Pulsed Hydrogen-deuterium Exchange Research Articles

A Model Study to Assess Fibrillation and Product Stability to Support Peptide Drug Design.

The fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and can accelerate product development by guiding the selection of solution-stable candidates and formulations. The studies reported here investigated the effects of structural modifications on the fibrillation of a 29-residue peptide (PepA) and two sequence modified variants (PepB, PepC). The C-terminus of PepA was amidated, whereas both PepB and PepC retained the carboxylate, and Ser16 in PepA and PepB was substituted with a helix-stabilizing residue, α-aminoisobutyric acid (Aib), in PepC. In thermal denaturation studies by far-UV CD spectroscopy and fibrillation kinetic studies by fluorescence and turbidity measurements, PepA and PepB showed heat-induced conformational changes and were found to form fibrils, whereas PepC did not fibrillate and showed only minor changes in the CD signal. Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed a high degree of protection from HD exchange in mature PepA fibrils and its proteolytic fragments, indicating that most of the sequence had been incorporated into the fibril structure and occurred nearly simultaneously throughout the sequence. The effects of the net peptide charge and formulation pH on fibrillation kinetics were investigated. In real-time stability studies of two formulations of PepA at pH's 7.4 and 8.0, analytical methods detected significant changes in the stability of the formulations at different time points during the study, which were not observed during accelerated studies. Additionally, PepA samples were withdrawn from real-time stability and subjected to additional stress (40 °C, continuous shaking) to induce fibrillation; an approach that successfully amplified oligomers or prefibrillar species previously undetected in a thioflavin T assay. Taken together, these studies present an approach to differentiate and characterize fibrillation risk in structurally related peptides under accelerated and real-time conditions, providing a model for rapid, iterative structural design to optimize the stability of therapeutic peptides.

Fibrillation of human insulin B-chain by pulsed hydrogen-deuterium exchange mass spectrometry

Insulin forms amyloid fibrils under slightly destabilizing conditions, and B-chain residues are thought to play an important role in insulin fibrillation. Here, pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS), far-UV circular dichroism spectroscopy, thioflavin T (ThioT) fluorescence, turbidity, and soluble fraction measurements were used to monitor the kinetics and mechanisms of fibrillation of human insulin B-chain (INSB) in acidic solution (1 mg/mL, pH 4.5) under stressed conditions (40°C, continuous shaking). Initially, INSB rapidly formed β-sheet-rich oligomers that were protected from HD exchange and showed weak ThioT binding. Subsequent fibril growth and maturation was accompanied by even greater protection from HD exchange and stronger ThioT binding. With peptic digestion of deuterated INSB, HDX-MS suggested early involvement of the N-terminal (1–11, 1–15) and central (12–15, 16–25) fragments in fibril-forming interactions, whereas the C-terminal fragment (25–30) showed limited involvement. The results provide mechanistic understanding of the intermolecular interactions and structural changes during INSB fibrillation under stressed conditions and demonstrate the application of pulsed HDX-MS to probe peptide fibrillation.

Using mass spectrometry-based methods to understand amyloid formation and inhibition of alpha-synuclein and amyloid beta.

Amyloid fibrils, insoluble β-sheets structures that arise from protein misfolding, are associated with several neurodegenerative disorders. Many small molecules have been investigated to prevent amyloid fibrils from forming; however, there are currently no therapeutics to combat these diseases. Mass spectrometry (MS) is proving to be effective for studying the high order structure (HOS) of aggregating proteins and for determining structural changes accompanying protein-inhibitor interactions. When combined with native MS (nMS), gas-phase ion mobility, protein footprinting, and chemical cross-linking, MS can afford regional and sometimes amino acid spatial resolution of the aggregating protein. The spatial resolution is greater than typical low-resolution spectroscopic, calorimetric, and the traditional ThT fluorescence methods used in amyloid research today. High-resolution approaches can struggle when investigating protein aggregation, as the proteins exist as complex oligomeric mixtures of many sizes and several conformations or polymorphs. Thus, MS is positioned to complement both high- and low-resolution approaches to studying amyloid fibril formation and protein-inhibitor interactions. This review covers basics in MS paired with ion mobility, continuous hydrogen-deuterium exchange (continuous HDX), pulsed hydrogen-deuterium exchange (pulsed HDX), fast photochemical oxidation of proteins (FPOP) and other irreversible labeling methods, and chemical cross-linking. We then review the applications of these approaches to studying amyloid-prone proteins with a focus on amyloid beta and alpha-synuclein. Another focus is the determination of protein-inhibitor interactions. The expectation is that MS will bring new insights to amyloid formation and thereby play an important role to prevent their formation.

