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.     

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.     

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.     

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.     

Conformational dynamics of the tetrameric hemoglobin molecule as revealed by hydrogen exchange: I. Effects of pH,temperature, and ligand binding

IR spectroscopy was used to study the rate of hydrogen-deuterium (H-D) exchange of peptide NH atoms in different forms of human hemoglobin (Hb) at pH 5–10 and temperatures of 10–63°C. The pH dependence of the H-D exchange rate fits the EX2 mechanism. At 10–30°C, there are two pH-dependent conformers of liganded Hb forms, the fluctuation probability being lower for the alkaline conformer. The differences between the conformers disappear at 40°C, where a third conformer, with a higher probability of local fluctuations, appears. Deoxyhemoglobin has no pH-dependent conformers in the pH range 6–9 at 20°C, and the probability of local fluctuations is considerably decreased compared to the acid conformer of liganded Hb. The destabilization of the liganded Hb structure by decreasing the pH to 5.0 at 20°C or increasing the temperature to 50–60°C at pH 7.1 enhances global fluctuations of the native structure ensuring the H-D exchange of slowly exchanging NH atoms. The mechanisms of local and high-temperature global fluctuations, as well as the possible similarity between the two pH-dependent conformers of liganded Hb and its functional R and R2 states revealed by X-ray analysis and NMR spectroscopy, are discussed.     

Torsional flexibility of B-DNA as revealed by conformational analysis.

The thermal fluctuations of a regular double helix belonging to the B-family were studied by means of atom-atomic potentials method. The winding angle fluctuation was found to be 2.4 degrees for poly(dA):poly(dT) and 3.0 degrees for poly(dG):poly(dC). The reasonable agreement of these estimations with those obtained experimentally reveals the essential role of the small-amplitude torsional vibrations of atoms in the mechanism of the double helix flexibility. The calculated equilibrium winding angle, tau 0, essentially depends on the degree of neutralization of phosphate groups, being about 35.5 degrees for the full neutralization. The deoxyribose pucker is closely related to the tau angle: while tau proceeds from 30 degrees to 45 degrees the pseudorotation phase angle, P, increases from 126 degrees to 164 degrees. Fluctuations of the angles TL and TW, which specify inclination of the bases to the helix axis, were evaluated to be 5 degrees-10 degrees. Possible correlation between conformational changes in the adjacent nucleotides is discussed.     

Individual breathing reactions measured in hemoglobin by hydrogen exchange methods.

Protein hydrogen exchange is generally believed to register some aspects of internal protein dynamics, but the kind of motion at work is not clear. Experiments are being done to identify the determinants of protein hydrogen exchange and to distinguish between local unfolding and accessibility-penetration mechanisms. Results with small molecules, polynucleotides, and proteins demonstrate that solvent accessibility is by no means sufficient for fast exchange. H-exchange slowing is quite generally connected with intramolecular H-bonding, and the exchange process depends pivotally on transient H-bond cleavage. At least in alpha-helical structures, the cooperative aspect of H-bond cleavage must be expressed in local unfolding reactions. Results obtained by use of a difference hydrogen exchange method appear to provide a direct measurement of transient, cooperative, local unfolding reactions in hemoglobin. The reality of these supposed coherent breathing units is being tested by using the difference H-exchange approach to tritium label the units one at a time and then attempting to locate the tritium by fragmenting the protein, separating the fragments, and testing them for label. Early results demonstrate the feasibility of this approach.     

Substrate binding and conformational changes of Clostridium glutamicum diaminopimelate dehydrogenase revealed by hydrogen/deuterium exchange and electrospray mass spectrometry.

C. glutamicum meso-diaminopimelate dehydrogenase is an enzyme of the L-lysine biosynthetic pathway in bacteria. The binding of NADPH and diaminopimelate to the recombinant, overexpressed enzyme has been analyzed using hydrogen/deuterium exchange and electrospray ionization/mass spectrometry. NADPH binding reduces the extent of deuterium exchange, as does the binding of diaminopimelate. Pepsin digestion of the deuterated enzyme and enzyme-substrate complexes coupled with liquid chromatography/mass spectrometry have allowed the identification of eight peptides whose deuterium exchange slows considerably upon the binding of the substrates. These peptides represent regions known or thought to bind NADPH and diaminopimelate. One of these peptides is located at the interdomain hinge region and is proposed to be exchangeable in the "open," catalytically inactive, conformation but nonexchangeable in the "closed," catalytically active conformation formed after NADPH and diaminopimelate binding and domain closure. Furthermore, the dimerization region has been localized by this method, and this study provides an example of detecting protein-protein interface regions using hydrogen/deuterium exchange and electrospray ionization.     

