Hydrogen-exchange mass spectrometry for the study of intrinsic disorder in proteins

Amide hydrogen/deuterium exchange detected by mass spectrometry (HXMS) is seeing wider use for the identification of intrinsically disordered parts of proteins. In this review, we discuss examples of how discovery of intrinsically disordered regions and their removal can aid in structure determination, biopharmaceutical quality control, the characterization of how post-translational modifications affect weak structuring of disordered regions, the study of coupled folding and binding, and the characterization of amyloid formation. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Dynamics of phosphodiesterase-induced cAMP dissociation from protein kinase A: Capturing transient ternary complexes by HDXMS

cAMP signaling is a fundamental cellular process necessary for mediating responses to hormonal stimuli. In contrast to cAMP-dependent activation of protein kinase A (PKA), an important cellular target, far less is known on termination in cAMP signaling, specifically how phosphodiesterases (PDEs) facilitate dissociation and hydrolysis of bound cAMP. In this study, we have probed the dynamics of a ternary complex of PKA and a PDE–RegA with an excess of a PDE-nonhydrolyzable cAMP analog, Sp-cAMPS by amide hydrogen/deuterium exchange mass spectrometry (HDXMS). Our results highlight how HDXMS can be used to monitor reactions together with mapping conformational dynamics of transient signaling complexes. Our results confirm a two-state model for active RegA-mediated dissociation of bound cAMP. Further, our results reveal that Sp-cAMPS and RegA mediate mutually exclusive interactions with the same region of PKA and at specific concentrations of Sp-cAMPS, RegA is capable of blocking Sp-cAMPS reassociation to PKA. This provides a molecular basis for how PDEs modulate levels of intracellular cAMP so that PKA is better suited to responding to fluxes rather than constant levels of cAMP. This study underscores how HDXMS can be a powerful tool for monitoring reactions together with mapping conformational dynamics in signaling proteins. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Mass spectrometry‐based methods to study protein architecture and dynamics

Mass spectrometry is now an indispensable tool in the armamentarium of molecular biophysics, where it is used for tasks ranging from protein sequencing and mapping of post‐translational modifications to studies of higher order structure, conformational dynamics, and interactions of proteins with small molecule ligands and other biopolymers. This mini‐review highlights several popular mass spectrometry‐based tools that are now commonly used for structural studies of proteins beyond their covalent structure with a particular emphasis on hydrogen exchange and direct electrospray ionization mass spectrometry.     

Native mass spectrometry: a bridge between interactomics and structural biology

Native mass spectrometry is an emerging technology that allows the topological investigation of intact protein complexes with high sensitivity and a theoretically unrestricted mass range. This unique tool provides complementary information to established technologies in structural biology, and also provides a link to high-throughput interactomics studies, which do not generate information on exact protein complex-composition, structure or dynamics. Here I review the current state of native mass spectrometry technology and discuss several important biological applications. I also describe current experimental challenges in native mass spectrometry, encouraging readers to contribute to solutions.     

Allosteric signaling in the biotin repressor occurs via local folding coupled to global dampening of protein dynamics

The biotin repressor is an allosterically regulated, site-specific DNA-binding protein. Binding of the small ligand bio-5′-AMP activates repressor dimerization, which is a prerequisite to DNA binding. Multiple disorder-to-order transitions, some of which are known to be important for the functional allosteric response, occur in the vicinity of the ligand-binding site concomitant with effector binding to the repressor monomer. In this work, the extent to which these local changes are coupled to additional changes in the structure/dynamics of the repressor was investigated using hydrogen/deuterium exchange coupled to mass spectrometry. Measurements were performed on the apo-protein and on complexes of the protein bound to four different effectors that elicit a range of thermodynamic responses in the repressor. Global exchange measurements indicate that binding of any effector to the intact protein is accompanied by protection from exchange. Mass spectrometric analysis of pepsin-cleavage products generated from the exchanged complexes reveals that the protection is distributed throughout the protein. Furthermore, the magnitude of the level of protection in each peptide from hydrogen/deuterium exchange correlates with the magnitude of the functional allosteric response elicited by a ligand. These results indicate that local structural changes in the binding site that occur concomitant with effector binding nucleate global dampening of dynamics. Moreover, the magnitude of dampening of repressor dynamics tracks with the magnitude of the functional response to effector binding.     

