Solution phase dynamics of the DNA repair enzyme spore photoproduct lyase as probed by H/D exchange

Spore photoproduct lyase (SPL) catalyzes the repair of the UV lesion spore photoproduct (SP) in a reaction dependent on S-adenosyl-l-methionine (SAM). We have utilized H/D exchange to show that in the presence of SAM, a significant reduction in H/D exchange is observed upon binding SPTpT or undamaged oligonucleotide, indicating a shift of 20 or 10 amide protons, respectively, from a rapidly-exchangable state to a fully-protected conformation. In the absence of SAM, neither the oligonucleotide nor the SPTpT produce a significant perturbation in H/D exchange, indicating SAM is a requisite binding partner. Performing the same experiments in aerobic conditions reduced the magnitude of ligand-induced structural changes, consistent with the importance of the oxygen-sensitive iron–sulfur cluster for SAM and substrate binding.     

Direct evidence by H/D exchange and ESI-MS for transient unproductive domain interaction in the refolding of an antibody scFv fragment

The refolding kinetics of a single-chain Fv (scFv) fragment, derived from a stabilized mutant of the phosphorylcholine binding antibody McPC603, was investigated by H/D exchange and ESI-MS and compared with the folding kinetics of its constituting domains V(H) and V(L). Both V(H) and V(L) adopt essentially native-like exchange protection within the dead time of the manual-mixing H/D exchange experiment (10 s) and in the case of V(L), which contains two cis-prolines in the native conformation, this fast protection is independent of proline cis/trans isomerization. At the earliest time point resolvable by manual mixing, fewer deuterons are protected in the scFv fragment than in the two isolated domains together, despite the fact that the scFv fragment is significantly more stable than V(L) and V(H). Full H/D exchange protection in the scFv fragment is gained on a time scale of minutes. This means that the domains in the scFv fragment do not refold independently. Rather, they associate prematurely and in nonnative form, a kinetic trap. Unproductive domain association is observed both after equilibrium- and short-term denaturation. For the equilibrium-denatured scFv fragment, whose native structure formation is dependent on a cis conformation of an interface proline in V(L), this cis/trans isomerization reaction proceeds about one order in magnitude more slowly than the escape from the trap to a conformation where full H/D exchange protection is already achieved. We interpret these data in terms of a general kinetic scheme involving intermediates with and without domain association.     

Different coordination modes of an aryl-substituted hydrotris(pyrazolyl)borate ligand in rhodium and iridium complexes

Complexes Tp^tolRh(C₂H₄)₂ (1a) and Tp^tolRh(CH₂C(Me)C(Me)CH₂) (1b) have been prepared by reaction of KTp^tol with the appropriate [RhCl(olefin)₂]₂ dimer (Tp^tol means hydrotris(3-p-tolylpyrazol-1-yl)borate). The two complexes show a dynamic behaviour that involves exchange between κ² and κ³ coordination modes of the Tp^tol ligand. The iridium analogue, Tp^tolIr(CH₂C(Me)CHCH₂) (2) has also been synthesized, and has been converted into the Ir(III) dinitrogen complex [(κ⁴-N,N’,N’’,C-Tp^tol)Ir(Ph)(N₂) (3) by irradiation with UV light under a dinitrogen atmosphere. Compound 3 constitutes a rare example of Ir(III)-N₂ complex structurally characterized by X-ray crystallography. Its N₂ ligand can be easily substituted by acetonitrile or ethylene upon heating and denticity changes in the Tp^tol ligand, from κ⁴-N,N’,N’’,C (monometallated Tp^tol, from now on represented as Tp^tol′) to κ⁵-N,N′,N″,C,C″ (dimetallated Tp^tol ligand, represented as Tp^tol″) have been observed. When complex 3 is heated in the presence of acetylene, dimerization of the alkyne takes place to yield the enyne complex [(κ⁵-N,N′,N′′,C,C′-Tp^tol)Ir(CH₂CHCCH), 7¸ in which the unsaturated organic moiety is bonded to iridium through the carbon-carbon double bond.     

