The conformational dynamic of the tetramer hemoglobin molecule as revealed by hydrogen exchange. I. Influence pH, temperature and ligand binding

The rate of the H-D exchange of the peptide NH atoms of the different forms of human Hb was studied at the range of pH 5-10 and temperature 10-63 degrees C by the IR spectroscopy. The pH-dependence of the H-D exchange rate is accordance with the EX2 mechanism. Two pH-dependent conformers of ligand forms of Hb existes at 10-30 degrees C with lower probability of local fluctuations of the alkaline conformer. The difference between two conformers vanishes at 40 degrees C with the appearance of the third conformer with higher probability of local fluctuations. The deoxyHb at 20 degrees C and pH range 6-9 has no pH-dependent conformers and the probability of local fluctuations is considerably reduced in comparison to the acid conformer of ligand Hb. Upon the destabilization of the ligand Hb structure by the pH decreasing to 5.0 at 20 degrees C or the temperature increasing up to 50-60 degrees C at pH 7.1 the global fluctuations of the native structure are intensified providing the H-D exchange of the slowest exchanging NH atoms. The nature of the local and global fluctuations and possible similarity between the two pH-dependent conformers of ligand Hb and its functional R and R2 states revealed by the X-ray analysis and NMR spectroscopy were discussed.     

Conformational dynamics of the tetrameric hemoglobin molecule as revealed by hydrogen exchange: 2. Effect of intersubunit contact cleavage

IR spectroscopy was used to study the rate of hydrogen-deuterium (H-D) exchange of peptide NH atoms in isolated α and β subunits of human hemoglobin (Hb) at pH 5.5–9.0 and 20°C. The H-D exchange occurs by the EX2 mechanism. The retardation factor of subunit exchange rate (P) is in a range of approximately 10²–10⁷. Compared to tetrameric Hb, the probability of local fluctuations (1/P) increases to a slightly greater extent in monomeric α subunits than in tetrameric β subunits. Unlike in the whole Hb molecule, oxygenation of its subunits has no effect on the probability of local fluctuations, and the subunits show no pH-dependent changes in 1/P values (observed for liganded Hb). Probable mechanisms accounting for overall intensification of local fluctuations upon the cleavage of contacts between subunits of the tetrameric Hb molecule are discussed with regard to structural crystallographic data.     

The conformational dynamic of the tetramer hemoglobin molecule as revealed by hydrogen exchange. II. Influence of the intersubunit contact splitting

The rate of the H-D exchange of the peptide NH atoms of the isolated alpha and beta subunits of human Hb were studied at the pH range 5.5-9.0 and 20 degrees C by the IR spectroscopy. The factor retardation of the exchange rate of subunits -P in the range -10(2)-10(7). In comparison with tetramer Hb the probability of local fluctuations (1/P) is increased to a slightly greater extent for the monomeric alpha subunits then for the tetramer beta subunits. Unlike Hb oxygenation of subunits does not influence on the probability of the local fluctuations and subunits have no the pH-dependent change of the value 1/P observable for the ligand Hb. The possible mechanisms of the overall intensification of the local fluctuations upon the splitting of the Hb tetrameric contacts between subunits are discussed with the inviting of the structural crystallographic data.     

Conformational dynamics of the tetrameric hemoglobin molecule as revealed by hydrogen exchange: III. The influence of heme removal

Two main types of conformational fluctuations, local and global, are characteristic of the native protein structure and are detectable by hydrogen exchange. The probability of such fluctuations changes to a different degree during hemoglobin (Hb) oxygenation, changes in pH, and splitting of the intersubunit contracts. For comparison with the effect of heme removal, the rate of the hydrogen-deuterium (H-D) exchange of peptide H atoms (PHs) of human apoHb was studied by IR spectroscopy at pH 5.5–9.0 and temperatures of 10–38°C. The removal of heme increased the H-D exchange rate for 80% of Hb PHs with the exchange retardation factor P ～ 10²-10⁸. For the majority of PHs, the probability of local fluctuations depended weakly on the temperature; changes in enthalpy upon such local conformational transitions were ΔH _op ^o = 0–15 kcal/M. Global fluctuations, displaying a stronger temperature dependence, did not arise with an increase in temperature to 38°C at pH 7.0, although apoHb began slowly denaturing and aggregating under these conditions. Destabilization of the apoHb structure with a concurrent decrease in pH to 5.5 and temperature to 10°C intensified global fluctuations in the native protein structure with ΔH _op ^o < 0. The mechanism underlying the overall intensification of local fluctuations upon the heme removal, the specific features of apoHb heat denaturation under conditions close to those of in vivo Hb self-assembly, and the analogies between low-temperature global fluctuations and cold denaturation of globular proteins are discussed.     