Insights into enzyme structure and dynamics elucidated by amide H/D exchange mass spectrometry

Conformational changes and protein dynamics play an important role in the catalytic efficiency of enzymes. Amide H/D exchange mass spectrometry (H/D exchange MS) is emerging as an efficient technique to study the local and global changes in protein structure and dynamics due to ligand binding, protein activation-inactivation by modification, and protein-protein interactions. By monitoring the selective exchange of hydrogen for deuterium along a peptide backbone, this sensitive technique probes protein motions and structural elements that may be relevant to allostery and function. In this report, several applications of H/D exchange MS are presented which demonstrate the unique capability of amide hydrogen/deuterium exchange mass spectrometry for examining dynamic and structural changes associated with enzyme catalysis.     

Pressure as a denaturing agent in studies of single-point mutants of an amyloidogenic protein human cystatin c

Recently, we presented a convenient method combining a deuterium‐hydrogen exchange and electrospray mass spectrometry for studying high‐pressure denaturation of proteins (Stefanowicz et al., Biosci Rep 2009; 30:91–99). Here, we present results of pressure‐induced denaturation studies of an amyloidogenic protein—the wild‐type human cystatin C (hCC) and its single‐point mutants, in which Val57 residue from the hinge region was substituted by Asn, Asp or Pro, respectively. The place of mutation and the substituting residues were chosen mainly on a basis of theoretical calculations. Observation of H/D isotopic exchange proceeding during pressure induced unfolding and subsequent refolding allowed us to detect differences in the proteins stability and folding dynamics. On the basis of the obtained results we can conclude that proline residue at the hinge region makes cystatin C structure more flexible and dynamic, what probably facilitates the dimerization process of this hCC variant. Polar asparagine does not influence stability of hCC conformation significantly, whereas charged aspartic acid in 57 position makes the protein structure slightly more prone to unfolding. Our experiments also point out pressure denaturation as a valuable supplementary method in denaturation studies of mutated proteins. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry

Oxidation of methionine residues in biopharmaceuticals is a common and often unwanted modification that frequently occurs during their manufacture and storage. It often results in a lack of stability and biological function of the product, necessitating continuous testing for the modification throughout the product shelf life. A major class of biopharmaceutical products are monoclonal antibodies (mAbs), however, techniques for their detailed structural analysis have until recently been limited. Hydrogen/deuterium exchange mass spectrometry (HXMS) has recently been successfully applied to the analysis of mAbs. Here we used HXMS to identify and localise the structural changes that occurred in a mAb (IgG1) after accelerated oxidative stress. Structural alterations in a number of segments of the Fc region were observed and these related to oxidation of methionine residues. These included a large change in the hydrogen exchange profile of residues 247–253 of the heavy chain, while smaller changes in hydrogen exchange profile were identified for peptides that contained residues in the interface of the C_H2 and C_H3 domains.     

Native ion mobility-mass spectrometry and related methods in structural biology

Mass spectrometry-based methods have become increasingly important in structural biology — in particular for large and dynamic, even heterogeneous assemblies of biomolecules. Native electrospray ionization coupled to ion mobility-mass spectrometry provides access to stoichiometry, size and architecture of noncovalent assemblies; while non-native approaches such as covalent labeling and H/D exchange can highlight dynamic details of protein structures and capture intermediate states. In this overview article we will describe these methods and highlight some recent applications for proteins and protein complexes, with particular emphasis on native MS analysis. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

High-sensitivity mass spectrometry for imaging subunit interactions: hydrogen/deuterium exchange

In recent years, advances in mass spectrometry have provided unprecedented knowledge of protein expression within cells. It has become apparent that many proteins function as macromolecular complexes. Structural genomics programs are determining the fold of these proteins at an increasing rate and electron microscopic tomography potentially provides a means to determine the location of these complexes within the cell. A complete understanding of the molecular mechanism of these proteins requires detailed information on the interactions and dynamics within the complex. Recent advances in mass spectrometry now make it possible to use hydrogen/deuterium exchange to detect intersubunit interfaces and dynamics within supramolecular complexes.     

