Hydrogen Exchange Studies Research Articles

Study of hydrogen exchange between the Zr–1%Nb alloy and the gaseous ambient through the oxidized surface under ion and atomic irradiation

In the article, a hydrogen exchange between the Zr–1%Nb alloy (E110) and the gas ambient was experimentally studied when the samples were irradiated with deuterium and argon plasma ions. It has been established that, upon irradiation with deuterium plasma ions with an energy of E = 650 eV/at, the enhanced absorption of deuterium exceeds the release of hydrogen initially contained in the samples, which leads to their loading with hydrogen isotopes. Adding 30 at.% oxygen to the plasma-forming gas or raising the sample temperature from T = 450 K to T = 600 K, significantly reduces the content of hydrogen isotopes in the sample. Based on the aggregate data obtained by atomic and ion irradiation, a mechanism of hydrogen exchange between zirconium alloy and gas ambient is proposed. The process includes three stages: reactions on the oxidized surface of the zirconium alloy (surface hydroxylation and formation of water molecules); reactions at the metal-oxide interface; transfer of hydrogen isotopes through the surface oxide layer in both directions due to hopping between neighboring oxygen ions. Surface reactions caused by irradiation of atoms and ions trigger the hydrogen exchange. The proposed model agrees with the experimental data on the irradiation of the E110 alloy with atoms and ions of hydrogen isotopes.

Solution NMR studies reveal novel CLIC4 chaperone activity with respect to profilin-1.

Despite its name, chloride intracellular channel 4 (CLIC4) is a soluble enzyme that can catalyze glutathione-dependent thiol transfers. It is unclear how this enzymatic activity relates to its biological role in remodeling the actin cytoskeleton. Previous studies have shown that CLIC4 interacts with profilin-1, a critical regulator of actin polymerization. We confirmed a direct interaction, 170 ± 20 μM by microscale thermophoresis. However, using solution nuclear magnetic resonance (NMR) spectroscopy, we demonstrate that CLIC4 has a higher affinity for profilin when it is unfolded. Amide hydrogen exchange studies demonstrate that the N- and C-terminal helices of profilin readily unfold, whereas the central β-beta sheet core remains relatively intact. Interaction with CLIC4 increases amide exchange rates throughout profilin, indicating preferential stabilization of partially unfolded intermediates. We note that profilin is glutathionylated in proteomic studies of cells exposed to oxidative stress. Profilin is unfolded by glutathionylation of buried cysteine residues in its N- or C-terminal helices, and this disrupts its ability to bind actin, as measured by pyrene-actin polymerization assay. We are currently attempting to demonstrate that CLIC4 can reverse glutathionylation of profilin-1 and promote its refolding in cells and in vitro.

Retrospective study for the universal applicability of the residue-based linear free energy relationship in the two-state exchange of protein molecules

Multiprobe measurements, such as NMR and hydrogen exchange studies, can provide the equilibrium constant, K, and rate constants for forward and backward processes, k and k′, of the two-state structural changes of a polypeptide on a per-residue basis. We previously found a linear relationship between log K and log k and between log K and log k′ for the topological exchange of a 27-residue bioactive peptide. To test the general applicability of the residue-based linear free energy relationship (rbLEFR), we performed a literature search to collect residue-specific K, k, and k′ values in various exchange processes, including folding-unfolding equilibrium, coupled folding and binding of intrinsically disordered peptides, and structural fluctuations of folded proteins. The good linearity in a substantial number of the log–log plots proved that the rbLFER holds for the structural changes in a wide variety of protein-related phenomena. Among the successful cases, the hydrogen exchange study of apomyoglobin folding intermediates is particularly interesting. We found that the residues that deviated from the linear relationship corresponded to the α-helix, for which transient translocation had been identified by other experiments. Thus, the rbLFER is useful for studying the structures and energetics of the dynamic states of protein molecules.

Measuring Solvent Hydrogen Exchange Rates by Multifrequency Excitation 15N CEST: Application to Protein Phase Separation.