The use of spin desalting columns in DMSO‐quenched H/D‐exchange NMR experiments

Dimethylsulfoxide (DMSO)‐quenched hydrogen/deuterium (H/D)‐exchange is a powerful method to characterize the H/D‐exchange behaviors of proteins and protein assemblies, and it is potentially useful for investigating non‐protected fast‐exchanging amide protons in the unfolded state. However, the method has not been used for studies on fully unfolded proteins in a concentrated denaturant or protein solutions at high salt concentrations. In all of the current DMSO‐quenched H/D‐exchange studies of proteins so far reported, lyophilization was used to remove D₂O from the protein solution, and the lyophilized protein was dissolved in the DMSO solution to quench the H/D exchange reactions and to measure the amide proton signals by two‐dimensional nuclear magnetic resonance (2D NMR) spectra. The denaturants or salts remaining after lyophilization thus prevent the measurement of good NMR spectra. In this article, we report that the use of spin desalting columns is a very effective alternative to lyophilization for the medium exchange from the D₂O buffer to the DMSO solution. We show that the medium exchange by a spin desalting column takes only about 10 min in contrast to an overnight length of time required for lyophilization, and that the use of spin desalting columns has made it possible to monitor the H/D‐exchange behavior of a fully unfolded protein in a concentrated denaturant. We report the results of unfolded ubiquitin in 6.0M guanidinium chloride.     

Activation and deactivation of a carbene containing non-classical ruthenium hydride complex in catalytic processes involving C-H bond cleavage

The carbene-containing non-classical ruthenium hydride complex [(PCy₃)Ru(H)₂(H₂)₂(IMes)] 1 (IMes=1,3-Bis-(2,4,6-trimethylphenyl)imidazol-2-ylidene) is an active catalyst for H/D exchange in aromatic ketones. It is also capable of combining sp² C-H bond activation with C-C bond formation. Comparing the chemo- and regio-selectivities of the H/D exchange process and the C-C bond formation clearly indicates that different intermediates are involved in the two processes. High pressure NMR studies provide strong evidence that the key intermediate for the C-C coupling reaction is analogous to that for other ruthenium catalysts reported previously. Catalytic turnover is limited by the instability of this intermediate in the presence of the olefinic coupling partner.     

Thermodynamic stability measurements on multimeric proteins using a new H/D exchange- and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based method

We recently reported on a new H/D exchange- and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based technique, termed SUPREX, that removes several important limitations associated with measuring the thermodynamic stability of proteins. In contrast to conventional spectroscopy-based techniques for characterizing the equilibrium unfolding behavior of proteins, SUPREX is amenable to the thermodynamic analysis of both purified and unpurified proteins using mg to ng quantities of material. Here we report on the application of SUPREX to the analysis of multimeric protein systems. Included in this work are the SUPREX results we obtained in studies on six model multimeric proteins including the GCN4p1 dimer, the coil-V(a)L(d) trimer, the 4-oxalocrotonate tautomerase (4-OT) hexamer, the Trp repressor (TrpR) dimer, the Arc repressor (ArcR) dimer, and an ArcR mutant (the (DOA20)ArcR) dimer which contained two destabilizing mutations including an Asp to Ala mutation at position 20 and an amide to ester bond mutation between amino acid (aa) residues 19 and 20. As part of the work described here, we present a new method for the analysis of SUPREX data that is generally applicable to both monomeric and multimeric protein systems. Our results on the model proteins in this study indicate that this new method can be used to determine folding free energies for proteins with the accuracy and precision of conventional spectroscopy-based methods.     