The conformational dynamics of the tetramer hemoglobin molecule as revealed by hydrogen exchange. III. Influence of the heme removal

Two main types of conformational fluctuations--local and global are characteristic of the native protein structure and revealed by hydrogen exchange. The probability of those fluctuations changes to a different extent upon hemoglobin oxygenation, changing of pH, splitting of the intersubunit contacts. To compare with the influence of the heme removal the rate of the H-D exchange of the peptide NH atoms of the human apoHb was studied at the pH range 5.5-9.0 and temperature 10-38 degrees C by the IR spectroscopy. The removal of the heme increases the rate of the H-D exchange of the 80% peptide NH atoms with the factor retardation of the exchange rate (P) in the range approximately 10(2)-10(8). For the most of the peptide NH atoms the probability of the local fluctuations weakly depends on the temperature, the enthalpy changes upon all such local conformational transitions deltaH(op) degrees are 0-15 kcal/M. Characterized by the stronger temperature dependence the global fluctuations are not arised upon the temperature increases up to 38 degrees C at pH 7.0 inspite of in these conditions the slow denaturation and aggregation of apoHb begin to occur. Upon the destabilization of the apoHb structure by the simultaneous decreasing of pH to 5.5 and temperature to 10 degrees C the global fluctuations of the apoHb native structure described by deltaH(op)o < 0 begin to intensify. The mechanism of the overall intensification of the local fluctuations upon the heme removal, the peculiarity of the heat denaturation of apoHb in conditions, close to that existing upon the selfassembly of Hb in vivo, and analogy between low temperature global fluctuations and cold denaturation of globular proteins are discussed.     

Hydrogen exchange and proteolytic degradation of ribonuclease A. Similarities and distinctions of the kinetic mechanisms

The studies by IR spectroscopy of the temperature dependence of the H-D exchange rate of the RNase A peptide NH atoms permit one to characterize two types of conformation fluctuations, local and global. A comparison with the temperature dependence of the proteolytic degradation rate of RNase A shows that similar in nature fluctuations allow for the H-D exchange of NH atoms and the splitting of peptide bonds of the native protein. In the low temperature region, both processes occur through local fluctuations, by way of the EX2 mechanism, and in the high temperature region, they occur through global fluctuations with the overall denaturation desorganization of the native structure, by way of the EX1 mechanism. The biphasic dependence of the rate of H-D exchange and proteolytic degradation of RNase A on urea concentration is also explained by the combination of local and global fluctuations.     

Structure and dynamics of the neutrophil defensins NP-2, NP-5, and HNP-1: NMR studies of amide hydrogen exchange kinetics

The exchange kinetics for the slowly exchanging amide hydrogens in three defensins, rabbit NP-2, rabbit NP-5, and human HNP-1, have been measured over a range of pH at 25°C using 1D and 2D NMR methods. These NHs have exchange rates 10² to 10⁵ times slower than rates from unstructured model peptides. The observed distribution of exchange rates under these conditions can be rationalized by intramolecular hydrogen bonding of the individual NHs, solvent accessibility of the NHs, and local fluctuations in structure. The temperature dependencies of NH chemical shifts (NH temperature coefficients) were measured for the defensins and these values are consistent with the defensin structure. A comparison is made between NH exchange kinetics, NH solvent accessibility, and NH temperature coefficients of the defensins and other globular proteins. Titration of the histidine side chain in NP-2 was examined and the results are mapped to the three-dimensional structure. © 1994 Wiley-Liss, Inc.     

