Hydrogen Exchange Rate Constants Research Articles

NMR Exchange Study of Z-DNA Duplexes Induced by the Zα Domain of Human ADAR1 Containing Mutation on the β-Hairpin

Left-handed Z-DNA forms in polymers of alternating d(CG)n sequence and is a higher energy conformation than B-DNA. Z-DNA is stabilized by high salt, negative supercoiling, and complex formation with Z-DNA binding proteins. The human double-stranded RNA deaminase I (ADAR1) contains two Z-DNA binding domains (Zα and Zβ) at the NH2-terminus. 4,5 The crystal structure of the Zα domain of human ADAR1 (ZαADAR1) complexed to Z-DNA showed that each of two Zα domains binds to one strand of a double-stranded DNA, yielding two-fold symmetry with respect to the DNA helical axis. This study also revealed that the intermolecular interaction is mediated by 5 residues in the α3 helix and 4 residues in the β-hairpin (Fig. 1). This is confirmed in solution by the significant backbone chemical shift changes of most residues in the α3 helix and β2-loop-β3 region upon binding to Z-DNA. These residues play important roles in Zα function and thus replacement of them with an alanine caused a dramatic decrease in Z-DNA binding affinity compared to wild-type ZαADAR1 (wt-ZαADAR1). 5 Previous NMR study suggested 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. This study also found that the K170A and R174A mutants significantly destabilize the G2·C5 base pair compared to wt-ZαADAR1, even though these mutants could efficiently change B-DNA to left-handed Z-DNA. However, the question of how the mutants on the β-hairpin affect the base pair stabilities of the Z-DNA remains. Here, we determined hydrogen exchange rate constants (kex) for the imino protons of the d(CG)3 in the complexes with ZαADAR1 containing mutation on the β-hairpin. Comparison of these results to previous studies of the mutant ZαADAR1-d(CG)3 complexes 8 provides the information required to identify the role of each residue in the β-hairpin during BZ transition of DNA duplexes.

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.

Hydrogen Exchange Study of Winter Flounder Type I Antifreeze Protein

Antifreeze proteins (AFPs) are found in various organisms, such as fish, insects, plants, bacteria, and fungi, to promote survival at subzero temperatures by decreasing the freezing point of bodily fluids. AFPs, which are isolated from Arctic and Antarctic fish, comprise several structurally diverse classes of proteins and four classes of structurally independent proteins are identified: type I AFPs, alanine-rich αhelical proteins of 3.3 to 4.5 kDa; type II AFPs, cysteinerich globular proteins containing five disulfide bonds; type III AFPs, 6-kDa globular proteins; and Type IV AFPs, glutamateand glutamine-rich α-helical proteins. The type I AFPs are alanine-rich amphiphilic α-helical proteins found in winter flounder, yellowtail flounder, Alaskan plaice, shorthorn sculpin, and Arctic sculpin. The winter flounder AFP isoform, HPLC6, which is the most extensively studied AFP, contains three 11-amino acid repeats of the sequence Thr-X2-Asx-X7, where X is generally alanine. X-ray crystallographic studies revealed that the type I AFP is completely α-helical in conformation, with the exception of the last residue (R37), which adopts a 310helix. The hydroxyl and methyl groups of four threonine residues, particularly of the central two residues, T13 and T24, play key roles in the ice-binding properties of the type I AFP. For example, T13S/T24S mutations of the type I AFP caused a 90-100% loss in activity relative to wild-type AFP. The mutants, A17L and A21L, where the substitution lies adjacent to the Thr-rich face, caused a significant decrement of the thermal hysteresis activity, whereas the mutants A19L and A20L exhibited wild-type activity. It was reported that termini of the HPLC6 isoform possess greater helix-stabilizing ability compared to the HPLC2 isoform, leading to a 50% difference in the thermal hysteresis activity between these two AFPs. These results suggested that helical stabilization of the N and C termini is the critical component for antifreeze activity of the type I AFP. It was recently reported that flexibility of the C-terminal region causes a loss of thermal hysteresis activity because its dynamic nature strongly prevents binding to the ice surface. Here, to further understand the correlation between the dynamic properties and function of type I AFPs, we performed NMR hydrogen exchange experiments on the winter flounder type I AFP (HPLC6 isoform) (Fig. 1, referred to as wt-AFP). The hydrogen exchange rate constants for the two mutants, A17Land A20L-AFP (Fig. 1), which exhibit no and wild-type activities in thermal hysteresis respectively, are also determined. Comparison of the wtAFP and mutant AFPs can provide valuable insights into the thermal hysteresis mechanism of type I AFPs.