Structural features of interferon-gamma aggregation revealed by hydrogen exchange

Using hydrogen-deuterium exchange (HX) and electrospray ionization mass spectrometry, we have investigated the stability and structural changes of recombinant human interferon-gamma (IFN-gamma) during aggregation induced by guanidine hydrochloride (GdnHCl) and potassium thiocyanate. First, HX labeling was initiated after the amorphous aggregates were formed to probe the tertiary structure of the aggregated state. Second, labeling was performed at low protein concentrations to assess stability under aggregation prone conditions. In 1 M GdnHCl, the stability of IFN-gamma was greatly reduced and much less protection from HX in solution was observed. Exchange under these conditions was slower in helix C than in the rest of the protein. Aggregates formed in 1 M GdnHCl showed a HX pattern consistent with a partially unfolded state with an intact helix C. Although aggregates formed in 0.3 M KSCN exhibited a HX pattern similar to those formed in GdnHCl, the solution phase HX pattern in 0.3 M KSCN was surprisingly comparable to that of the native state. Varying the aggregation time before performing HX revealed that KSCN first precipitated native protein and then facilitated partial unfolding of the precipitated protein. These results show that helix C, which forms the hydrophobic core of the IFN-gamma dimer, is highly protected from HX under native conditions, is more stable in GdnHCl than the rest of the protein and remains intact in both GdnHCl- and KSCN-induced aggregates. This suggests that native-state HX patterns may presage regions of the protein susceptible to unfolding during aggregation.     

The coupling of catalytically relevant conformational fluctuations in subtilisin BPN' to solution viscosity revealed by hydrogen isotope exchange and inhibitor binding

We have measured the tritium outexchange of subtilisin BPN'. A consistent and rather small group of hydrogens was isolated by their sensitivity to inhibitor binding. The viscosity dependence of exchange from these inhibitor protected hydrogens was then examined in 0.05 M MES buffer, pH 6.5 and 10 degrees C. The viscosity of the reaction medium was varied by added glycerol and ethylene glycol. The exchange rates were corrected to be compared at identical hydroxyl ion and water activity. The salient observation is the strikingly similar viscosity coupling behavior when compared to the deacylation step of ester hydrolysis catalyzed by the same enzyme (Ng and Rosenberg, Biophysical Chemistry, 39 (1991) 57). We have obtained a viscosity coupling constant of 0.68 -/+ 0.18 for hydrogen exchange in glycerol (cf. 0.65 -/+ 0.11 for deacylation in glycerol, sucrose, glucose and fructose); 1.67 -/+ 0.07 for outexchange (cf. 1.92 -/+ 0.09 for deacylation), in the presence of ethylene glycol. The two reactions are very chemically dissimilar, yet they show very similar viscosity coupling behavior. This together with the well established role of structural fluctuations in hydrogen exchange implies a similar role of structural fluctuations in the deacylation step of subtilisin BPN' catalyzed ester hydrolysis.     

Discussion to II. Light-induced conformational changes of the rhodopsin molecule

European Biophysics Journal -     

NAD-binding mode and the significance of intersubunit contact revealed by the crystal structure of Mycobacterium tuberculosis NAD kinase-NAD complex

NAD kinase is a key enzyme in NADP biosynthesis. We solved the crystal structure of polyphosphate/ATP-NAD kinase from Mycobacterium tuberculosis (Ppnk) complexed with NAD (Ppnk-NAD) at 2.6A resolution using apo-Ppnk structure solved in this work, and revealed the details of the structure and NAD-binding site. Superimposition of tertiary structures of apo-Ppnk and Ppnk-NAD demonstrated a substantial conformational difference in a loop (Ppnk-flexible loop). As a quaternary structure, these Ppnk structures exhibited tetramer as in solution condition. Notably, the Ppnk-flexible loop was involved in the intersubunit contact and probably related to the NAD-binding of the other subunit. Furthermore, the two residues (Asp189, His226) substantially contributed to creating NAD-binding site on the other subunit. The two residues and the residues involved in NAD-binding were conserved. However, residues corresponding to the Ppnk-flexible loop were not conserved, making us to speculate that the Ppnk-flexible loop may be Ppnk-specific.     

Structural and dynamic characteristics of a partially folded state of ubiquitin revealed by hydrogen exchange mass spectrometry

Structural and dynamic properties of a partially folded conformation (A-state) of ubiquitin are studied using amide hydrogen exchange in solution (HDX) and mass spectrometric detection. A clear distinction between the native state of the protein and the A-state can be made when HDX is carried out in a semicorrelated regime. Convoluted exchange patterns are interpreted with the aid of HDX simulations in a three-state system (highly structured, partially unstructured, and fully unstructured states). The data clearly indicate a highly dynamic character of the non-native state. Furthermore, combination of HDX and protein ion fragmentation in the gas phase [by means of collision-induced dissociation (CAD)] is used to evaluate the conformational stability of various protein segments specifically in the molten globular state. Chain flexibility appears to be distributed very unevenly in this non-native conformation. The highest degree of structural disorder is displayed by the C-terminal segment (Gly(53)-Gly(76)), which was previously suggested to form a transient alpha-helix. The least dynamic segment of ubiquitin in the A-state is Thr(9)-Glu(18) (which was previously suggested to form a stable nativelike beta-strand), with the adjacent segments exhibiting somewhat diminished conformational stability. The study also demonstrates the power of mass spectrometry as a tool in providing conformer-specific information about the structure and dynamics of both native and non-native protein states coexisting in solution under equilibrium.     