Volume-activated Na/H exchange activity in fetal and adult pig red cells: Inhibition by cyclic AMP

Summary Hyposmotic swelling of pig red cells leads to a selective increase in K permeability, whereas hyperosmotic cell shrinkage augments the Na permeability. In this regard, the ouabain-resistant (OR) Na flux of red cells of newborn and adult pigs is characterized in detail. A reduction in cell volume by approximately 18% leads to an increase in the OR Na efflux of fetal and adult cells by 15-and fourfold, respectively. The OR Na influx in both cell types is equally influenced by cell shrinkage. Depletion of cellular K does not influence the volume-activated OR Na efflux. Nor does OR Na influx require external K. Both OR Na efflux and influx activated by shrinkage are inhibited by the diuretics furosemide and amiloride. The rank order of decreasing anion sensitivity for diuretic-sensitive Na efflux was acetate > chloride > gluconate > nitrate. Cell shrinkage induced by the addition of hypertonic salts results in an acidification of the unbuffered and CO₂-free media, provided that both Na and DIDS are present. The qcidification process can be reversed by either of the diuretic agents. These findings suggest that the shrinkageactivated OR Na flux is primarily mediated by a Na/H exchanger rather than by a Na/K/Cl cotransporter. Once loaded with either cAMP or cGMP, cell swelling can no longer activate the Na/H exchanger. The Na/H exchanger activity is detectable in the fetal cells of normal volume but quiescent in adult cells, indicating that the exchanger undergoes a developmental change during the transition from the fetal to adult stage.     

Deuterium exchange and mass spectrometry reveal the interaction differences of two synthetic modulators of RXRalpha LBD

Protein amide hydrogen/deuterium (H/D) exchange was used to compare the interactions of two antagonists, UVI 2112 and UVI 3003, with that of the agonist, 9-cis-retinoic acid, upon binding to the human retinoid X receptor alpha ligand-binding domain (hRXRalpha LBD) homodimer. Analysis of the H/D content by mass spectrometry showed that in comparison to 9-cis-retinoic acid, the antagonists provide much greater protection toward deuterium exchange-in throughout the protein, suggesting that the protein-antagonist complex adopts a more restricted conformation or ensemble of conformations in which solvent accesses to amide protons are reduced. A comparison between the two antagonists shows that UVI 3003 is more protective in the C-terminal region due to the extra hydrophobic interactions derived from the atoms in the benzene ring of the carboxylic acid chain. It was less protective within regions comprising peptides 271-278 and 326-330 due to differences in conformational orientation, and/or shorter carboxylic acid chain length. Decreased deuterium exchange-in in the segment 234-239 where the residues do not involve interactions with the ligand was observed with the two antagonists, but not with 9-cis-RA. The amide protons of helix 12 of the agonist- or antagonist-occupied protein in solution have the same deuterium exchange rates as the unliganded protein, supporting a suggestion made previously that helix 12 can cover the occupied binding cavity only with the cofactor present to adjust its location.     

Potassium/proton exchange in brush-border membrane of rat ileum

Summary These experiments were designed to determine whether proton-driven⁸⁶Rb uptake was present in apical membrane vesicles prepared from rat ileum. The uptake of⁸⁶Rb was approximately 300 to 350% greater in the presence of a 100-fold H⁺ gradient than in its absence and was greater at 1, 2 and 5 minutes (overshoot) than that at 90 minutes. Proton-driven⁸⁶Rb uptake was decreased by 20% in TMA-nitrate compared to that in TMA-gluconate. 0.3mm amiloride did not significantly inhibit proton-driven⁸⁶Rb uptake; in contrast, proton-driven²²Na uptake was significantly inhibited by 0.3mm amiloride by 34%. Similarly, 25mm KCl inhibited proton-driven⁸⁶Rb uptake more than that of²²Na, while the inhibition of proton-driven²²Na uptake by 25mm NaCl was greater than that of⁸⁶Rb. In additional studies intravesicular acidification measured by acridine orange fluorescence was demonstrated in the presence of an out-wardly directed K gradient. These studies demonstrate that a proton gradient stimulates⁸⁶Rb uptake and a K gradient induces intravesicular acidification; and that these fluxes are mediated by a K/H exchange distinct from Na/H exchange which is also present in this membrane. We conclude that a specific exchange process for K/H is located in ileal apical membrane vesicles.     