Solvent exchange rates provide important information about the structural and dynamical properties of biomolecules. A large number of NMR experiments have been developed to measure such rates in proteins, the great majority of which quantify the buildup of signals from backbone amides after initial perturbation of water magnetization. Here we present a different approach that circumvents the main limitations that result from these classical hydrogen exchange NMR experiments. Building on recent developments that enable rapid recording of chemical exchange saturation transfer (CEST) pseudo-3D data sets, we describe a 15N-based CEST scheme for measurement of solvent exchange in proteins that exploits the one-bond 15N deuterium isotope shift. The utility of the approach is verified with an application to a 236 residue intrinsically disordered protein domain under conditions where it phase separates and a second application involving a mutated form of the domain that does not phase separate, establishing very similar hydrogen exchange rates for both samples. The methodology is well suited for studies of hydrogen exchange in any 15N-labeled biomolecule. A discussion of the merits of the CEST experiment in relation to the popular CLEANEX-PM scheme is presented.

Cytochrome c folds through foldon-dependent native-like intermediates in an ordered pathway

Previous hydrogen exchange (HX) studies of the spontaneous reversible unfolding of Cytochrome c (Cyt c) under native conditions have led to the following conclusions. Native Cyt c (104 residues) is composed of five cooperative folding units, called foldons. The high-energy landscape is dominated by an energy ladder of partially folded forms that differ from each other by one cooperative foldon unit. The reversible equilibrium unfolding of native Cyt c steps up through these intermediate forms to the unfolded state in an energy-ordered sequence, one foldon unit at a time. To more directly study Cyt c intermediates and pathways during normal energetically downhill kinetic folding, the present work used HX pulse labeling analyzed by a fragment separation-mass spectrometry method. The results show that 95% or more of the Cyt c population folds by stepping down through the same set of foldon-dependent pathway intermediates as in energetically uphill equilibrium unfolding. These results add to growing evidence that proteins fold through a classical pathway sequence of native-like intermediates rather than through a vast number of undefinable intermediates and pathways. The present results also emphasize the condition-dependent nature of kinetic barriers, which, with less informative experimental methods (fluorescence, etc.), are often confused with variability in intermediates and pathways.

Estimation of Hydrogen-Exchange Protection Factors from MD Simulation Based on Amide Hydrogen Bonding Analysis.

Hydrogen exchange (HX) studies have provided critical insight into our understanding of protein folding, structure, and dynamics. More recently, hydrogen exchange mass spectrometry (HX-MS) has become a widely applicable tool for HX studies. The interpretation of the wealth of data generated by HX-MS experiments as well as other HX methods would greatly benefit from the availability of exchange predictions derived from structures or models for comparison with experiment. Most reported computational HX modeling studies have employed solvent-accessible-surface-area based metrics in attempts to interpret HX data on the basis of structures or models. In this study, a computational HX-MS prediction method based on classification of the amide hydrogen bonding modes mimicking the local unfolding model is demonstrated. Analysis of the NH bonding configurations from molecular dynamics (MD) simulation snapshots is used to determine partitioning over bonded and nonbonded NH states and is directly mapped into a protection factor (PF) using a logistics growth function. Predicted PFs are then used for calculating deuteration values of peptides and compared with experimental data. Hydrogen exchange MS data for fatty acid synthase thioesterase (FAS-TE) collected for a range of pHs and temperatures was used for detailed evaluation of the approach. High correlation between prediction and experiment for observable fragment peptides is observed in the FAS-TE and additional benchmarking systems that included various apo/holo proteins for which literature data were available. In addition, it is shown that HX modeling can improve experimental resolution through decomposition of in-exchange curves into rate classes, which correlate with prediction from MD. Successful rate class decompositions provide further evidence that the presented approach captures the underlying physical processes correctly at the single residue level. This assessment is further strengthened in a comparison of residue resolved protection factor predictions for staphylococcal nuclease with NMR data, which was also used to compare prediction performance with other algorithms described in the literature. The demonstrated transferable and scalable MD based HX prediction approach adds significantly to the available tools for HX-MS data interpretation based on available structures and models.

