Hydrogen-deuterium Exchange Behavior Research Articles

Ion Mobility Spectrometry-Mass Spectrometry Coupled with Gas-Phase Hydrogen/Deuterium Exchange for Metabolomics Analyses.

Ion mobility spectrometry-mass spectrometry (IMS-MS) in combination with gas-phase hydrogen/deuterium exchange (HDX) and collision-induced dissociation (CID) is evaluated as an analytical method for small-molecule standard and mixture characterization. Experiments show that compound ions exhibit unique HDX reactivities that can be used to distinguish different species. Additionally, it is shown that gas-phase HDX kinetics can be exploited to provide even further distinguishing capabilities by using different partial pressures of reagent gas. The relative HDX reactivity of a wide variety of molecules is discussed in light of the various molecular structures. Additionally, hydrogen accessibility scoring (HAS) and HDX kinetics modeling of candidate (in silico) ion structures is utilized to estimate the relative ion conformer populations giving rise to specific HDX behavior. These data interpretation methods are discussed with a focus on developing predictive tools for HDX behavior. Finally, an example is provided in which ion mobility information is supplemented with HDX reactivity data to aid identification efforts of compounds in a metabolite extract. Graphical Abstract ᅟ.

Comprehensive Gas-Phase Peptide Ion Structure Studies Using Ion Mobility Techniques: Part 2. Gas-Phase Hydrogen/Deuterium Exchange for Ion Population Estimation.

Gas-phase hydrogen/deuterium exchange (HDX) using D2O reagent and collision cross-section (CCS) measurements are utilized to monitor the ion conformers of the model peptide acetyl-PAAAAKAAAAKAAAAKAAAAK. The measurements are carried out on a home-built ion mobility instrument coupled to a linear ion trap mass spectrometer containing electron transfer dissociation (ETD) capabilities. ETD is utilized to obtain per-residue deuterium uptake data for select ion conformers, and a new algorithm is presented for interpreting the HDX data. Using molecular dynamics (MD) production data and a hydrogen accessibility scoring (HAS)-number of effective collisions (NEC) model, hypothetical HDX behavior is attributed to various in-silico candidate (CCS match) structures. The HAS-NEC model is applied to all candidate structures, and non-negative linear regression is employed to determine structure contributions resulting in the best match to deuterium uptake. The accuracy of the HAS-NEC model is tested with the comparison of predicted and experimental isotopic envelopes for several of the observed c-ions. It is proposed that gas-phase HDX can be utilized effectively as a second criterion (after CCS matching) for filtering suitable MD candidate structures. In this study, the second step of structure elucidation, 13 nominal structures were selected (from a pool of 300 candidate structures) and each with a population contribution proposed for these ions. Graphical Abstract ᅟ.

Probing Electrospray Ionization Dynamics Using Differential Mobility Spectrometry: The Curious Case of 4-Aminobenzoic Acid

Here, we present the separation of two ions that differ only by the site of protonation of the analyte molecule using differential mobility spectrometry (DMS). Protonated 4-aminobenzoic acid molecules (4-ABA) generated by positive-mode electrospray ionization [ESI(+)] can exist with the proton residing on either the amine nitrogen (N-protonated) or the carboxylic acid oxygen (O-protonated), and the protonation site can differ on the basis of the solvent system used. In this study, we demonstrate the identification and separation of N- and O-protonated 4-ABA using DMS, with structural assignments verified by: (1) the presence of distinct peaks in the DMS ionogram, (2) the observed effects resulting from altering the ESI(+) solvent system, (3) the observed (13)C NMR chemical shifts arising from altering the solvent system, (4) the observation of distinct MS/MS fragmentation patterns for the two DMS-separated ions, (5) the unique hydrogen-deuterium exchange behavior for these ions, and (6) the fundamental behavior of these two ions within the DMS cell, linked back to the structural differences between the two protonated forms.