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy

Atom-resolved real-time studies of kinetic processes in proteins have been hampered in the past by the lack of experimental techniques that yield sufficient temporal and atomic resolution. Here we present band-selective optimized flip-angle short transient (SOFAST) real-time 2D NMR spectroscopy, a method that allows simultaneous observation of reaction kinetics for a large number of nuclear sites along the polypeptide chain of a protein with an unprecedented time resolution of a few seconds. SOFAST real-time 2D NMR spectroscopy combines fast NMR data acquisition techniques with rapid sample mixing inside the NMR magnet to initiate the kinetic event. We demonstrate the use of SOFAST real-time 2D NMR to monitor the conformational transition of alpha-lactalbumin from a molten globular to the native state for a large number of amide sites along the polypeptide chain. The kinetic behavior observed for the disappearance of the molten globule and the appearance of the native state is monoexponential and uniform along the polypeptide chain. This observation confirms previous findings that a single transition state ensemble controls folding of alpha-lactalbumin from the molten globule to the native state. In a second application, the spontaneous unfolding of native ubiquitin under nondenaturing conditions is characterized by amide hydrogen exchange rate constants measured at high pH by using SOFAST real-time 2D NMR. Our data reveal that ubiquitin unfolds in a gradual manner with distinct unfolding regimes.

Hydrogen Exchange and Hydration Dynamics in Gelatin Gels

Gelatin, derived from the collagen triple helix, is the most widely used functional biopolymer and a prototype for studies of physical gels. Gelatin gels have also served as models for soft biological tissue in efforts to elucidate the molecular basis of the magnetic relaxation phenomena that govern magnetic resonance image contrast. Yet, the microstructure, hydration, and magnetic relaxation behavior of gelatin gels are not well understood. To address these issues, we report here the water 2H and 17O magnetic relaxation dispersion (MRD) profiles from gelatin gels over wide ranges of resonance frequency and pH. For the global analysis of this extensive data set, we use a generalized relaxation theory that remains valid for arbitrarily slow molecular dynamics. The strong pH dependence in the 2H profiles can be rationalized quantitatively as the result of exchange with bulk water of labile hydrogens in gelatin side chains. The global analysis of the MRD data yields hydrogen-exchange rate constants, acid dissociation constants, and orientational order parameters in agreement with independent structural, thermodynamic, and kinetic data. The MRD analysis reveals a highly mobile hydration layer at the surface of the gelatin triple helix and a small number of trapped water molecules with residence times on the order of 10(-8) s, presumably associated with structural defects and branch points in the gel. The MRD data also indicate that approximately 20% of the gelatin residues belong to flexible polypeptide chains, rather than to rigid triple-helical segments. By identifying the molecular species and motions responsible for the 2H and 17O dispersion profiles, this study takes a significant step toward a quantitative understanding of water relaxation in aqueous gels and biological tissue.

Hydrogen exchange rates in proteins from water (1)H transverse magnetic relaxation.

Access to the fast exchange kinetics of labile protein hydrogens in solution is provided by exchange broadening of the water 1H NMR line. We analyzed the chemical shift modulation contribution of labile hydrogens in bovine pancreatic trypsin inhibitor (BPTI) to the transverse 1H spin relaxation rate, R2, of the bulk solvent. Both the experimental pH dependence and the CPMG dispersion of R2 could be quantitatively accounted for on the basis of known chemical shifts, exchange rates, and ionization constants for BPTI. This analysis provided, for the first time, the hydrogen exchange rate constants for Lys and Arg side chains in a protein and pointed to an internal catalysis of the N-terminal amino protons in BPTI by a salt bridge. The method can be used for mapping the hydrogen exchange rates in protein solutions and biomaterials, which may be important for the control of relaxation-weighted contrast in biological MRI.

Assignment of Gas-Phase Dipeptide Amide Hydrogen Exchange Rate Constants by Site-Specific Substitution: GlyGly

Amide hydrogen deuterium exchange is widely used to probe conformations of gas-phase biomacromolecular ions. Meaningful interpretation of such experiments depends on relating the experimentally determined H/D exchange rate constants to specific exchangeable hydrogen sites on the molecule. Here, we present the first definitive assignment of H/D exchange rate constants for the N-terminal ammonium, amide hydrogen, and carboxyl hydrogens in diglycine, based on comparison of site-specific gas-phase H/D exchange rate constants for diglycine (GlyGly) and its selectively methylated homologues (GlySar, GlyGly-OMe, and GlySar-OMe). The present method may be extended to identification of site-specific H/D exchange rate constants for other dipeptides, to provide insight into H/D exchange reaction mechanisms.