Watching the components of photosynthetic bacterial membranes and their in situ organisation by atomic force microscopy

The atomic force microscope has developed into a powerful tool in structural biology allowing information to be acquired at submolecular resolution on the protruding structures of membrane proteins. It is now a complementary technique to X-ray crystallography and electron microscopy for structure determination of individual membrane proteins after extraction, purification and reconstitution into lipid bilayers. Moving on from the structures of individual components of biological membranes, atomic force microscopy has recently been demonstrated to be a unique tool to identify in situ the individual components of multi-protein assemblies and to study the supramolecular architecture of these components allowing the efficient performance of a complex biological function.Here, recent atomic force microscopy studies of native membranes of different photosynthetic bacteria with different polypeptide contents are reviewed. Technology, advantages, feasibilities, restrictions and limits of atomic force microscopy for the acquisition of highly resolved images of up to 10 Å lateral resolution under native conditions are discussed. From a biological point of view, the new insights contributed by the images are analysed and discussed in the context of the strongly debated organisation of the interconnected network of membrane-associated chlorophyll-protein complexes composing the photosynthetic apparatus in different species of purple bacteria.     

Hydrogen/deuterium exchange mass spectrometric analysis of conformational changes accompanying the assembly of the yeast prion Ure2p into protein fibrils

The Ure2 protein from baker's yeast (Saccharomyces cerevisiae) has prion properties. In vitro, at neutral pH, soluble Ure2p forms long, twisted fibrils. Two models have been proposed to account for Ure2p polymerization. The first postulates that a segment of 70 amino acid residues in the flexible N-terminal domain from different Ure2p molecules forms a parallel superpleated beta-structure running along the fibrils. The second hypothesizes that assembly of full-length Ure2p is driven by limited conformational rearrangements and non-native inter- and intramolecular interactions. The knowledge of the three-dimensional structure of the fibrillar form of Ure2p is critical for understanding the molecular events leading to the polymerization of soluble Ure2p into fibrils and hence for the design of inhibitors that might have therapeutic potential as yeast prions possessing domains rich in N and Q residues, similar to huntingtin. Solvent-accessibility studies using hydrogen/deuterium exchange monitored by mass spectrometry (HXMS) can provide insights into the structure of the fibrillar form of Ure2p and characterize at the molecular level the conformational rearrangements that occur upon assembly, in particular through the identification of protected regions and their localization in the overall structure of the protein. We have analyzed the changes in Ure2p structure associated with its assembly into fibrils using HXMS. The deuterium incorporation profile along the sequence allows the identification of the regions that exhibit the most important conformational change. Our data reveal that Ure2p undergoes minor structural changes upon assembly. While polypeptides [82-92] and [13-37] exhibit significant increased and decreased exposure to the solvent, respectively, no marked change was observed for the rest of the protein upon assembly. Our results afford new insights into the conformational rearrangements that lead to the assembly of Ure2p into fibrils and the propagation of the [URE3] element in yeast.     

Conformational Dynamics of the Escherichia coli DNA Polymerase Manager Proteins UmuD and UmuD′

The expression of Escherichia coli umuD gene products is upregulated as part of the SOS response to DNA damage. UmuD is initially produced as a 139-amino-acid protein, which subsequently cleaves off its N-terminal 24 amino acids in a reaction dependent on RecA/single-stranded DNA, giving UmuD′. The two forms of the umuD gene products play different roles in the cell. UmuD is implicated in a primitive DNA damage checkpoint and prevents DNA polymerase IV-dependent − 1 frameshift mutagenesis, while the cleaved form facilitates UmuC-dependent mutagenesis via formation of DNA polymerase V (UmuD′₂C). Thus, the cleavage of UmuD is a crucial switch that regulates replication and mutagenesis via numerous protein-protein interactions. A UmuD variant, UmuD3A, which is noncleavable but is a partial biological mimic of the cleaved form UmuD′, has been identified. We used hydrogen-deuterium exchange mass spectrometry (HXMS) to probe the conformations of UmuD, UmuD′, and UmuD3A. In HXMS experiments, backbone amide hydrogens that are solvent accessible or not involved in hydrogen bonding become labeled with deuterium over time. Our HXMS results reveal that the N-terminal arm of UmuD, which is truncated in the cleaved form UmuD′, is dynamic. Residues that are likely to contact the N-terminal arm show more deuterium exchange in UmuD′ and UmuD3A than in UmuD. These observations suggest that noncleavable UmuD3A mimics the cleaved form UmuD′ because, in both cases, the arms are relatively unbound from the globular domain. Gas-phase hydrogen exchange experiments, which specifically probe the exchange of side-chain hydrogens and are carried out on shorter timescales than solution experiments, show that UmuD′ incorporates more deuterium than either UmuD or UmuD3A. This work indicates that these three forms of the UmuD gene products are highly flexible, which is of critical importance for their many protein interactions.     