Gas-phase hydrogen deuterium exchange behavior of lysine and its homologs

The gas-phase hydrogen/deuterium exchange behavior for protonated lysine and its three shorter homologs, ornithine (Orn), 2,4-diamino butanoic acid (Daba), and 2,3-diamino propanoic acid (Dapa) were studied in a modified LCQ-DECA ion trap mass spectrometer. The HDX behavior of protonated lysine has been studied previously by several groups. It undergoes five equivalent exchanges, corresponding to the hydrogen atoms attached to the protonated lysine side chain and amine terminus nitrogen, and one slower exchange corresponding to the COOH hydrogen. The shorter homologs also exchange all six labile hydrogen atoms but at different rates. Relative to protonated lysine, DapaH+ exchanges two times faster, DabaH+ exchanges slightly slower, and OrnH+ exchanges nearly 30 times more slowly. High-level hybrid density functional theory calculations attribute this difference to the increased barrier for the relay-type exchange mechanism in OrnH+, relative to the other homologs.

Hydrogen Exchange Mass Spectrometry of Bacteriorhodopsin Reveals Light-Induced Changes in the Structural Dynamics of a Biomolecular Machine

Many proteins act as molecular machines that are fuelled by a nonthermal energy source. Examples include transmembrane pumps and stator-rotor complexes. These systems undergo cyclic motions (CMs) that are being driven along a well-defined conformational trajectory. Superimposed on these CMs are thermal fluctuations (TFs) that are coupled to stochastic motions of the solvent. Here we explore whether the TFs of a molecular machine are affected by the occurrence of CMs. Bacteriorhodopsin (BR) is a light-driven proton pump that serves as a model system in this study. The function of BR is based on a photocycle that involves trans/cis isomerization of a retinal chromophore, as well as motions of transmembrane helices. Hydrogen/deuterium exchange (HDX) mass spectrometry was used to monitor the TFs of BR, focusing on the monomeric form of the protein. Comparative HDX studies were conducted under illumination and in the dark. The HDX kinetics of BR are dramatically accelerated in the presence of light. The isotope exchange rates and the number of backbone amides involved in EX2 opening transitions increase roughly 2-fold upon illumination. In contrast, light/dark control experiments on retinal-free protein produced no discernible differences. It can be concluded that the extent of TFs in BR strongly depends on photon-driven CMs. The light-induced differences in HDX behavior are ascribed to protein destabilization. Specifically, the thermodynamic stability of the dark-adapted protein is estimated to be 5.5 kJ mol(-1) under the conditions of our work. This value represents the free energy difference between the folded state F and a significantly unfolded conformer U. Illumination reduces the stability of F by 2.2 kJ mol(-1). Mechanical agitation caused by isomerization of the chromophore is transferred to the surrounding protein scaffold, and subsequently, the energy dissipates into the solvent. Light-induced retinal motions therefore act analogously to an internal heat source that promotes the occurrence of TFs. Overall, our data highlight the potential of HDX methods for probing the structural dynamics of molecular machines under "engine on" and "engine off" conditions.

Hydrogen/deuterium exchange mass spectrometry and optical spectroscopy as complementary tools for studying the structure and dynamics of a membrane protein

The hydrophobic nature of membrane proteins poses considerable challenges for the application of traditional analytical techniques. Hydrogen/deuterium exchange (HDX) with electrospray mass spectrometry (ESI-MS) detection is a potentially very powerful approach for investigating the structure and dynamics of membrane proteins, but thus far this technique has been applied mostly to soluble species. By using ESI-MS in conjunction with low temperature size exclusion chromatography (SEC) the current study explores the global HDX behavior of the integral membrane protein bacteriorhodopsin (BR) under various conditions. The experiments are complemented by UV–vis absorption measurements that report on the environment of the protein's retinal chromophore and fluorescence spectroscopy which probes the extent of resonance energy transfer between Trp residues and retinal. In agreement with previous reports, our HDX data imply that amide hydrogens in the transmembrane helices of native BR are highly protected, whereas most sites in solvent-exposed loops are readily exchangeable. Low temperature SEC/ESI-MS allows the labile Schiff-base linkage between protein and retinal to be completely preserved, such that the HDX properties of co-existing protein populations can be monitored individually. Solubilization of BR in sodium dodecyl sulfate (SDS) induces major structural changes and hydrolytic loss of retinal. The SDS-denatured protein appears to retain a significant degree of stable helical structure (around 25%, compared to the native state value of 74%). Bicelle-mediated refolding of SDS-denatured bacterioopsin in the presence of free retinal regenerates a native-like BR conformation with a yield close to 0.9. The HDX/SEC/ESI-MS approach developed in this work should be applicable to other membrane protein systems as well.