Comparison of the backbone dynamics of wild-type Hydrogenobacter thermophilus cytochrome c(552) and its b-type variant.

Cytochrome c552 from the thermophilic bacterium Hydrogenobacter thermophilus is a typical c-type cytochrome which binds heme covalently via two thioether bonds between the two heme vinyl groups and two cysteine thiol groups in a CXXCH sequence motif. This protein was converted to a b-type cytochrome by substitution of the two cysteine residues by alanines (Tomlinson and Ferguson in Proc Natl Acad Sci USA 97:5156–5160, 2000a). To probe the significance of the covalent attachment of the heme in the c-type protein, 15N relaxation and hydrogen exchange studies have been performed for the wild-type and b-type proteins. The two variants share very similar backbone dynamic properties, both proteins showing high 15N order parameters in the four main helices, with reduced values in an exposed loop region (residues 18–21), and at the C-terminal residue Lys80. Some subtle changes in chemical shift and hydrogen exchange protection are seen between the wild-type and b-type variant proteins, not only for residues at and neighbouring the mutation sites, but also for some residues in the heme binding pocket. Overall, the results suggest that the main role of the covalent linkages between the heme group and the protein chain must be to increase the stability of the protein.

Temperature dependent hydrogen exchange study of DNA duplexes containing binding sites for Arabidopsis TCP transcription factors

The TCP domain is a DNA-binding domain present in plant transcription factors and plays important roles in various biological functions. The hydrogen exchange rate constants of the imino protons were determined for the three DNA duplexes containing the DNA-binding sites for the TCP11, TCP15, and TCP20 transcription factors using NMR spectroscopy. The M11 duplex displays unique hydrogen exchange property of the five base pairs in the first binding site (5'-GTGGG-3'). However, the M15 and M20 duplexes lead to clear changes in thermal stabilities of these five base pairs. The unique dynamic features of the five base pairs in the first binding site might play crucial roles in the sequence-specific DNA binding of the class I TCP transcription factors.

Rational stabilization of helix 2 of the prion protein prevents its misfolding and oligomerization.

Designed stabilization of helix 2 of the mouse prion protein is shown to lead to an increase in global stability of the protein. Studies of hydrogen exchange coupled to mass spectrometry confirm that the increase in stability is confined primarily to helix 2, and that it accounts for the global stabilization of the protein. Importantly, such localized stabilization of the protein can completely inhibit its ability to form oligomers and slows down amyloid fibril formation.

Non-Native Structure Appears in Microseconds during the Folding of E. coli RNase H

The folding pathway of Escherichia coli RNase H is one of the best experimentally characterized for any protein. In spite of this, spectroscopic studies have never captured the earliest events. Using continuous-flow microfluidic mixing, we have now observed the first several milliseconds of folding by monitoring the tryptophan fluorescence lifetime (60μs dead time). Two folding intermediates are observed, the second of which is the previously characterized Icore millisecond intermediate. The new earlier intermediate is likely on-pathway and appears to have long-range non-native structure, providing a rare example of such non-native structure formation in a folding pathway. The tryptophan fluorescence lifetimes also suggest a deviation from native packing in the second intermediate, Icore. Similar results from a fragment of RNase H demonstrate that only half of the protein is significantly involved in this early structure formation. These studies give us a view of the formation of tertiary structure on the folding pathway, which complements previous hydrogen-exchange studies that monitored only secondary structure and observed sequential native structure formation. Our results provide detailed folding information on both a timescale and a size-scale accessible to all-atom molecular dynamics simulations of protein folding.