Long-lived conformational changes in active Gsα revealed in atomistic detail by integrating HDX-MS with enhanced molecular dynamics simulations

Heterotrimeric guanine nucleotide-binding proteins (G proteins) play major roles in intracellular signal transduction as their activation by G protein-coupled receptors triggers a variety of biological responses. Although several features of the mechanisms underlying this process have been elucidated using various biochemical and biophysical techniques, atomic-level structural and kinetic information of the conformational changes accompanying G protein activation has been difficult to attain. Here, we present a novel computational strategy that combines Bayesian inference, maximum caliber, and transition path theory (TPT) to obtain atomic details of metastable states of the active (GTP-bound) α subunit of G protein Gs (Gsα), as well as details of the kinetic transition network connecting them, from pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) measurements integrated with enhanced molecular dynamics simulations.

Pulsed Hydrogen-Deuterium Exchange Reveals Altered Structures and Mechanisms in the Aggregation of Familial Alzheimer's Disease Mutants.

Mutations of the Amyloid Precursor Protein, from which the amyloid β peptide Aβ42 is cleaved, are associated with familial Alzheimer's disease. The disease-relevant familial mutations include the Arctic (E22G), Iowa (D23N), Italian (E22K), Dutch (E22Q), Japanese (D7N), English (D6R), and Flemish (A21G) variants. A detailed mechanistic understanding of the aggregation behavior of the mutant peptides at the residue level is, however, still lacking. We report here a study of the aggregation kinetics of these mutants in vitro by pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) to obtain a temporally and sequence resolved picture of their self-assembly. For all variants, HDX occurs to give a bimodal distribution representing two soluble classes of aggregates, one protected and one solvent-exposed. There is no evidence of other classes of structural intermediates within the detection limits of the HDX approach. The fractional changes in the bimodal exchange profiles for several regions of Aβ42 reveal that the central and C-terminal peptides gain protection upon fibril formation, whereas the N-terminal regions remain largely solvent-accessible. For these mutants, all peptide fragments follow the same kinetics, acquiring solvent protection at the same time, further supporting that there are no significant populations of intermediate species under our experimental conditions. The results demonstrate the potential of pulsed HDX-MS for resolving the region-specific aggregation behavior of Aβ42 isoforms in solution where X-ray crystallography and solid-state NMR (ssNMR) are challenged.

Pulsed Hydrogen-Deuterium Exchange Illuminates the Aggregation Kinetics of α-Synuclein, the Causative Agent for Parkinson's Disease.

α-Synuclein (aS) forms toxic intermediates ranging from small oligomers and protofibrils to large amyloid fibrils. Understanding the time course of aS fibril formation and the role played by its regions is critical for therapeutic intervention. Here, we used pulsed hydrogen-deuterium exchange and mass spectrometry (HDX-MS) for the first time to probe kinetic intermediates of the full aS aggregation in vitro, achieving kinetic snapshots containing spatially resolved protein information about critical stages. Monitoring the resultant mass shifts shows distinct binomial abundances for two main exchange profiles: one that represents a fast-exchanging, solvent-accessible species and another with a more protected nature. We show using a series of proteolytic peptides from the full protein that self-association is most pronounced in the non-amyloid-β-component region and less so for either terminus. The N-terminus, however, shows a minor protected population at mid- and late times, whereas the C-terminus shows predominantly unimodal HDX, indicating that these regions are devoid of any large conformational rearrangements. Focusing on the hydrophobic core, we confirmed and modeled the different isotopic distributions and calculated their relative fractions to discern their individual contributions. The data fitting reports respective t1/2 values, which are nearly identical and do not depend on location. We followed the aggregation by complementary transmission electron microscopy to observe the morphology of aggregates and circular dichroism to assess changes in secondary structure. Our results provide a detailed picture of aS aggregation in vitro and demonstrate that HDX-MS offers unique spatially resolved, coexisting kinetic intermediates in solution. This new platform is suitable for testing promising inhibitors of aS aggregation.