Dehydrogenation of alkanes catalyzed by an iridium phosphinito PCP pincer complex

The novel phosphinito iridium PCP pincer complex, IrH₄{C₆H₃-2,6-(OPPrⁱ₂)₂} can be conveniently synthesized and isolated in 74% overall yield. The complex catalyzes the dehydrogenation of linear alkanes to alkenes at the rate of 13 turnovers min⁻¹ at 200 °C. This catalytic activity is slightly higher than that exhibited by iridium complexes of analogous bis(phosphino) PCP ligands. The bis(phosphinito) complex is resistant to decomposition for periods up to 7 days at temperatures as high as 200 °C thus matching the thermal stability of the bis(phosphino) pincer complexes.     

Inclusion of carvone enantiomers in cyclomaltoheptaose (beta-cyclodextrin): thermal behaviour and H-->D and D-->H exchange

The inclusion compounds of carvone enantiomers in cylcomaltoheptaose (beta-cyclodextrin, betaCD) are studied at defined temperatures above room temperature and in relation to H-->D and D-->H exchanges. Loss of water molecules and release of carvone molecules from the betaCD cavity are caused by increase of temperature above room temperature and are measured by the integrated intensities of the O-H and C-H Raman stretching bands, respectively. In turn, H-->D and D-->H exchanges are monitored by the integrated intensities of the O-H and O-D Raman stretching bands, respectively. All of these processes were followed in real time with a Raman spectrometer equipped with CCD detection. The results indicate that distinct carvone enantiomers lead to the formation of different betaCD inclusion hydrates that have different water content and hydration structures. In particular, the results suggest that SCarv-betaCD has a greater water content, dehydrates strongly for temperatures above room temperature, and exchanges protons faster than the RCarv-betaCD complex.     

Kinetic studies on the first dihydrogen aquacomplex, [Ru(H2)(H2O)5]: Formation under H2 pressure and catalytic H/D isotope exchange in water

The ruthenium(II) hexaaqua complex [Ru(H₂O)₆]²⁺ reacts with dihydrogen under pressure to give the η²-dihydrogen ruthenium(II) pentaaqua complex [Ru(H₂)(H₂O)₅]²⁺.The complex was characterized by ¹H, ²H and ¹⁷O NMR: δ_H = −7.65 ppm, J_HD = 31.2 Hz, δ_O = −80.4 ppm (trans to H₂) and δ_O = −177.4 ppm (cis to H₂).The H-H distance in coordinated dihydrogen was estimated to 0.889 Å from J_HD, which is close to the value obtained from DFT calculations (0.940 Å).Kinetic studies were performed by ¹H and ²H NMR as well as by UV-Vis spectroscopy, yielding the complex formation rate and equilibrium constants: k_f = (1.7 ± 0.2) × 10⁻³ kg mol⁻¹ s⁻¹ and K_eq = 4.0 ± 0.5 mol kg⁻¹.The complex formation rate with dihydrogen is close to values reported for other ligands and thus it is assumed that the reaction with dihydrogen follows the same mechanisn (I_d).In deuterated water, one can observe that [Ru(H₂)(H₂O)₅]²⁺ catalyses the hydrogen exchange between the solvent and the dissolved dihydrogen.A hydride is proposed as the intermediate for this exchange.Using isotope labeling, the rate constant for the hydrogen exchange on the η²-dihydrogen ligand was determined as k₁ = (0.24 ± 0.04) × 10⁻³ s⁻¹.The upper and lower limits of the pK_a of the coordinated dihydrogen ligand have been estimated:3 < pK_a < 14.     