NMR study of the Z-DNA binding mode and B–Z transition activity of the Zα domain of human ADAR1 when perturbed by mutation on the α3 helix and β-hairpin

The Zα domains of human ADAR1 (ZαADAR1) bind to Z-DNA via interaction mediated by the α3-core and β-hairpin. Five residues in the α3 helix and four residues in the β-hairpin play important roles in Zα function, forming direct or water-mediated hydrogen bonds with DNA backbone phosphates or interacting hydrophobically with DNA bases. To understand the roles of these residues during B–Z transition of duplex DNA, we performed NMR experiments on complexes of various ZαADAR1 mutants with a 6-bp DNA duplex at various protein-to-DNA molar ratios. Our study suggests that single mutations at residues K169, N173, or Y177 cause unusual conformational changes in the hydrophobic faces of helices α1, α2, and α3, which dramatically decrease the Z-DNA binding affinity. 1D imino proton spectra and chemical shift perturbation showed that single mutations at residues K170, R174, T191, P192, P193, or W195 slightly affected the Z-DNA binding affinity. A hydrogen exchange study proved that the K170A- and R174A-ZαADAR1 proteins could efficiently change B-DNA to left-handed Z-DNA via an active B–Z transition pathway, whereas the G2·C5 base pair was significantly destabilized compared to wild-type ZαADAR1.

Hydrogen Exchange Study of DNA Duplexes Containing the Consensus Binding Site for Arabidopsis thaliana SPL14 Transcription Factor

To understand the DNA binding mechanism ofAtSPL14 protein, the imino proton exchange rates weremeasured for the DNA duplex containing the consensusDNA-binding site for the AtSPL14 transcription factor(referred to as wt-SPL14 duplex, Fig. 1). To further under-stand the correlation between the base pair stability/dynamicsand DNA binding affinity of the AtSPL14, the exchange rateconstants of the imino protons for the wt-SPL14 duplexwere compared with those of the mutant SPL14 duplexes(see Fig. 1), which display different binding affinities forAtSPL14 protein. Experimental SectionAll DNA oligonucleotides were purchased from M-bio-tech Co. (Seoul, Korea). The oligonucleotides were purifiedby reverse-phase HPLC and desalted by Sephadex G-25 column.DNA duplexes were prepared by dissolving two strands at a1:1 stoichiometric ratio in an NMR buffer (90% H

NMR Study of Hydrogen Exchange in the DNA Decamer Duplexes Containing the p53 Response Element

E-mail: byongseok.choi@kaist.ac.kr Received October 5, 2011, Accepted November 24, 2011Key Words : NMR, Hydrogen exchange, p53 response element, CancerP53 is the major tumor suppressor and is a transcriptionfactor involved in various cellular metabolisms, includingDNA repair, regulation of cell cycle, apoptotic cell death.

Hydrogen Exchange Study on the Hydroxyl Groups of Serine and Threonine Residues in Proteins and Structure Refinement Using NOE Restraints with Polar Side-Chain Groups

We recently developed new NMR methods for monitoring the hydrogen exchange rates of tyrosine hydroxyl (Tyr-OH) and cysteine sulfhydryl (Cys-SH) groups in proteins. These methods facilitate the identification of slowly exchanging polar side-chain protons in proteins, which serve as sources of NOE restraints for protein structure refinement. Here, we have extended the methods for monitoring the hydrogen exchange rates of the OH groups of serine (Ser) and threonine (Thr) residues in an 18.2 kDa protein, EPPIb, and thus demonstrated the usefulness of NOE restraints with slowly exchanging OH protons for refining the protein structure. The slowly exchanging Ser/Thr-OH groups were readily identified by monitoring the (13)C(β)-NMR signals in an H(2)O/D(2)O (1:1) mixture, for the protein containing Ser/Thr residues with (13)C, (2)H-double labels at their β carbons. Under these circumstances, the OH groups exist in equilibrium between the protonated and deuterated isotopomers, and the (13)C(β) peaks of the two species are resolved when their exchange rate is slower than the time scale of the isotope shift effect. In the case of EPPIb dissolved in 50 mM sodium phosphate buffer (pH 7.5) at 40 °C, one Ser and four Thr residues were found to have slowly exchanging hydroxyl groups (k(ex) < ~40 s(-1)). With the information for the slowly exchanging Ser/Thr-OH groups in hand, we could collect additional NOE restraints for EPPIb, thereby making a unique and important contribution toward defining the spatial positions of the OH protons, and thus the hydrogen-bonding acceptor atoms.