Metal-free Superoxide Dismutase-1 and Three Different Amyotrophic Lateral Sclerosis Variants Share a Similar Partially Unfolded β-Barrel at Physiological Temperature

The structure and unfolding of metal-free (apo) human wild-type SOD1 and three pathogenic variants of SOD1 (A4V, G93R, and H48Q) that cause familial amyotrophic lateral sclerosis have been studied with amide hydrogen/deuterium exchange and mass spectrometry. The results indicate that a significant proportion of each of these proteins exists in solution in a conformation in which some strands of the beta-barrel (i.e. beta2) are well protected from exchange at physiological temperature (37 degrees C), whereas other strands (i.e. beta3 and beta4) appear to be unprotected from hydrogen/deuterium exchange. Moreover, the thermal unfolding of these proteins does not result in the uniform incorporation of deuterium throughout the polypeptide but involves the local unfolding of different residues at different temperatures. Some regions of the proteins (i.e. the "Greek key" loop, residues 104-116) unfold at a significantly higher temperature than other regions (i.e. beta3 and beta4, residues 21-53). Together, these results show that human wild-type apo-SOD1 and variants have a partially unfolded beta-barrel at physiological temperature and unfold non-cooperatively.

Conformational Dynamics of Free and Catalytically Active Thermolysin Are Indistinguishable by Hydrogen/Deuterium Exchange Mass Spectrometry

Conformational dynamics are thought to be a prerequisite for the catalytic activity of enzymes. However, the exact relationship between structural fluctuations and function is not well understood. In this work hydrogen/deuterium exchange (HDX) and electrospray ionization mass spectrometry (ESI-MS) are used for exploring the conformational dynamics of thermolysin. Amide HDX reflects the internal mobility of proteins; regions that undergo frequent unfolding-refolding show faster exchange than segments that are highly stable. Thermolysin is a zinc protease with an active site that is located between two lobes. Substrate turnover is associated with hinge bending that leads to a closed conformation. Product release regenerates the open form, such that steady-state catalysis involves a continuous closing/opening cycle. HDX/ESI-MS with proteolytic peptide mapping in the absence of substrate shows that elements in the periphery of the two lobes are most mobile. A comparison with previous X-ray data suggests that these peripheral regions undergo quite pronounced structural changes during the catalytic cycle. In contrast, active site residues exhibit only a moderate degree of backbone flexibility, and the central zinc appears to be in a fairly rigid environment. The presence of both rigid and moderately flexible elements in the active site may reflect a carefully tuned balance that is required for function. Interestingly, the HDX behavior of catalytically active thermolysin is indistinguishable from that of the free enzyme. This result is consistent with the view that catalytically relevant motions preexist in the resting state and that enzyme function can only be performed within the limitations given by the intrinsic dynamics of the protein. The data presented in this work indicate the prevalence of stochastic elements in the function of thermolysin, rather than supporting a deterministic mechanism.

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).

Tracking lysozyme unfolding during salt-induced precipitation with hydrogen exchange and mass spectrometry

We utilized electrospray ionization mass spectrometry (ESI-MS) and hydrogen-deuterium exchange (HX) to detect unfolding of hen egg white lysozyme during salt-induced precipitation. Deuterated lysozyme was dissolved in protonated buffer at pH 2.16 and precipitated with ammonium sulfate, sodium chloride, and potassium thiocyanate. ESI-MS was used to detect mass differences in lysozyme due to the loss of deuterons for solvent protons, providing insight on the conformational history of the protein during the labeling experiment. Precipitation with ammonium sulfate and sodium chloride did not unfold lysozyme, consistent with the known stabilizing effects of kosmotropic salts. Potassium thiocyanate, an aggressive chaotrope, was an effective precipitant at 0.2 M, but also disrupted lysozyme structure and caused the formation of precipitate fractions that did not readily redissolve into aqueous solution without the use of a chemical denaturant. Precipitation with 1.0 M thiocyanate resulted in faster rates of unfolding and larger amounts of the insoluble precipitate. The unfolding kinetics were biphasic, exhibiting a slow phase after a few hours that presumably reflected a smaller propensity for lysozyme to unfold in the precipitated state. Bimodal mass distributions in the ESI-MS spectra for the thiocyanate precipitates indicate two states for lysozyme in this system, a native and a molten globule-like partially unfolded state. ESI-MS analysis of the insoluble precipitates indicated that they consisted primarily of protein molecules that had unfolded. Investigation of the HX behavior of lysozyme in a KSCN solution at low protein concentrations confirmed the destabilizing effect of the salt on the protein structure, even when there was almost no solid phase present. The HX/ESI-MS results provide insight into the mechanism combining precipitation and denaturation for such a system, both in terms of obtaining quantitative kinetic and stability information and the identification of the conformers present.