Core and heterogeneity of beta2-microglobulin amyloid fibrils as revealed by H/D exchange

Dialysis-related amyloidosis, which occurs in the patients receiving a long-term hemodialysis with high frequency, accompanies the deposition of amyloid fibrils composed of beta(2)-microglobulin (beta2-m). In vitro, beta2-m forms two kinds of fibrous structures at acidic pH. One is a rigid "mature fibril", and the other is a flexible thin filament often called an "immature fibril". In addition, a 22-residue peptide (K3 peptide) corresponding to Ser20 to Lys41 of intact beta2-m forms rigid amyloid-like fibrils similar to mature fibrils. We compared the core of these three fibrils at single-residue resolution using a recently developed hydrogen/deuterium (H/D) exchange method with the dissolution of fibrils by dimethylsulfoxide (DMSO). The exchange time-course of these fibrils showed large deviations from a single exponential curve showing that, because of the supramolecular structures, the same residue exists in different environments from molecule to molecule, even in a single fibril. The exchange profiles revealed that the core of the immature fibril is restricted to a narrow region compared to that of the mature fibril. In contrast, all residues were protected from exchange in the K3 fibril, indicating that a whole region of the peptide is engaged in the beta-sheet network. These results suggest the mechanism of amyloid fibril formation, in which the core beta-sheet formed by a minimal sequence propagates to form a rigid and extensive beta-sheet network.     

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.     

Regulation of cyclooxygenase catalysis by hydroperoxides

Activation of cyclooxygenase catalysis in prostaglandin H synthase-1 and -2 by peroxide-dependent formation of a tyrosyl radical is emerging as an important part of regulating cellular production of bioactive prostanoids. This review discusses the mechanism of tyrosyl radical formation and the influence of peroxide, fatty acid, peroxidase cosubstrate, and protein structure on the activation process and cyclooxygenase catalysis.     

Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry

MAb1, a human IgG1 monoclonal antibody produced in a NS0 cell line, exhibits charge heterogeneity because of the presence of variants formed by processes such as N-terminal glutamate cyclization, C-terminal lysine truncation, deamidation, aspartate isomerization and sialylation in the carbohydrate moiety. Four major charge variants of MAb1 were isolated and the conformations of these charge variants were studied using hydrogen/deuterium exchange mass spectrometry, including the H/D exchange time course (HX-MS) and the stability of unpurified proteins from rates of H/D exchange (SUPREX) techniques. HX-MS was used to evaluate the conformation and solution dynamics of MAb1 charge variants by measuring their deuterium buildup over time at the peptide level. The SUPREX technique evaluated the unfolding profile and relative stability of the charge variants by measuring the exchange properties of globally protected amide protons in the presence of a chemical denaturant. The H/D exchange profiles from both techniques were compared among the four charge variants of MAb1. The two techniques together offered extensive understanding about the local and subglobal/global unfolding of the charge variants of MAb1. Our results demonstrated that all four charge variants of MAb1 were not significantly different in conformation, solution dynamics and chemical denaturant-induced unfolding profile and stability, which aids in understanding the biofunctions of the molecules. The analytical strategy used for conformational characterization may also be applicable to comparability studies done for antibody therapeutics.     

Regulation of intracellular pH in LLC-PK1 cells by Na+/H+ exchange

Summary Suspensions of LLC-PK₁ cells (a continuous epitheliod cell line with renal characteristics) are examined for mechanisms of intracellular pH regulation using the fluorescent probe BCECF. Initial experiments determine suitable calibration procedures for use of the BCECF fluorescent signal. They also determine that the cell suspension contains cells which (after 4 hr in suspension) have Na⁺ and K⁺ gradients comparable to those of cells in monolayer culture. The steady-state intracellular pH (7.05±0.01,n=5) of cells which have recovered in (pH 7.4) Na⁺-containing medium is not affected over several minutes by addition of 100 M amiloride or removal of extracellular Na⁺ (Na _o ⁺ /H _i ⁺ and Na _i ⁺ /H _o ⁺ exchange reactions are functionally inactive (compared to cellular buffering capacity). In contrast, Na _o ⁺ /H _i ⁺ exchange is activated by an increased cellular acid load. This activation may be observed directly either as a stimulation of net H⁺ efflux or net Na⁺ influx with decreasing intracellular pH. The extrapolation of this latter data suggests a set point of Na⁺/H⁺ exchange of approximately pH 7.0, consistent with the observed resting intracellular pH of approximately 7.05.