Rates of structural fluctuations of lysozyme in the range of thermal unfolding transition

The hydrogen–deuterium exchange reaction for the tryptophan residues in lysozyme have been followed in 4.5M LiBr at pH 7.2 in the temperature range of the unfolding transition by measuring the transmittance change at 293 nm. The exchange reaction proceeded in three phases at low temperature for native protein. The first and the second phases were ascribed to the H-D exchange reactions of three relatively exposed tryptophan residues on the molecular surface. The third phase corresponded to the H-D exchange reaction of the three tryptophan residues buried in the interior of the molecule. The H-D exchange reaction proceeded in two phases near the melting temperature and in a single phase at high temperature, where almost all molecules are unfolded. The H-D exchange of three tryptophan residues buried in folded molecules was caused by fluctuation between the folded and unfolded structure of the protein molecule. The rates of such a fluctuation were determined from the rates of the exchange reaction at various temperatures. These rates agreed very well with those determined from the temperature-jump method. This means that a protein molecule in solution fluctuates between the N- and D-states at every temperature within the transition region, where the N-form is the tightly folded native structure and the D-form the randomly coiled chain. From measurements of thermal unfolding of ester-108-lysozyme and the binding constant of (NAG)₃ to ester-108-lysozyme, it was found that almost all cross-linked molecules are in the folded state near 50°C and pH 7.2 in 4.5M LiBr, where intact molecules are unfolded. We also studied the H-D exchange reaction of ester-108-lysozyme. In the temperature region of 43–50°C, about 70% of the exchangeable tryptophan residues of ester-108-lysozyme were exchanged within 1 s immediately after the mixing of D₂O, in spite of the fact that almost all molecules are in the folded state. This was considered the premelting of the surface of a corss-linked molecule.     

Solubility of hemoglobin S by sedminetation, equilibrium, and antisickling compounds.

The effect of the compounds guanidine, arginine, lysine, and aspartic acid and the salt arginine aspartate on the solubility of deoxyhemoglobin S (Hb S) was studied by sedimentation equilibrium at 20–22 °C. Guanidine and arginine were found to be most effective, whereas aspartic acid and lysine had only a small effect. The effectiveness of these compounds in solubilizing Hb S is relatively pH independent. It is unlikely that the small effect of lysine and aspartic acid on the solubility of Hb S can account for the antisickling properties of lysine and aspartic acid previously reported (Sophianopoulos, A. J., et al. (1974) Clin. Biochem.7, 112–118). The effect of guanidine and arginine is large enough to account for a large part of such antisickling properties (Sophianopoulos et al. (1974). The nonideality of concentrated hemoglobin solutions (up to 0.3 g cm^?3) has been studied in detail. By using the liganded as well as the unliganded forms of both Hb S and Hb A, it was found that the magnitude of the virial (nonideality) coefficients can change with varying solution conditions. A comparison of pure Hb S with hemolysates is made using viscosity and sedimentation velocity.     

Enhanced Unfolding of Bovine β-Lactoglobulin Structure Using Microwave Treatment: a Multi-Spectroscopic Study

Commercial whey protein hydrolysates containing bovine β-lactoglobulin (β-Lg) may have residual allergenicity due to the inaccessibility of some sequential epitopes to proteases. Microwave may enhance unfolding pathways in protein structure due to its non-thermal effects. This research compared the effects of microwave heating (MW) and conventional heating (CH) on the unfolding in the secondary and tertiary structures of β-Lg over a temperature range of 40-90 °C using circular dichroism (CD), fluorescence spectroscopy, and two dimensional (2D) ¹H nuclear magnetic resonance (NMR) spectroscopy. Above 50 °C, β-sheet and α-helical secondary structures decreased during MW and CH, with a higher decrease being observed during MW. The near-UV spectra of MW β-Lg showed lower intensity suggesting higher tertiary structure loss than in CH β-Lg at all temperatures. The fluorescence spectra of MW β-Lg showed increased exposure of tryptophan residues to solvent as compared to CH β-Lg and suggested greater unfolding in tertiary structure in MW β-Lg at 60 °C than in CH β-Lg at 70 °C. 2D ¹H NMR spectra confirmed more extensive H-D exchange in MW β-Lg explained by the exposure of β-sheets (C, G, and H) at 50 °C under microwave treatment, which are thermally resistant to H-D exchange up to 75 °C during conventional heating. These results revealed a substantial enhancing effect of microwave treatment on the thermal unfolding and exposure of buried amide groups in β-Lg compared to conventional heating. Microwave processing could be a promising alternative to produce hydrolysates with lower allergenicity and improved bioactivity through structure modification.     

