Mapping the structure of folding cores in TIM barrel proteins by hydrogen exchange mass spectrometry: the roles of motif and sequence for the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus

Abstract

To test the roles of motif and amino acid sequence in the folding mechanisms of TIM barrel proteins, hydrogen-deuterium exchange was used to explore the structure of the stable folding intermediates for the of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS). Previous studies of the urea denaturation of sIGPS revealed the presence of an intermediate that is highly populated at approximately 4.5 M urea and contains approximately 50% of the secondary structure of the native (N) state. Kinetic studies showed that this apparent equilibrium intermediate is actually comprised of two thermodynamically distinct species, I(a) and I(b). To probe the location of the secondary structure in this pair of stable on-pathway intermediates, the equilibrium unfolding process of sIGPS was monitored by hydrogen-deuterium exchange mass spectrometry. The intact protein and pepsin-digested fragments were studied at various concentrations of urea by electrospray and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, respectively. Intact sIGPS strongly protects at least 54 amide protons from hydrogen-deuterium exchange in the intermediate states, demonstrating the presence of stable folded cores. When the protection patterns and the exchange mechanisms for the peptides are considered with the proposed folding mechanism, the results can be interpreted to define the structural boundaries of I(a) and I(b). Comparison of these results with previous hydrogen-deuterium exchange studies on another TIM barrel protein of low sequence identify, alpha-tryptophan synthase (alphaTS), indicates that the thermodynamic states corresponding to the folding intermediates are better conserved than their structures. Although the TIM barrel motif appears to define the basic features of the folding free energy surface, the structures of the partially folded states that appear during the folding reaction depend on the amino acid sequence. Markedly, the good correlation between the hydrogen-deuterium exchange patterns of sIGPS and alphaTS with the locations of hydrophobic clusters defined by isoleucine, leucine, and valine residues suggests that branch aliphatic side-chains play a critical role in defining the structures of the equilibrium intermediates.

Scheme 1

Figure 1

Representative mass spectra of the +25 charge state of sIGPS equilibrated in 0-8 M urea followed by pulse-labeling in urea/²H₂O for 5 s at 25 °C and pH 7.8. Isotope exchange was quenched on ice with TFA/acetonitrile at pH 2.7, and samples were analyzed by HPLC ESI-MS. The m/z values of ions representing the native, N, intermediate, I, and unfolded, U, sIGPS are 1027, 1030 and 1032.2, respectively, and the corresponding peaks are labeled.

Figure 2

Amino acid sequence of sIGPS displaying secondary structure (above) and peptide coverage (below). Regions of the sequence corresponding to β-strands are colored blue, and α-helical segments are colored fuchsia. More highly-protected peptides (≥ 49% average protection) are underlined in blue, those with lower protection (23-27% average protection) are underlined in green, and unprotected peptides (≥ 11% average protection) at 5 M urea are underlined in red. The dashed gray region is the unstructured loop region linking β2 and α2, whose protection against HD exchange cannot be measured even in the native state.

Figure 3

Representative mass spectra of peptides displaying the four different types of exchange behavior observed at 5 M urea and at 25 °C, pH 7.8: (a) unprotected at 5 M urea, residues 211-220, (b) EX1 mechanism with lower average protection (23-27%), residues 180-196, (c) EX2 mechanism with higher average protection (≥49%), residues 143-151, and (d) EX1 mechanism with higher average protection (≥49%), residues 107-120. The isotope envelopes representing the unfolded reference at higher m/z in 8 M urea and the folded reference at lower m/z in 0 M urea are shown as dotted and dashed lines, respectively

Figure 4

Comparison of the HD average protection pattern for sIGPS pulse-labeled at 5 M urea with the position of hydrophobic clusters of ILV residues for sIGPS. (a) Structured regions in the equilibrium intermediate determined from the HD exchange mass spectrometry experiment. More highly protected regions (≥49%) are shown in blue, regions with lesser protection (∼25%) are shown in green, and unprotected regions (≤ 11%) are shown in red. (b) Four hydrophobic clusters formed by ILV residues obtained using a 4.2 Å cut-off: cluster 1 (orange); cluster 2 (magenta); cluster 3 (cyan) and cluster 4 (yellow). Structures were generated using Pymol and Protein Data Bank entry 2C3Z.pdb.

Figure 5

Two-dimensional contact map at 4.2 Å highlighting pairwise hydrophobic contacts for sIGPS. The ILV to ILV contacts are shown with colored squares according to the color scheme for the clusters in Figure 4b, the ILV to FMY contacts are shown with open circles, and the FMY to FMY contacts are shown with closed black circles. sIGPS contains no tryptophan side chains. The blue box encompasses the most strongly protected region and represents structured regions in the I_a intermediate, and the green box encompasses regions that are at least moderately protected and represents structured regions in the I_b intermediate.