NMR Study of Hydrogen Exchange in the DNA Decamer Duplexes Containing the -10 galP1 Promoter Sequence

Most Escherichia coli promoters are consists of two 6-bp sequence elements which are centered at ~10 and 35 nucleotides upstream of the transcription initiation point. The conserved region 4.2 and 2.4 of the RNA polymerase σ subunit recognize −10 and −35 promoter elements, respectively. The open complex between σ subunit and promoter elements, in which the promoter DNA is locally melted, is competent to initiate RNA synthesis according to the DNA sequence of template strand. Promoter opening is temperature-dependent; the most −10/−35 promoter complexes display closed forms below 15 C, whereas promoter complexes on the extended −10 galP1 promoter remain open at 5 C. The reason for this unusual behavior of the −10 galP1 promoter is not completely understood. The conserved A·T base pair at the −11 position of the promoters plays very important role in the initiation of E. coli transcription. The in vitro transcription with the −10 galP1 promoter was not initiated when this A base was substituted to other bases. To understand the function of the A-11 base in the transcription initiation process, the imino proton exchange rates were measured for the DNA decamer duplex containing the wild-type −10 galP1 promoter sequence (position: −14 ~ −5) (referred to as wt galP1, Fig. 1). The exchange rate constants of the imino protons for the wt galP1 duplex were compared with those of the modified galP1 promoters at the −11 position (see Fig. 1). This comparison indicates that some differences in the base pair dynamics are correlated with function of the −10 galP1 promoter during transcription initiation.

Base pair opening kinetics study of the aegPNA:DNA hydrid duplex containing a site-specific GNA-like chiral PNA monomer

Peptide nucleic acids (PNA) are one of the most widely used synthetic DNA mimics where the four bases are attached to a N-(2-aminoethyl)glycine (aeg) backbone instead of the negative-charged phosphate backbone in DNA. We have developed a chimeric PNA (chiPNA), in which a chiral GNA-like γ3T monomer is incorporated into aegPNA backbone. The base pair opening kinetics of the aegPNA:DNA and chiPNA:DNA hybrid duplexes were studied by NMR hydrogen exchange experiments. This study revealed that the aegPNA:DNA hybrid is much more stable duplex and is less dynamic compared to DNA duplex, meaning that base pairs are opened and reclosed much more slowly. The site-specific incorporation of γ3T monomer in the aegPNA:DNA hybrid can destabilize a specific base pair and its neighbors, maintaining the thermal stabilities and dynamic properties of other base pairs. Our hydrogen exchange study firstly revealed the unique kinetic features of base pairs in the aegPNA:DNA and chiPNA:DNA hybrids, which will provide an insight into the development of methodology for specific DNA recognition using PNA fragments.

NMR study of hydrogen exchange during the B–Z transition of a DNA duplex induced by the Zα domains of yatapoxvirus E3L

The Yaba-like disease viruses (YLDV) are members of the Yatapoxvirus family and have double-stranded DNA genomes. The E3L protein, which is essential for pathogenesis in the vaccinia virus, consists of two domains: an N-terminal Z-DNA binding domain and a C-terminal RNA binding domain. The crystal structure of the E3L orthologue of YLDV (yabZα E3L) bound to Z-DNA revealed that the overall structure of yabZα E3L and its interaction with Z-DNA are very similar to those of hZα ADAR1. Here we have performed NMR hydrogen exchange experiments on the complexes between yabZα E3L and d(CGCGCG) 2 with a variety of protein-to-DNA molar ratios. This study revealed that yabZα E3L could efficiently change the B-form helix of the d(CGCGCG) 2 to left-handed Z-DNA via the active-mono B–Z transition pathway like hZα ADAR1.