Understanding curli amyloid-protein aggregation by hydrogen–deuterium exchange and mass spectrometry

Bacteria within Curli biofilms are protected from environmental pressures (e.g., disinfectants, antibiotics), and this is responsible in part for intractable infections. Understanding aggregation of the major protein component of Curli, CsgA, may uncover disease-associated amyloidogenesis mechanisms. Here, we report the application of pulsed hydrogen–deuterium exchange and mass spectrometry (HDX-MS) to study CsgA aggregation, thereby obtaining region-specific information. By following time-dependent peptide signal depletion, presumably a result of insoluble fibril formation, we acquired sigmoidal profiles that are specific for regions (region-specific) of the protein. These signal-depletion profiles not only provide an alternative aggregation measurement, but also give insight on soluble species in the aggregation. The HDX data present as bimodal isotopic distributions, one representing a highly disordered species whereas the other a well-structured one. Although the extents of deuterium uptake of the two species remain the same with time, the relative abundance of the lower mass, less-exchanged species increases in a region-specific manner. The same region-specific aggregation properties also pertain to different aggregation conditions. Although CsgA is an intrinsically disordered protein, within the fibril it is thought to consist of five imperfect β-strand repeating units (labeled R1–R5). We found that the exterior repeating units R1 and R5 have higher aggregation propensities than do the interior units R2, R3, and R4. We also employed TEM to obtain complementary information of the well-structured species. The results provide insight on aggregation and a new approach for further application of HDX-MS to unravel aggregation mechanisms of amyloid proteins.

Continuous and pulsed hydrogen-deuterium exchange and mass spectrometry characterize CsgE oligomerization.

We report the use of hydrogen-deuterium amide exchange coupled to mass spectrometry (HDX-MS) to study the interfaces of and conformational changes accompanying CsgE oligomerization. This protein plays an important role in enteric bacteria biofilm formation. Biofilms provide protection for enteric bacteria from environmental extremes and raise concerns about controlling bacteria and infectious disease. Their proteinaceous components, called curli, are extracellular functional amyloids that initiate surface contact and biofilm formation. The highly regulated curli biogenesis involves a major subunit, CsgA, a minor subunit CsgB, and a series of other accessory proteins. CsgE, possibly functioning as oligomer, is a chaperonin-like protein that delivers CsgA to an outer-membrane bound oligomeric CsgG complex. No higher-order structure, or interfaces and dynamics of its oligomerization, however, are known. In this work, we determined regions involved in CsgE self-association by continuous HDX, and, on the basis of that, prepared a double mutant W48A/F79A, derived from interface alanine scan, and verified that it exists as monomer. Using pulsed HDX and MS, we suggest there is a structural rearrangement occurring during the oligomerization of CsgE.

Time-Resolved Pulsed Hydrogen/Deuterium Exchange Mass Spectrometry Probes Gaseous Proteins Structural Kinetics

A pulsed hydrogen/deuterium exchange (HDX) method has been developed for rapid monitoring of the exchange kinetics of protein ions with D2O a few milliseconds after electrospray ionization (ESI). The stepwise gradual evolution of HDX of multiply charged protein ions was monitored using the pulsed HDX mass spectrometry technique. Upon introducing a very short pulse of D2O (in the μs to ms time scale) into the linear ion trap (LIT) of a time-of-flight (TOF) mass spectrometer, bimodal distributions were detected for the ions of cytochrome c and ubiquitin. Mechanistic details of HDX reactions for ubiquitin and cytochrome c in the gas phase were uncovered and the structural transitions were followed by analyzing the kinetics of HDX.

Pulsed hydrogen–deuterium exchange mass spectrometry probes conformational changes in amyloid beta (Aβ) peptide aggregation

Probing the conformational changes of amyloid beta (Aβ) peptide aggregation is challenging owing to the vast heterogeneity of the resulting soluble aggregates. To investigate the formation of these aggregates in solution, we designed an MS-based biophysical approach and applied it to the formation of soluble aggregates of the Aβ42 peptide, the proposed causative agent in Alzheimer's disease. The approach incorporates pulsed hydrogen-deuterium exchange coupled with MS analysis. The combined approach provides evidence for a self-catalyzed aggregation with a lag phase, as observed previously by fluorescence methods. Unlike those approaches, pulsed hydrogen-deuterium exchange does not require modified Aβ42 (e.g., labeling with a fluorophore). Furthermore, the approach reveals that the center region of Aβ42 is first to aggregate, followed by the C and N termini. We also found that the lag phase in the aggregation of soluble species is affected by temperature and Cu(2+) ions. This MS approach has sufficient structural resolution to allow interrogation of Aβ aggregation in physiologically relevant environments. This platform should be generally useful for investigating the aggregation of other amyloid-forming proteins and neurotoxic soluble peptide aggregates.

