Structural and Dynamic Characteristics of a Partially Folded State of Ubiquitin Revealed by Hydrogen Exchange Mass Spectrometry

Abstract

Structural and dynamic properties of a partially folded conformation (A-state) of ubiquitin are studied using amide hydrogen exchange in solution (HDX) and mass spectrometric detection. A clear distinction between the native state of the protein and the A-state can be made when HDX is carried out in a semicorrelated regime. Convoluted exchange patterns are interpreted with the aid of HDX simulations in a three-state system (highly structured, partially unstructured, and fully unstructured states). The data clearly indicate a highly dynamic character of the non-native state. Furthermore, combination of HDX and protein ion fragmentation in the gas phase [by means of collision-induced dissociation (CAD)] is used to evaluate the conformational stability of various protein segments specifically in the molten globular state. Chain flexibility appears to be distributed very unevenly in this non-native conformation. The highest degree of structural disorder is displayed by the C-terminal segment (Gly 53 -Gly 76 ), which was previously suggested to form a transient R-helix. The least dynamic segment of ubiquitin in the A-state is Thr 9 -Glu 18 (which was previously suggested to form a stable nativelike -strand), with the adjacent segments exhibiting somewhat diminished conformational stability. The study also demonstrates the power of mass spectrometry as a tool in providing conformer-specific information about the structure and dynamics of both native and non-native protein states coexisting in solution under equilibrium.Molten globule is a term that is used to describe a class of compact non-native protein states that are often thought to be general intermediates in protein folding, which are characterized by a substantial degree of secondary structure and a nearly complete absence of a native tertiary fold (1). Extensive studies of molten globular states of a variety of proteins were initially catalyzed by the realization that elucidating their structural and dynamic features may provide important clues for solving the protein folding problem (2,3). Since the kinetic folding intermediates are populated only transiently during the protein folding process, properties of molten globular states are often studied using the equilibrium models. In fact, it is the partially unfolded equilibrium intermediates that were initially proposed to belong to a common physical state of globular proteins, for which the term molten globular state was coined. More recently, renewed interest in the properties and behavior of molten globular states was instigated by a growing understanding that structural disorder is quite ubiquitous in vivo and often plays an important role in a variety of physiological processes (4). Such partially unstructured protein states have been implicated in a variety of processes ranging from protein translocation through membranes to ordered oligomerization, recognition, signaling, and even catalysis.The experimental tools employed in the studies of molten globular states include circular dichroism spe...

Characterization of acid-induced protein conformational changes and noncovalent complexes in solution by using coldspray ionization mass spectrometry

“…Below pH 2.3, another distribution centered at ϩ11 appeared in the CSI mass spectra (Supplementary Figure S3a). Notably, a new distribution with main peaks at ϩ8 and ϩ9 was observed when the pH values decrease to 1.7 (Figure 4c), indicating the presence of an acid-induced unfolded ubiquitin intermediate [22,57,58]. Figure 4d shows that the maximum at pH 7.0 in the ESI mass spectrum remains at ϩ7 but the charge-state distribution is broadened.…”

Conformer-specific characterization of nonnative protein states using hydrogen exchange and top-down mass spectrometry

“…Ub populates a partially structured state (the socalled A-state) in the presence of alcohol in acidic solution at low salt (30), and this state was postulated to exist under a variety of other conditions (31), including those that favor correlated exchange regimes, where a distinction between this conformer and the native protein can be made based on markedly different levels of deuterium incorporation in HDX MS measurements (32). The A-state of Ub, the structure of which has been extensively characterized using NMR (33)(34)(35)(36)(37)(38) and other spectroscopic techniques (39), largely maintains the native secondary structure within the N-terminal part, whereas significant conformational changes within the C-terminal part eliminate the native fold and transform it into a transient helix.…”

“…To interrogate structural features of a nonnative Ub conformer coexisting at equilibrium with other protein states, top-down MS measurements were carried out following HDX in a solution with high alcohol content at neutral pH (see condition II in Table 1). Ub is known to populate more than one conformation under these conditions (31,32), one of which was previously hypothesized to be the classical A-state of Ub (the commonly accepted way to generate the A-state of Ub uses an acidic solution with high alcohol content). As illustrated in Fig.…”

Vapor Treatment of Electrospray Droplets: Evidence for the Folding of Initially Denatured Proteins on the Sub-Millisecond Time-Scale

“…In our previous work with neutral pH solutions [43], the leak-in of weak acids resulted in a modest increase in q ave (+7.8 and +7.3 for acetic and formic acids, respectively) relative to the nano-ESI mass spectrum of a neutral pH solution (q ave =+6.8), whereas the leak-in of the strong acids TFA and HCl resulted in a distribution corresponding to the folded ubiquitin A-State or even a folding back to a more native state (N-state) with an observed CSD from +4 to +8 (q ave =+5.9) [61]. In this work, for ubiquitin prepared at pH= 2.8, the CSD shifted from +4 to +13 (q ave =+6.9) prior to vapor leak-in (Figure 4a) to a CSD from +4 to +8 (q ave =+5.6) with the introduction of ammonia (Figure 4b) to one from +4 to +5 (q ave =+5.0) with the introduction of piperidine vapors (Figure 4c).…”

“…The charge state distributions of cytochrome c [9,59] and ubiquitin [60][61][62] have been extensively studied [63]. Bovine cytochrome c is a globular protein containing 104 amino acids (23 basic and 12 acidic amino acids) and one covalently attached heme group [64].…”