Hierarchy of local structural and dynamics perturbations due to subdenaturing urea in the native state ensemble of DLC8 dimer

Local structural and dynamic modulations due to small environmental perturbations reflect the adaptability of the protein to different interactors. We have investigated here the preferential local perturbations in Dynein light chain protein (DLC8), a cargo adapter, by sub-denaturing urea concentrations. Equilibrium unfolding experiments by optical spectroscopic methods indicated a two state like unfolding of DLC8 dimer, with the transition mid-point occurring around 8.6 M urea. NMR studies identified the β3 and β4 strands, N-, C- terminal regions, loops connecting β1 to α1, α1 to α2 and β3 to β4 as the soft targets of urea perturbation and thus indicated potential unfolding initiation sites. Native-state hydrogen exchange studies suggested the unfolding to traverse from the edges towards the centre of the secondary structural elements. At 6 M urea the whole protein chain acts like a cooperative unit. These observations are expected to have important implications for the protein's multiple functions.

Investigation of amide hydrogen back-exchange in Asp and His repeats measured by hydrogen (1H/2H) exchange mass spectrometry

Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry (HDX-MS) has become a valuable tool in studies of protein dynamics and protein interactions. Isotopic exchange is typically initiated by diluting a protein solution into deuterated buffer at physiological conditions. For MS analysis, the exchange reaction is quenched by acidification and cooling and the labeled protein (or a digest thereof) is analyzed by mass spectrometry. An inevitable deuterium loss occurs during quench conditions (i.e., back-exchange). The primary structure of the peptide influences the back-exchange rate due to steric hindrance by bulky side groups and by inductive and charge effects. Here we show that the back-exchange in histidine repeats (His6) at HDX-MS quench conditions is complete within a few seconds using either acetic acid or formic acid in the quench solution, while aspartic acid repeats (Asp6) retain deuterons for several minutes using formic acid. We employ electron transfer dissociation to obtain residue-specific deuterium levels of the Asp repeat in K2D6IIKIIK using a hybrid linear ion trap-Orbitrap mass spectrometer. Our results show an unexpected uneven distribution of deuterium in the Asp repeat. The implication of the rapid back-exchange of His repeats for HDX-MS protein hydrogen exchange studies is discussed. We also discuss the implications of retained deuterons in the Asp repeat of K2D6IIKIIK when this peptide is used as a probe for the occurrence of hydrogen scrambling.

The Structural Characteristics of Nonspecific Lipid Transfer Proteins Explain Their Resistance to Gastroduodenal Proteolysis

The structure and stability of the allergenic nonspecific lipid transfer protein (LTP) of peach were compared with the homologous LTP1 of barley and its liganded form LTP1b. All three proteins were resistant to gastric pepsinolysis and were only slowly digested at 1 to 2 out of 14 potential tryptic and chymotryptic cleavage sites under duodenal conditions. Peach LTP was initially cleaved at Tyr79-Lys80 and then at Arg39-Thr40 (a site lost in barley LTP1). Molecular dynamics simulations of the proteins under folded conditions showed that the backbone flexibility is limited, explaining the resistance to duodenal proteolysis. Arg39 and Lys80 side chains were more flexible in simulations of peach compared with barley LTP1. This may explain differences in the rates of cleavage observed experimentally for the two proteins and suggests that the flexibility of individual amino acid side chains could be important in determining preferred proteolytic cleavage sites. In order to understand resistance to pepsinolysis, proteins were characterized by NMR spectroscopy at pH 1.8. This showed that the helical regions of both proteins remain folded at this pH. NMR hydrogen exchange studies confirmed the rigidity of the structures at acidic pH, with barley LTP1 showing some regions with greater protection. Collectively, these data suggest that the rigidity of the LTP scaffold is responsible for their resistance to proteolysis. Gastroduodenal digestion conditions do not disrupt the 3D structure of peach LTP, explaining why LTPs retain their ability to bind IgE after digestion and hence their allergenic potential.