Crystal Structure of a Thermophilic GrpE Protein: Insight into Thermosensing Function for the DnaK Chaperone System

A homodimeric GrpE protein functions as a nucleotide exchange factor of the eubacterium DnaK molecular chaperone system. The co-chaperone GrpE accelerates ADP dissociation from, and promotes ATP binding to, DnaK, which cooperatively facilitates the DnaK chaperone cycle with another co-chaperone, DnaJ. GrpE characteristically undergoes two-step conformational changes in response to elevation of the environmental temperature. In the first transition at heat-shock temperatures, a fully reversible and functionally deficient structural alteration takes place in GrpE, and then the higher temperatures lead to the irreversible dissociation of the GrpE dimer into monomers as the second transition. GrpE is also thought to be a thermosensor of the DnaK system, since it is the only member of the DnaK system that changes its structure reversibly and loses its function at heat-shock temperatures of various organisms. We here report the crystal structure of GrpE from Thermus thermophilus HB8 (GrpE_Tth) at 3.23 Å resolution. The resolved structure is compared with that of GrpE from mesophilic Escherichia coli (GrpE_Eco), revealing structural similarities, particularly in the DnaK interaction regions, and structural characteristics for the thermal stability of GrpE_Tth. In addition, the structure analysis raised the possibility that the polypeptide chain in the reported GrpE_Eco structure was misinterpreted. Comparison of these two GrpE structures combined with the results of limited proteolysis experiments provides insight into the protein dynamics of GrpE_Tth correlated with the shift of temperature, and also suggests that the localized and partial unfolding at the plausible DnaK interaction sites of GrpE_Tth causes functional deficiency of nucleotide exchange factor in response to the heat shock.     

Time-Dependent Changes in Side-Chain Solvent Accessibility during Cytochrome c Folding Probed by Pulsed Oxidative Labeling and Mass Spectrometry

The current work employs a novel approach for characterizing structural changes during the refolding of acid-denatured cytochrome c (cyt c). At various time points (ranging from 10 ms to 5 min) after a pH jump from 2 to 7, the protein is exposed to a microsecond hydroxyl radical (·OH) pulse that induces oxidative labeling of solvent-exposed side chains. Most of the covalent modifications appear as + 16-Da adducts that are readily detectable by mass spectrometry. The overall extent of labeling decreases as folding proceeds, reflecting dramatic changes in the accessibility of numerous residues. Peptide mapping and tandem mass spectrometry reveal that the side chains of C14, C17, H33, F46, Y48, W59, M65, Y67, Y74, M80, I81, and Y97 are among the dominant sites of oxidation. Temporal changes in the accessibility of these residues are consistent with docking of the N- and C-terminal helices as early as 10 ms. However, structural reorganization at the helix interface takes place up to at least 1 s. Initial misligation of the heme iron by H33 leads to distal crowding, giving rise to low solvent accessibility of the displaced (native) M80 ligand and the adjacent I81. W59 retains a surprisingly high level of accessibility long into the folding process, indicating the presence of packing defects in the hydrophobically collapsed core. Overall, the results of this work are consistent with previous hydrogen/deuterium exchange studies that proposed a foldon-mediated mechanism. The structural data obtained by ·OH labeling monitor the packing and burial of side chains, whereas hydrogen/deuterium exchange primarily monitors the formation of secondary structure elements. Hence, the two approaches yield complementary information. Considering the very short time scale of pulsed oxidative labeling, an extension of the approach used here to sub-millisecond folding studies should be feasible.     