Protein Folding Mechanisms Studied by Time-Resolved Electrospray Mass Spectrometry

Mass spectrometry (MS) owes its remarkable rise as a bioanalytical tool to the development of two “soft” ionization techniques: matrix-assisted laser desorption/ ionization (MALDI) (1,2) and electrospray ionization (ESI) (3). The salient feature of soft ionization is that a wide range of analytes, from small metabolites to very large biomolecular systems, can be transferred intact into the gas phase, thus making them amenable to mass spectrometric analysis. For MALDI-MS, the sample is embedded into a crystalline matrix prior to analysis. For ESI, in contrast, ionization is initiated directly from the liquid phase by spraying analyte solution from the tip of an electrically charged capillary. The small droplets formed by the ESI emitter undergo rapid evaporation and fi ssion, ultimately releasing the analyte as multiply protonated (or deprotonated) species (4).

Pulsed Hydrogen/Deuterium Exchange MS/MS for Studying the Relationship between Noncovalent Protein Complexes in Solution and in the Gas Phase after Electrospray Ionization

Electrospray ionization mass spectrometry (ESI-MS) has become a standard method for monitoring noncovalent protein-protein interactions. Studies employing this approach tend to operate on the premise that the ionic species observed in the mass spectrum directly reflect the corresponding solution-phase protein quaternary structures. However, dissociation or clustering events taking place during ESI may lead to disparities between the ions observed in the mass spectrum and the protein binding state in bulk solution. Recognizing the occurrence of dissociation or clustering artifacts is not straightforward, leading to possible ambiguities in the interpretation of ESI-MS data. This work employs on-line pulsed hydrogen-deuterium exchange (HDX) for probing the origin of various species in the ESI mass spectrum of hemoglobin. In addition to the canonical hemoglobin tetramer, ESI-MS reveals the presence of monomers, dimers, hexamers, and octamers. Tandem mass spectrometry (MS/MS) is used for extracting HDX levels in a subunit-specific manner. Dimeric species exhibit exchange levels that are significantly above those of the tetramer. Monomeric hemoglobin subunits are labeled to an even greater extent. This HDX pattern implies that monomers and dimers do not represent dissociation artifacts generated during ESI. Instead, they are derived from preexisting solution-phase structures. In contrast, hexamers and octamers exhibit HDX levels that resemble those of the tetramer, thus identifying these larger species as nonspecific clustering artifacts. Overall, it appears that the pulsed HDX MS/MS approach introduced in this work represents a widely applicable tool for deciphering the relationship between ESI mass spectra and protein quaternary structures in solution.

Protein-folding kinetics and mechanisms studied by pulse-labeling and mass spectrometry.

The "protein-folding problem" refers to the question of how and why a denatured polypeptide chain can spontaneously fold into a compact and highly ordered conformation. The classical description of this process in terms of reaction pathways has been complemented by models that describe folding as a biased conformational diffusion on a multidimensional energy landscape. The identification and characterization of short-lived intermediates provide important insights into the mechanism of folding. Pulsed hydrogen/deuterium exchange (HDX) methods are among the most powerful tools for studying the properties of kinetic intermediates. Analysis of pulse-labeled proteins by mass spectrometry (MS) provides information that is complementary to that obtained in nuclear magnetic resonance (NMR) studies; NMR data represent an average of entire protein ensembles, whereas MS can detect co-existing protein species. MS-based pulse-labeling experiments can distinguish between folding scenarios that involve parallel pathways, and those where folding is channeled through obligatory intermediates. The proteolytic digestion/MS technique provides spatially resolved information on the HDX pattern of folding intermediates. This method is especially important for proteins that are too large to be studied by NMR. Although traditional pulsed HDX protocols are based on quench-flow techniques, it is also possible to use electrospray (ESI) MS to analyze the reaction mixture on-line and "quasi-instantaneously" after labeling. This approach allows short-lived protein conformations to be studied by their HDX level, their ESI charge-state distribution, and their ligand-binding state. Covalent labeling of free cysteinyl residues provides an alternative approach to pulsed HDX experiments. Another promising development is the use of synchrotron X-rays to induce oxidation at specific sites within a protein for studying their solvent accessibility during folding.