An NMR-based quenched hydrogen exchange investigation of model amyloid fibrils formed by cold shock protein A.

Acid-denatured cold shock protein A (CspA) self-assembles into polymers with properties typical of amyloid fibrils. In the present work, a quenched hydrogen exchange experiment was used to probe the interactions of CspA fibrils with solvent. Exchange was initiated by incubating suspensions of fibrils in D2O, and quenched by flash freezing. Following lyophilization, exchange-quenched samples were dissolved in 90% DMSO/10% D2O, giving DMSO-denatured monomers. Intrinsic exchange rates for denatured CspA in 90% DMSO/10% D2O (pH* 4.5) were sufficiently slow (approximately 1 x 10(-3) min-1) to enable quantification of NMR signal intensity decays due to H/D exchange in the fibrils. Hydrogen exchange rate constants for CspA fibrils were found to vary less than 3-fold from a mean value of 5 x 10(-5) min-1. The uniformity of rate constants suggests that exchange is in the EX1 limit, and that the mechanism of exchange involves a cooperative dissociation of CspA monomers from fibrils, concomitant with unfolding. Previous studies indicated that the highest protection factors in native CspA are approximately 10(3), and that protection factors for the acid-denatured monomer precursors of CspA fibrils are close to unity. Because exchange in is in the EX1 regime, it is only possible to place a lower limit of at least 10(5) on protection factors in CspA fibrils. The observation that all amide protons are protected from exchange indicates that the entire CspA polypeptide chain is structured in the fibrils.

Human recombinant [C22A] FK506‐binding protein amide hydrogen exchange rates from mass spectrometry match and extend those from NMR

Hydrogen/deuterium exchange behavior of human recombinant [C22A] FK506 binding protein (C22A FKBP) has been determined by protein fragmentation, combined with electrospray Fourier transform ion cyclotron resonance mass spectrometry (MS). After a specified period of H/D exchange in solution, C22A FKBP was digested by pepsin under slow exchange conditions (pH 2.4, 0 degree C), and then subjected to on-line HPLC/MS for deuterium analysis of each proteolytic peptide. The hydrogen exchange rate of each individual amide hydrogen was then determined independently by heteronuclear two-dimensional NMR on 15N-enriched C22A FKBP. A maximum entropy method (MEM) algorithm makes it possible to derive the distributions of hydrogen exchange rate constants from the MS-determined deuterium exchange-in curves in either the holoprotein or its proteolytic segments. The MEM-derived rate constant distributions of C22A FKBP and different segments of C22A FKBP are compared to the rate constants determined by NMR for individual amide protons. The rate constant distributions determined by both methods are consistent and complementary, thereby validating protein fragmentation/mass spectrometry as a reliable measure of hydrogen exchange in proteins.

Conformational stability of ribonuclease T1 determined by hydrogen-deuterium exchange.

The hydrogen-deuterium exchange kinetics of 37 backbone amide residues in RNase T1 have been monitored at 25, 40, 45, and 50 degrees C at pD 5.6 and at 40 and 45 degrees C at pD 6.6. The hydrogen exchange rate constants of the hydrogen-bonded residues varied over eight orders of magnitude at 25 degrees C with 13 residues showing exchange rates consistent with exchange occurring as a result of global unfolding. These residues are located in strands 2-4 of the central beta-pleated sheet. The residues located in the alpha-helix and the remaining strands of the beta-sheet exhibited exchange behaviors consistent with exchange occurring due to local structural fluctuations. For several residues at 25 degrees C, the global free energy change calculated from the hydrogen exchange data was over 2 kcal/mol greater than the free energy of unfolding determined from urea denaturation experiments. The number of residues showing this unexpected behavior was found to increase with temperature. This apparent inconsistency can be explained quantitatively if the cis-trans isomerization of the two cis prolines, Pro-39 and Pro-55, is taken into account. The cis-trans isomerization equilibrium calculated from kinetic data indicates the free energy of the unfolded state will be 2.6 kcal/mol higher at 25 degrees C when the two prolines are cis rather than trans (Mayr LM, Odefey CO, Schutkowski M, Schmid FX. 1996. Kinetic analysis of the unfolding and refolding of ribonuclease T1 by a stopped-flow double-mixing technique. Biochemistry 35: 5550-5561). The hydrogen exchange results are consistent with the most slowly exchanging hydrogens exchanging from a globally higher free energy unfolded state in which Pro-55 and Pro-39 are still predominantly in the cis conformation. When the conformational stabilities determined by hydrogen exchange are corrected for the proline isomerization equilibrium, the results are in excellent agreement with those from an analysis of urea denaturation curves.