The cold denatured state is compact but expands at low temperatures: hydrodynamic properties of the cold denatured state of the C-terminal domain of L9

A point mutation of a small globular protein, the C-terminal domain of L9 destabilizes the protein and leads to observable cold-denaturation at temperatures above zero. The cold denatured state is in slow exchange with the native state on the NMR time scale, and this allows the hydrodynamic properties of the cold unfolded state and the native state to be measured under identical conditions using pulsed-field gradient NMR diffusion measurements. This provides the first experimental measurement of the hydrodynamic properties of a cold unfolded protein and its folded form under identical conditions. Hydrodynamic radii of the cold-induced unfolded states were measured for a set of temperatures ranging from 2 °C to 25 °C at pD 6.6 in the absence of denaturant. The cold unfolded state is compact compared to the urea or acid unfolded state and a trend of increasing radii of hydration is observed as the temperature is lowered. These observations are confirmed by experiments on the same protein at pD 8.0, where it is more stable, in the presence of a modest concentration of urea. The expansion of the cold-denatured state at lower temperatures is consistent with the temperature dependence of hydrophobic interactions.     

Solution structure and backbone dynamics of human Raf-1 kinase inhibitor protein

Human Raf-1 kinase inhibitor protein (hRKIP) is a small multi-functional protein of 187 residues. It contains a conserved pocket, which binds a wide range of ligands from various small molecules to distinct proteins. To provide a structural basis for the ligand diversity of RKIP, we herein determined the solution structure of hRKIP, and analyzed its structural dynamics. In solution, hRKIP mainly comprises two antiparallel β sheets, two α helices and two 3₁₀ helices. NMR dynamic analysis reveals that the overall structure of hRKIP is rigid, but its C-terminal helix which is close to the ligand-binding site is mobile. In addition, residues around the ligand-binding pocket exhibit significant conformational exchange on the μs–ms timescale. Conformational flexibility may allow the ligand-binding pocket and the C-terminal helix to adopt various conformations to interact with different substrates. This work may shed light on the underlying molecular mechanisms of how hRKIP recognizes and binds diverse substrate ligands.     

Functional switching of a novel prokaryotic 2-Cys peroxiredoxin (PpPrx) under oxidative stress

Many proteins have been isolated from eukaryotes as redox-sensitive proteins, but whether these proteins are present in prokaryotes is not clear. Redox-sensitive proteins contain disulfide bonds, and their enzymatic activity is modulated by redox in vivo. In the present study, we used thiol affinity purification and mass spectrometry to isolate and identify 19 disulfide-bond-containing proteins in Pseudomonas putida exposed to potential oxidative damages. Among these proteins, we found that a typical 2-Cys Prx-like protein (designated PpPrx) displays diversity in structure and apparent molecular weight (MW) and can act as both a peroxidase and a molecular chaperone. We also identified a regulatory factor involved in this structural and functional switching. Exposure of pseudomonads to hydrogen peroxide (H₂O₂) caused the protein structures of PpPrx to convert from high MW complexes to low MW forms, triggering a chaperone-to-peroxidase functional switch. This structural switching was primarily guided by the thioredoxin system. Thus, the peroxidase efficiency of PpPrx is clearly associated with its ability to form distinct protein structures in response to stress.     

Structural comparison of recombinant human macrophage colony stimulating factor beta and a partially reduced derivative using hydrogen deuterium exchange and electrospray ionization mass spectrometry

Hydrogen deuterium exchange, monitored by electrospray ionization mass spectrometry, has been employed to characterize structural features of a derivative of recombinant human macrophage colony stimulating factor beta (rhm-CSFbeta) in which two of the nine disulfide bridges (Cys157/Cys159-Cys'157/Cys'159) were selectively reduced and alkylated. Removal of these two disulfide bridges did not affect the biological activity of the protein. Similarities between CD and fluorescence spectra for rhm-CSFbeta and its derivative indicate that removing the disulfide bonds did not strongly alter the overall three-dimensional structure of rhm-CSFbeta. However, differences between deuterium exchange data of the intact proteins indicate that more NHs underwent fast deuterium exchange in the derivative than in rhm-CSFbeta. Regions located near the disulfide bond removal site were shown to exhibit faster deuterium exchange behavior in the derivative than in rhm-CSFbeta.