Cooperative alpha-helix unfolding in a protein-DNA complex from hydrogen-deuterium exchange

We present experimental evidence for a cooperative unfolding transition of an alpha-helix in the lac repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogen-deuterium (H-D) exchange experiments monitored by NMR spectroscopy. In the EX1 limit, observed exchange rates become pH-independent and exclusively sensitive to local structure fluctuations that expose the amide proton HN to exchange. Close to this regime, we measured decay rates of individual backbone HN signals in D2O, and of their mutual HN-HN NOE by time-resolved two-dimensional (2D) NMR experiments. The data revealed correlated exchange at the center of the lac headpiece recognition helix, Val20-Val23, and suggested that the correlation breaks down at Val24, at the C terminus of the helix. A lower degree of correlation was observed for the exchange of Val9 and Ala10 at the center of helix 1, while no correlation was observed for Val38 and Glu39 at the center of helix 3. We conclude that HN exchange in the recognition helix and, to some extent, in helix 1 is a cooperative event involving the unfolding of these helices, whereas the HN exchange in helix 3 is dominated by random local structure fluctuations.     

pH-dependent effects of sodium tungstate on the steroid-binding properties of hen oviduct progesterone receptor

Effects of sodium tungstate on the steroid-binding properties of hen oviduct progesterone receptor were examined and were found to be pH-dependent. When freshly prepared hen oviduct cytosol containing progesterone receptor was heated at 37°C for 20 min, its ability to bind [³H]progesterone decreased to 20% level of unheated samples. At pH 7, presence of 2–3 mM tungstate during the above incubation period reduced this loss of binding. At higher tungstate concentrations (>5 mM), this stabilizing effect was gradually abolished. Similar results were obtained with preparations that contained [³H]progesterone-receptor complexes; 70–80% of which remained after a 20 min incubation at 37°C in the presence of 2–3 mM tungstate at pH 7. At pH 8, presence of tungstate (1–10 mM) during the 37°C incubation stabilized both the steroid-bound and the unoccupied progesterone receptor in a concentration-dependent manner. The extent of steroid binding by the receptor at 4°C remained unchanged in the presence of up to 10 mM tungstate at both pH 7 and pH 8 assay conditions while presence of 20 mM tungstate lowered this binding capacity. These results indicate that tungstate effects may be mediated via its interaction with the progesterone receptor.     

Conformational study on ketamine by circular dichroism and ultraviolet spectroscopy

Since the enantiomers of the N‐methyl‐D ‐aspartate (NMDA) receptors antagonist ketamine have different pharmacological profiles, CD and UV spectroscopy were applied for the study of conformer equilibrium and pH dependence in ketamine solutions. The assignment of the configurations and conformations was performed on the basis of the “octant rule” and UV spectra. In accord with published data, it was established that, on protonation, the phenyl group of the ketamine molecule occupies an axial position, while for the base form, the ratio of conformers containing axial/equatorial aryl moieties is strongly solvent‐dependent. The CD and UV spectra indicate the presence of an intramolecular H‐bond C=O····H—N in the conformer with axial aryl moiety. Chirality 11:280–285, 1999. © 1999 Wiley‐Liss, Inc.     

Purification,Characterization, and Crystallization of Crocodylus siamensis Hemoglobin

Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and β chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form. The unpolymerized Hb (composed of two α chains and two β chains) showed high oxygen affinity at 3.13 mmHg (P₅₀) and 1.96 (n value), and a small Bohr effect (δH⁺ = ?0.29) at a pH of 6.9–8.4. Adenosine triphosphate did not affect the oxygenation properties, whereas bicarbonate ions strongly depressed oxygen affinity. Crude C. siamensis Hb solutions were showed high O₂ affinity at P₅₀ of 2.5 mmHg which may assure efficient utilization of the lung O₂ reserve during breath holding and diving. The purified Hbs were changed to cyanmethemoglobin forms prior crystallization. Rod- and plate-shaped crystals were obtained by the sitting-drop vapor-diffusion method at 5 °C using equal volumes of protein solution (37 mg/ml) and reservoir [10–13 % (w/v) PEG 4000, with 0.1 M Tris buffer in present of 0.2 M MgCl₂·6H₂O] solution at a pH of 7.0–8.5.     