The bimolecular hydrogen-deuterium exchange behavior of protonated alkyl dipeptides in the gas phase

As part of an ongoing characterization of the intrinsic chemical properties of peptides, thermal hydrogen-deuterium exchange has been studied for a series of fast-atom-bombardment-generated protonated alkyldipeptides and related model compounds in the reaction with D2O, CH3OD, and ND3 in a Fourier transform ion cyclotron resonance mass spectrometer. Despite the very large basicity difference between the dipeptides and the D2O and CH3OD exchange reagents, efficient exchange of all active hydrogen atoms occurs. From the kinetic data it appears that exchange of the amino, amide, and hydroxyl hydrogens proceeds with different efficiencies, which implies that the proton in thermal protonated dipeptides is immobile. The selectivity of the exchange at the different basic sites is governed by the nature of both the dipeptide and the exchange reagent. The results indicate that reversible proton transfer in the reaction complexes, which effectuates the deuterium incorporation, is assisted by formation of multiple hydrogen bonds between the reagents. Exchange is considered to proceed via the intermediacy of different competing intermediate complexes, each of which specifically leads to deuterium incorporation at different basic sites. The relative stabilization of the competing intermediate complexes can be related to the relative efficiencies of deuterium incorporation at different basic sites in the dipeptide. For all protonated dipeptides studied, the exchange in the reaction with ND3 proceeds with unit efficiency, whereas all active hydrogen atoms are exchanged equally efficiently. Evidently specific multiple hydrogen bond formations are far less important in the reversible proton transfers with the relatively basic ammonia, which allows effective randomization of all active hydrogen atoms in the reaction complexes.

Fourier transform infrared investigation of the Escherichia coli methionine aporepressor.

This study represents the first physicochemical analysis of the recently cloned methionine repressor protein (Met aporepressor) from Escherichia coli. Infrared spectrometry was used to investigate the secondary structure and the hydrogen-deuterium exchange behavior of the E. coli Met aporepressor. The secondary structure of the native bacterial protein was derived by analysis of the amide I mode. The amide I band contour was found to consist of five major component bands (at 1625, 1639, 1653, 1665, and 1676 cm-1) which reflect the presence of various substructures. The relative areas of these component bands are consistent with a high alpha-helical content of the peptide chain secondary structure in solution (43%) and a small amount of beta-sheet structure (7%). The remaining substructure is assigned to turns (10%) and to unordered (or less ordered) structures (40%). The temperature dependence of the infrared spectra of native Met aporepressor in D2O medium over the temperature interval 20-80 degrees C indicates that there are two discrete thermal events: the first thermal event, centered at 42 degrees C, is associated with the hydrogen-deuterium exchange of the hard-to-exchange alpha-helical peptide bonds accompanied by a partial denaturation of the protein, while the second event, centered around 50 degrees C, represents the irreversible thermal denaturation of the protein.

Measurement and calibration of peptide group hydrogen-deuterium exchange by ultraviolet spectrophotometry

An exceedingly simple and convenient method is described for measuring the hydrogen-deuterium exchange behavior of peptide bond-containing molecules by ultraviolet spectrophotometry. The exchange reaction is initiated by diluting a sample from H 2O into D 2O, or the reverse, and can be followed by an easily observable optical density change in the region of peptide absorbance. The method, unlike infrared and magnetic resonance approaches, requires only small amounts of material and, unlike the tritium-Sephadex method, is not restricted to the study of large molecules. Calibrations are provided for exchange rate as a function of pD and temperature and for the change in absorbance per mole peptide group. With this information, the exchange curve to be expected for any peptide group exposed to solvent can be predicted. Comparison with the measured data can then identify peptide-group hydrogen bonding and can also give a measure of the stability of the hydrogen-bonded structure.