primary determinant of the folding free energy surface for the productive folding reaction. The early misfolding reaction must involve aspects of non-native structure not detected by the Gō model simulation.

Figure 1

The peptide map of sIGPS for HX-MS analysis. The elements of secondary structure and the 19 peptides resulting from pepsin digestion at pH 2.7 are displayed on the amino acid sequence of IGPS from S. sulfataricus. The α-helices are shown in magenta, and the β-strands in blue above the sequence, and the peptides are shown as black lines below the sequence. This figure was taken from Gu et al., (2007) with minor modifications.

Figure 2

Representative MALDI-TOF spectra of four peptides displaying different types of behavior over the first few hundred milliseconds of folding of sIGPS: (A) peptide 36–47; protection appears to decrease with time (B) peptide 100–115; protection increases with time (C) peptide 143–151; protection is largely independent of time and (D) peptide 197–203; no protection is apparent at any of these folding times. The spectra obtained at various refolding time intervals prior to quenched-flow labeling and both the folded and unfolded reference spectra for each peptide are shown in each panel. The refolding reaction was initiated from 8.0 M urea by 11 times dilution to 10 mM potassium phosphate buffer, pH 7.8, at 25 °C.

Figure 3

Percent protection against exchange of amide hydrogens for solvent deuterium for 19 peptides of sIGSP derived from pepsin digestion at pH 2.7 and 0 °C as a function of refolding time. The estimated errors are ±5%.

Figure 4

Percent protection for 18 of the 19 peptic peptides derived from sIGPS for quenched-flow labeling of the burst-phase intermediate with a 50 ms pulse at pH 9.5 after 75 ms of folding (green), manual-mixing labeling of the equilibrium intermediates, I_a and I_b, at 5 M urea with a 5 s pulse of deuterated buffer at pH 7.8 (black), manual-mixing labeling of the I_a intermediate highly-populated after 5 s of folding with a 5 s pulse of deuterated buffer at pH 7.8 (red), quenched-flow labeling of the equilibrium intermediates, I_a and I_b, at 5 M D₄-urea with a 50 ms pulse of protonated buffer at pH 9.5 (blue) and Gō-model simulation (cyan). The data for peptide 59–68 were not included because this peptide does not display protection against HX under any conditions.

Figure 5

(A) The fluorescence-detected refolding reaction of sIGPS via stopped-flow mixing from 8 M to 1.2 M urea at pH 7.8 and 25 °C. The trace was best fit by three exponentials with time constants and relative amplitudes of 0.6 s and −47%, 7.3 s and 28% and 80 s and 72%; the amplitudes were normalized to the sum of the two phases of increasing intensity. The residuals are shown in the lower part of panel A. The data density was reduced by 10-fold to illustrate the quality of the fit. (B) A chevron plot of the refolding (open symbols) and unfolding (filled symbols) time constants as a function of the final urea concentration. Stopped-flow fluorescence detected phases have symbols with cross-marks inside. The CD data (symbols without cross-marks) are taken from Figure 7 in Forsyth et al., (2002). The assignments of the time constants to individual steps in the folding mechanism, Scheme 2, are also shown. The unfolding reactions were initiated in 0 M urea and ended at the indicated urea concentrations; the refolding jumps were initiated at 8 M urea and ended at the indicated urea concentrations. The buffer in all experiments contained 10 mM phosphate buffer at pH 7.8 and 25 °C.

Figure 6

Implied structures and protection patterns for the off-pathway I_bp specie at 75 ms, on-pathway I_a (5s) and I_b species are displayed on ribbon diagrams along with the folding scheme of sIGPS. Regions with strong percentage protection, ≥ 40%, are shown in blue, regions with moderate percentage protection, 10% < protection < 40% are shown in green, regions with no significant protection, ≤10%, are shown in red, the disordered region (peptide 59–68) are shown in grey, and regions with no peptide coverage are shown in orange. For reference, the unfolded and native structures are shown in all red and all blue respectively.

Figure 7

Gō-model simulation trajectory of sIGPS folding. (A) Number of native contacts, Q, at various time steps in folding for a fast productive trajectory (black), slow productive trajectory (green), and an unproductive trajectory (yellow). (B) Probability of refolding trajectories sampling each value of Q. The grey lines indicate the one standard deviation level of uncertainty.

Figure 8

Structural profiles of all 30 sIGPS simulation trajectories in folding stages A–F, displayed as the fraction folded in each of the peptic fragments. Trajectories with a bias of folded contacts in the center are highlighted in black, trajectories with a bias of folded contacts at the N-terminus are highlighted in red and, trajectories with a bias of folded contacts at the C-terminus are highlighted in blue. The 7 unproductive trajectories which did not reach the native state within the time of the simulation are highlighted in yellow.

Figure 9

Average fraction folded of the 23 productive trajectories in folding stages B (orange), C (blue), D (green) and E (black). The fraction folded was normalized such that stage A (unfolded) values were set to 0 and stage F values (folded) were set to 1. Errors indicate standard deviations. The locations of the 6 primary active site residues are highlighted with purple vertical lines.

Figure 10

Contact map of the 2c3z sIGPS structure displaying each of the 571 contacts (black squares). Backtracking contacts, which decrease in probability as the overall Q progresses from folding stages D to E, are shown as yellow squares. For reference, secondary structure elements are shown along each axis.

scheme 1

scheme 2