Determination of the Activation Volume of the Uncatalyzed Hydrogen Exchange Reaction between N-Methylacetamide and Water

The amide−water hydrogen exchange rates of the individual backbone amide protons are very sensitive to the local environment of the protons and can be used to probe fluctuations in the local conformations of proteins. Hydrogen exchange between N-methylacetamide (NMA) and water is important in that it serves as a model for the amide−water hydrogen exchange in peptides and proteins. The rate constants for amide−water hydrogen exchange between 16 mol % NMA and water at 66 °C were measured at pressures ranging from 1 bar to 5 kbar at three different pH values using the NMR magnetization transfer technique, and the apparent activation volumes of hydrogen exchange were obtained at each pH. The activation volume of the acid-catalyzed hydrogen exchange reaction was calculated to be +1.7 cm3 mol-1, and the activation volume of the base-catalyzed hydrogen exchange reaction was calculated to be +11.0 cm3 mol-1. The hydrogen exchange rate constants were also measured as a function of concentration and pH at ambient p...

Hydrogen Exchange and Protein Hydration: The Deuteron Spin Relaxation Dispersions of Bovine Pancreatic Trypsin Inhibitor and Ubiquitin

Water deuteron ( 2H) spin relaxation was used to study hydrogen exchange hydration, and protein dynamics in aqueous soltuions of the globular proteins bovine pancreatic trypsin inhibitor (BPTI) and ubiquitin. The frequency dispersion of the longitudinal 2H relaxation rate was measured in the pD range 2 to 11 at 27°C. In contrast to the previously reported water 17O relaxation dispersion from the same samples, the 2H dispersion depends strongly on pD. This pD dependence is due to labile protein deuterons in acidic side-chains and surface peptide groups, which exchange rapidly with water deuterons. The pD dependence of the 2H relaxation in BPTI solutions could be quantitatively accounted for in terms of known p Kvalues and hydrogen exchange rate constants. For ubiquitin, labile protein deuterons contribute importantly to the 2H relaxation dispersion even in the neutral pD range. The 2H relaxation data also provided information about the orientational order and internal motion of OD and ND bonds in side-chains and surface peptides. A comparison of the water contribution to the 2H dispersion with the 17O dispersion indicates that one of the four internal water molecules of BPTI, presumably the deeply buried water molecule W122, exchanges more slowly (10 −6to 10 −4second) than the other three (10 −8to 10 −6second).

Hydrogen kinetics of peptide amide protons at the bovine pancreatic trypsin inhibitor protein-solvent interface

Hydrogen exchange rate constants of the 25 most rapidly exchanging peptide amide protons in bovine pancreatic trypsin inhibitor have been determined over a range of pH that spans pH min, the pH of minimum rate. Most of these are on the protein surface, exposed to solvent and not hydrogen bonded in the crystal structure. Contrary to commonly held assumptions, the exchange kinetics of surface NH groups are not equivalent to the kinetics of NH groups in peptides in the extended configuration. All surface NH groups exchange more slowly than NH groups in model peptides, with rate constants distributed over a range of more than two orders of magnitude. In addition, their pH min values vary widely. For most of the surface NH groups, pH min is lower than in model compounds and, for several, pH min is < 1. These results indicate that the local environment of the surface peptide groups when the exchange event occurs is very different from that of extended peptides. Analysis based on consideration of an O-protonation mechanism for acid catalysis and of electrostatic effects on exchange kinetics further indicates (see the accompanying paper) that, in general, exchange of surface NH groups occurs from a conformation of the protein approximated by the crystal structure. The 1H- 2H exchange rate constants were measured from 300 MHz nuclear magnetic resonance spectra in which assigned surface N 1H resonances are resolved by the use of partially deuterated protein samples. A marked pH dependence of the chemical shifts observed in the pH range 1 to 4.5 for several surface NH groups reflects the titration of nearby carboxyl groups.

