Native state dynamics drive the unfolding of the SH3 domain of PI3 kinase at high denaturant concentration

Abstract

Little is known about the role of protein dynamics in directing protein unfolding along a specific pathway and about the role played by chemical denaturants in modulating the dynamics and the initiation of unfolding. In this study, deuterium-hydrogen exchange (HX) detected by electrospray ionization mass spectrometry (ESI-MS) was used to study the unfolding of the SH3 domain of the PI3 kinase. Unfolding on the principal unfolding pathway occurs in 2 steps, both in the absence and in the presence of 1.8 M guanidine hydrochloride (GdnHCl). In both cases, the first step leads to the formation of an intermediate, I N, with 5 fewer protected amide hydrogen sites than in N. In the second step, IN loses the structure protecting the remaining 14 amide hydrogen sites from HX as it unfolds completely. ESI-MS analysis of fragments of the protein created by proteolytic digestion, after completion of the HX reaction, shows that I N has lost protection against HX in the same segments of native structure during unfolding in the absence and presence of 1.8 M GdnHCl. Hence, GdnHCl does not appear to play a direct active role in the initiation of unfolding. However, at higher GdnHCl concentrations, a second unfolding pathway is shown to compete effectively with the N 7 IN 7 U pathway. In this way, the denaturant modulates the energy landscape of unfolding.hydrogen exchange ͉ mass spectrometry ͉ protein unfolding ͉ partially unfolded conformation ͉ native-state exchange F olded proteins possess dynamic structures, and the lowest energy conformation of a folded protein exists at equilibrium with many high energy conformational substates, which are Boltzmann-distributed in the folded protein ensemble (1-4). Fluctuations in protein structure appear not to be random. They may be directed toward enhancing function (3-7), but their role in preferentially directing protein folding and unfolding reactions on to specific pathways is poorly understood (8). Even less is known about how the thermal fluctuations that lead to protein unfolding are perturbed by denaturants such as guanidine hydrochloride (GdnHCl) and urea. Protein denaturants may act indirectly by disrupting the structure of water, thereby making hydrophobic groups more readily solvated (9, 10), or directly by interacting more strongly than water with the protein backbone and side chains (11-13). To understand how denaturants act, it is necessary to determine whether the mechanism of unfolding of a protein, as well as the thermal fluctuations that enable unfolding, are the same in the absence of a chemical denaturant and in the presence of a high concentration of the denaturant.Structural characterization of protein unfolding in the presence, as well as absence, of denaturant becomes possible when the hydrogen exchange (HX) experiment is carried out in conjunction with NMR spectroscopy or MS. HX-NMR experiments have been carried out on many different proteins under conditions where HX is rate-limited by the intrinsic exchange rate constant (k int ) of the fully solvent-exposed a...

Evidence for Initial Non-specific Polypeptide Chain Collapse During the Refolding of the SH3 Domain of PI3 Kinase

“…In these conditions, the unfolding reaction occurs in multiple steps. 55 Importantly, the final unfolding of an intermediate to the fully unfolded state does not occur in an all-ornone manner. These conditions are identical with the strongly stabilizing folding conditions utilized in that study, so the initial stage of refolding would be expected to be the reverse of the last stage of unfolding.…”

“…Very recently, a partially folded intermediate, which forms at a late stage during the folding of the PI3K SH3 domain, has been identified. 54 Studies of the unfolding of the PI3K SH3 domain, using hydrogen exchange detected by mass spectrometry, 55 in the absence and in low concentrations of denaturant also suggested that the dissolution of structure during unfolding occurs in many steps; hence, refolding too must occur in many steps in the absence and in low concentrations of denaturant. Nevertheless, earlier refolding studies were not able to detect any intermediate on the refolding pathway, probably because a suitable high-resolution probe was not used.…”

Development of the Structural Core and of Conformational Heterogeneity during the Conversion of Oligomers of the Mouse Prion Protein to Worm-like Amyloid Fibrils

“…43 It should be noted that the bimodal isotope distribution patterns observed in three fragments of the aggregates are not attributable to uncorrelated HDX occurring in local structural regions in aggregate molecules under the quench conditions (pH 2.5, 4°C), because such patterns are not observed for the same three fragments derived from native moPrP or deuterated moPrP that were also subjected to exactly the same quench conditions as were the aggregates (Fig. 5).…”

Characterization of deamidation of barstar using electrospray ionization quadrupole time‐of‐flight mass spectrometry, which stabilizes an equilibrium unfolding intermediate

“…8,9 It has been postulated, 10,11 and has also been experimentally shown for many proteins, [12][13][14] 16 or by mutagenesis of the protein sequence [17][18][19] or by using a more sensitive detection method to probe the folding reaction. [20][21][22] Identifying and populating intermediate states during folding and unfolding, 23 as well as subsequent characterization of their kinetic role, 24 remain a major goal in protein folding studies. Deamidation of glutamyl and asparaginyl residues is an important nonenzymatic post-translational modification of proteins and peptides.…”