Structure of -lactalbumin and its fluctuation

The kinetics of the hydrogen-deuterium exchange reaction in bovine α-lactalbumin have been followed, by infrared absorption measurement, in aqueous solutions at various pH values and at various temperatures. A thermal transition which takes place at about 60 °C has been examined by ultraviolet absorption measurement and circular dichroism measurement.Outlines of the exchange kinetics and the thermal transition are quite similar to those observed for hen egg-white lysozyme, the amino acid sequence of which is known to be very similar to that of α-lactalbumin. Between these two proteins, however, differences have been found in the following respects. (1) The number of slowly exchanging peptide hydrogen atoms (35 in α-lactalbumin compared with 44 in egg-white lysozyme). (2) Kinetic profile of the slow exchange reaction. (3) The midpoint of the thermal transition (54 °C in water and 58 °C in deuterium oxide for α-lactalbumin, compared with 76 °C in both water and deuterium oxide for egg-white lysozyme). (4) The enthalpy and entropy changes in the transition (72 kcal/mol and 220 e.u., respectively, for α-lactalbumin, compared with 127 kcal/mol and 364 e.u. for egg-white lysozyme). (5) The circular dichroic spectrum of the “unfolded” molecule. (6) The effective amount of the unfolded forms estimated from the kinetic measurement at temperatures slightly lower than the transition temperature. (7) The effect of pH on the exchange kinetics.These differences between the proteins are interpreted in terms of the molecular structures and their fluctuations.     

Anesthetic stabilization of protein intermediates: myoglobin and halothane

Halothane, an inhaled anesthetic, destabilizes the folded structure of myoglobin. To determine whether this is due to preferential interactions with less stable folded conformers of myoglobin versus the completely unfolded state, we used photoaffinity labeling, hydrogen exchange, fluorescence spectroscopy, and circular dichroism spectroscopy. Apomyoglobin was used as a model of a less stable conformer of myoglobin. Halothane destabilizes myoglobin and binds with low affinity and stoichiometry but stabilizes and binds with higher affinity to apomyoglobin. The same halothane concentration has no effect on cytochrome c stability. The apomyoglobin/halothane complex is favored at pH 6.5 as compared to pH 4.5 or pH 2.5. Halothane photoincorporates into several sites in apomyoglobin, some allosteric to the heme pocket. Guanidinium unfolding of myoglobin, monitored by CD spectroscopy, shows destabilization at less than 1.3 M Gdm but stabilization at greater than 1.3 M Gdm, consistent with the hypothesis that less stable conformers of myoglobin bind halothane preferentially. We suggest the structural feature underlying preferential binding to less stable conformers is an enlarged cavity volume distribution, since myoglobin has several intermediate-sized cavities, while cytochrome c is more well packed and has no cavities detected by GRASP. Specific binding to less stable intermediates may underlie anesthetic potentiation of protein activity.     

Hydrogen-exchange kinetics of the indole NH proton of the buried tryptophan in the constant fragment of the immunoglobulin light chain

The constant fragment of the immunoglobulin light chain (type lambda) has two tryptophyl residues at positions 150 and 187. Trp-150 is buried in the interior, and Trp-187 lies on the surface of the molecule. The hydrogen-deuterium exchange kinetics of the indole NH proton of Trp-150 were studied at various pH values at 25 degrees C by 1H nuclear magnetic resonance. Exchange rates were approximately first order in hydroxyl ion dependence above pH 8, were relatively independent of pH between pH 7 and 8, and decreased below pH 7. On the assumption that the exchange above pH 8 proceeds through local fluctuations of the protein molecule, the exchange rates between pH 7 and 8 through global unfolding were estimated. The exchange rate constant within this pH range at 25 degrees C thus estimated was consistent with that of the global unfolding of the constant fragment under the same conditions as those reported previously [Kikuchi, H., Goto, Y., & Hamaguchi, K. (1986) Biochemistry 25, 2009-2013]. The activation energy for the exchange process at pH 7.8 was the same as that for the unfolding process by 2 M guanidine hydrochloride. The exchange rates of backbone NH protons were almost the same as that of the indole NH proton of Trp-150 at pH 7.1. These observations also indicated that the exchange between pH 7 and 8 occurs through global unfolding of the protein molecule and is rate-limited by the unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentration-dependent hydrogen exchange kinetics of 3H-labeled S-peptide in ribonuclease S.