Staphylococcal nuclease folding intermediate characterized by hydrogen exchange and NMR spectroscopy.

Pulsed hydrogen-deuterium exchange during refolding was used to probe the protection of backbone amide hydrogens from solvent exchange of the staphylococcal nuclease Pro117-->Gly variant. The extent of exchange for 39 residues was determined by two-dimensional proton NMR after refolding for 5 ms to 10 s. Three kinetic phases are inferred. Modest protection of amides in the early refolding intermediate composed of two beta-sheets formed by local sequence interactions was observed after a 5-ms refolding period. Protection factors were determined by varying the high pH labeling pulse after refolding for 100 ms. The intermediate state has modest, yet significant, protection for residues in the beta-sheets (protection factors of 10-60) and almost no protection in the alpha-helices (protection factors of < 10). The pattern of labeling is consistent with a role for beta-turns and beta-hairpins in the formation of the early intermediate.

The molten globule intermediate of apomyoglobin and the process of protein folding

The molten globule model for the beginning of the folding process, which originated with Kuwajima's studies of alpha-lactalbumin (Kuwajima, K., 1989, Proteins Struct. Funct. Genet. 6, 87-103, and references therein), states that, for those proteins that exhibit equilibrium molten globule intermediates, the molten globule is a major kinetic intermediate near the start of the folding pathway. Pulsed hydrogen-deuterium exchange measurements confirm this model for apomyoglobin (Jennings, P.A. & Wright, P.E., in prep.). The energetics of the acid-induced unfolding transition, which have been determined by fitting a minimal three-state model (N<-->I<-->U; N = native, I = molten globule intermediate, U = unfolded) show that I is more stable than U at neutral pH (Barrick, D. & Baldwin, R.L., 1993, Biochemistry 32, in press), which provides an explanation for why I is formed from U at the start of folding. Hydrogen exchange rates measured by two-dimensional NMR for individual peptide NH protons, taken together with the CD spectrum of I, indicate that moderately stable helices are present in I at the locations of the A, G, and H helices of native myoglobin (Hughson, F.M., Wright, P.E., & Baldwin, R.L., 1990, Science 249, 1544-1548). Directed mutagnesis experiments indicate that the interactions between the A, G, and H helices in I are loose (Hughson, F.M., Barrick, D., & Baldwin, R.L., 1991, Biochemistry 30, 4113-4118), which can explain why I is formed rapidly from U at the start of folding.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection and characterization of an early folding intermediate of T4 lysozyme using pulsed hydrogen exchange and two-dimensional NMR.

Two-dimensional 1H-15N NMR techniques combined with pulsed hydrogen-deuterium exchange have been used to characterize the folding pathway of T4 lysozyme. In the unfolded state, there is little differential protection of the various amides from hydrogen exchange. In the native folded structure, 84 amides of the 164 residues are sufficiently spectrally resolved and protected from solvent exchange to serve as probes of the folding pathway. These probes are located in both the N-terminal and C-terminal domains of the native folded structure of the protein. The studies described here show that at least one intermediate is formed early during refolding at low denaturant concentrations. This intermediate (or intermediates) forms very rapidly (within the 10-ms temporal resolution of our mixing device) under the conditions used and is completed at least 10 times faster than the overall folding event. The intermediate(s) protect(s) from exchange a subset of amides in the N-terminal and C-terminal regions of the protein. In the final folded states these protected regions correspond to two alpha-helices and a beta-sheet region. These amides are protected from exchange by factors between 20 and 200 as compared to the fully unfolded protein. Protection of this magnitude is consistent with the formation of somewhat exposed secondary structure in these regions and could represent a "molten globule"-like or a "framework"-like structure for the intermediate(s) in which specific parts of the sequence form isolated secondary structures that are not stabilized by extensive tertiary interactions.