Mechanism of surface peptide proton exchange in bovine pancreatic trypsin inhibitor salt effects and O-protonation

The acid-catalyzed hydrogen exchange rate constants k H, and the base-catalyzed rate constants k OH, have been determined (in the preceding paper) for the 25 most rapidly exchanging NH groups of bovine pancreatic trypsin inhibitor. Most of these NH groups are at the protein-solvent interface. The correlation of k H, but not k OH, with the static accessibility and hydrogen bonding of the peptide carbonyl O atom indicates that the mechanism of acid catalysis in proteins involves O-protonation. Agreement between the ionic strength dependence observed for k H and k OH and the ionic strength dependence calculated for an O-protonation mechanism supports this conclusion. N-protonation for acid catalysis, as well as N-deprotonation for base catalysis, have traditionally been assumed in the mechanism of the chemical step in peptide amide proton exchange. A preference for the alternative O-protonation mechanism has far-reaching implications in the interpretation of protein hydrogen exchange kinetics. With an O-protonation mechanism, acid-catalyzed rates of surface NH groups are primarily a function of the average solvent accessibility of the carbonyl O atoms in the dynamic solution structure, while base-catalyzed rates of surface NH groups measure solvent accessibility of the peptide N. The relative dynamic accessibilities of peptide O atoms, as measured by relative values of k H (corrected for electrostatic effects), correlate with O static accessibilities in the crystal structure. A lower correlation of static accessibility of N atoms with k OH is observed for surface NH groups in peptide groups in which the carbonyl O is not hydrogen bonded. For some surface NH groups, the observed pH of minimum rate, pH min, deviates widely from the pH min of model compounds. This is explained as the combined result of electrostatic effects and of the differences in accessibility of the carbonyl O and N atoms that result in a change in the relative values of k H and k OH as compared to those of model peptides. A mechanism whereby exchange of interior sites is catalyzed by interactions of catalysis ions with protein surface atoms via charge transfer is suggested.

Fluctuations of protein structure as expressed in the distribution of hydrogen exchange rate constants.

AbstractHydrogen exchange kinetics of proteins provide information about the dynamics of their structure. The interpretation of experiments has been limited by difficulty in identifying individual rate constants. In order to invert the kinetic data, and to gain information about the individual reacting sites, we introduce a Laplace inversion technique and obtain the distribution function of rate constants by which the intricate reaction proceeds. A series of carefully overlapped experiments were performed on lysozyme at 25°C, an exchange profile obtained, and a distribution function extracted. This function was composed of a product of two terms, indicating two parallel pathways. The first, a power‐law term, was attributed to exchange from the native state. This part of the distribution function thus describes the scope of the conformational fluctuations in proteins at constant temperature and pressure. The second, an exponential, was seen to be associated with the pathway involving thermal unfolding and subsequent free exchange with the solvent. The influence of trichlorethanol and glycerol on the total distribution function was measured. Trichloroethanol selectively increased the contribution from the thermal unfolding pathway, whereas glycerol, besides decreasing this type of contribution, increased the width of the distribution function attributed to structural fluctuations.

Structure and fluctuation of a Streptomyces subtilisin inhibitor

The kinetics of the hydrogen-deuterium exchange reaction in a subtilisin inhibitor from Streptomyces albogriseolus has been examined by infrared absorption measurement in aqueous solutions at various pH values and temperatures. In the analysis of each piece of kinetic data, it was assumed that the total 104 peptide hydrogen atoms are classified into three kinetic classes A, B 1, and B 2, and that the sizes of these classes are 72, 15, and 17, respectively at every pH and at every temperature examined. On the basis of the peak position determined for the amide II band in each stage of the exchange reaction, an approximate assignment was suggested of the A, B 1, and B 2 respectively to an unordered structure, a β-structure, and an α-helical structure in the molecule. This assignment was supported by infrared absorption measurement of a film of this protein and by circular dichroic study of the solutions. On the basis of the temperature effect on the hydrogen-exchange rate constants and on the basis of ultraviolet absorption study in the higher temperature region (40 to 90 °C), a discussion has been made on the nature of the fluctuation of the molecular structure of this protein.

The kinetics and mechanism of the electrophilic substitution of heteroaromatic compounds. Part XXXIX. Transmission of substituent effects in the pyridine system: Hammett-like treatment of hydrogenexchange data

Hydrogen-exchange rate constants for pyridines and for benzenes are correlated by the same equation provided that interactions between substituents are allowed for by ΣΠσ+. The derived equation permits the calculation of rate constants for polysubstituted benzenes and pyridines with reactivities covering 17 log units; the significance of this equation as representing something more than an empirical relationship is however in considerable doubt.

Acidity of niccolocene in dimethyl sulfoxide and methanol solutions

The acidity of niccolocene in dimethyl sulfoxide-methanol solution during potassium methoxide catalysis was determined by measuring the isotopic hydrogen-exchange rate constants of 9-phenylxanthene and niccolocene in this mixture of solvents.