The hydrogen exchange kinetics of the S-peptide in ribonuclease S can be measured by first tritiating the S-peptide in the absence of S-protein and then allowing it to recombine rapidly with S-protein. Afterwards the exchange reactions of this specific segment of ribonuclease S can be studied. The exchange kinetics of bound S-peptide are complex, indicating that different protons exchange at markedly different rates. The terminal exchange reaction, involving at least five highly protected protons, has been studied as a function of pH.At low concentrations of ribonuclease S the exchange kinetics become concentration-dependent, owing to the dissociation of the S-peptide. Although the fraction of free S-peptide is always very small, its rate of exchange is several orders of magnitude faster than that of bound S-peptide, and the concentration dependence of the exchange kinetics is readily measurable. It provides a highly sensitive method for determining small dissociation constants (K_D). Values of K_D ranging from 10^?6m at pH 2.7, 0 °C, to 2 × 10^?10m at pH 7.0, 0 °C, are reported here. Our value for K_D at pH 7.0, 0 °C, confirms the data and extrapolation to 0 °C of Hearn et al. (1971).At high concentrations of ribonuclease S the terminal exchange reaction is independent of concentration. It probably results from a local unfolding reaction of the bound S-peptide. Above pH 4 the strong pH dependence of K_D closely resembles that of the apparent equilibrium constant for this local unfolding reaction. The latter may be one step in the dissociation process and we present such a model for ribonuclease S dissociation.Measurement of concentration-dependent exchange kinetics should provide a useful method of determining small dissociation constants in other systems: for example, in studies of protein-nucleic acid interactions.     

Effect of temperature on the interaction of cisplatin with the model protein hen egg white lysozyme

The products of the reaction between cisplatin (CDDP) and the model protein hen egg white lysozyme (HEWL) at 20, 37 and 55 °C in pure water were studied by UV–Vis absorption spectroscopy, intrinsic fluorescence and circular dichroism, dynamic and electrophoretic light scattering and inductively coupled plasma mass spectrometry. X-ray structures were also solved for the adducts formed at 20 and 55 °C. Data demonstrate that high temperature facilitates the formation of CDDP-HEWL adducts, where Pt atoms bind ND1 atom of His15 or NE2 atom of His15 and NH1 atom of Arg14. Our study suggests that high human body temperature (fever) could increase the rate of drug binding to proteins thus enhancing possible toxic side effects related to CDDP administration.     

Construction of an improved amperometric acrylamide biosensor based on hemoglobin immobilized onto carboxylated multi-walled carbon nanotubes/iron oxide nanoparticles/chitosan composite film

A method is described for construction of an improved amperometric acrylamide biosensor based on covalent immobilization of hemoglobin (Hb) onto nanocomposite of carboxylated multi-walled carbon nanotubes (cMWCNT) and iron oxide nanoparticles (Fe₃O₄NPs) electrodeposited onto Au electrode through chitosan (CHIT) film. The Hb/cMWCNT-Fe₃O₄NP/CHIT/Au electrode was characterized by scanning electron microscopy, Fourier transform infra-red spectroscopy, electrochemical impedance spectroscopy, and differential pulse voltammetry at different stages of its construction. The biosensor was based on interaction between acrylamide and Hb, which led to decrease in the electroactivity of Hb, i.e., current generated during its reversible conversion [Fe(II)/Fe(III)]. The biosensor showed optimum response within 8 s at pH 5.0 and 30 °C. The linear working range for acrylamide was 3–90 nM, with a detection limit of 0.02 nM and sensitivity of 36.9 μA/nM/cm². The biosensor was evaluated and employed for determination of acrylamide in potato crisps.