Inactivation of annexin II tetramer by S-nitrosoglutathione.

We investigated the effect of nitric oxide (NO) donors on the activities of annexin II tetramer (AIIt), a member of the Ca2+- dependent phospholipid-binding protein family. Incubation of purified AIIt with S-nitrosoglutathione (GSNO) led to the inhibition of AIIt-mediated liposome aggregation. This effect was dose-dependent with an IC50 of approximately 100 micro m. Sodium nitroprusside, another NO donor also inhibited AIIt-mediated liposome aggregation, whereas reduced glutathione, nitrate, or nitrite had no effects. GSNO also inhibited AIIt-mediated membrane fusion, but not the binding of AIIt to the membrane. GSNO only has a modest effect on liposome aggregation mediated by annexins I, III or IV. The binding of AIIt to the membrane protected the reactive sites of GSNO on AIIt. GSNO did not inhibit AIIt-mediated liposome aggregation in the presence of dithiothreitol. Taken together, our results suggest that GSNO inactivates AIIt possibly via S-nitrosylation and/or the formation of disulfide bonds.     

Allosteric sensitivity in hemoglobin at the alpha-subunit N-terminus studied by hydrogen exchange

Allosteric structure change in human hemoglobin was studied by hydrogen-tritium-exchange methods. The functional labeling method used takes advantage of the change in H-exchange rate at allosterically involved sites to selectively label, with tritium, H-exchange sites that are fast in one protein state and slow in another. The position of the labeled sites can then be located by the medium-resolution fragmentation-separation method. These methods reveal 5 allosterically sensitive, H-bonded, peptide NH's within the first 12 residues of the alpha chain. All five exchange with solvent protons at similar rates in deoxyhemoglobin (T form), and all shift to a new rate, about 30-fold faster, in the liganded protein (R) form. This indicates a decrease in structural stability at the alpha-chain N-terminus in going from the T to the R form, consistent with the loss of stabilizing interactions in that segment. The results indicate a loss of perhaps 2 kcal/mol in stabilization free energy and thus document a significant role for changes at the alpha-chain N-terminus in the allosteric transition.     

The conformational dynamics of a metastable serpin studied by hydrogen exchange and mass spectrometry

Serpins are a class of protease inhibitors that initially fold to a metastable structure and subsequently undergo a large conformational change to a stable structure when they inhibit their target proteases. How serpins are able to achieve this remarkable conformational rearrangement is still not understood. To address the question of how the dynamic properties of the metastable form may facilitate the conformational change, hydrogen/deuterium exchange and mass spectrometry were employed to probe the conformational dynamics of the serpin human alpha(1)-antitrypsin (alpha(1)AT). It was found that the F helix, which in the crystal structure appears to physically block the conformational change, is highly dynamic in the metastable form. In particular, the C-terminal half of the F helix appears to spend a substantial fraction of time in a partially unfolded state. In contrast, beta-strands 3A and 5A, which must separate to accommodate insertion of the reactive center loop (RCL), are not conformationally flexible in the metastable state but are rigid and stable. The conformational lability required for loop insertion must therefore be triggered during the inhibition reaction. Beta-strand 1C, which anchors the distal end of the RCL and thus prevents transition to the so-called latent form, is also stable, consistent with the observation that alpha(1)AT does not spontaneously adopt the latent form. A surprising degree of flexibility is seen in beta-strand 6A, and it is speculated that this flexibility may deter the formation of edge-edge polymers.     

Conformational differences between arrestin2 and pre-activated mutants as revealed by hydrogen exchange mass spectrometry

Arrestins are regulatory proteins that bind specifically to ligand-activated phosphorylated G protein-coupled receptors to terminate G protein-mediated signaling, cause the internalization of the receptor-arrestin complex, and initiate additional intracellular signaling cascades. Multiple lines of evidence suggest that arrestin normally exists in an inactive basal state and undergoes conformational activation in the process of receptor binding. "Pre-activated" phosphorylation-independent arrestin mutants display increased binding to ligand-activated but unphosphorylated receptors. The mutations are believed to expose key receptor-binding regions, allowing the mutants to mimic, to some extent, the transition of arrestin to its active state. In the present study, amide hydrogen exchange (HX) and mass spectrometry (MS) were used to examine the inactive conformation of wild-type arrestin2 and compare its solution conformation with two pre-activated mutants (R169E and 3A (I385A, V386A, F387A)). The results suggest an unexpected level of structural organization within arrestin elements containing clathrin and adaptin2-binding sites that were previously believed to be completely disordered. Increased deuterium incorporation was observed in both mutant forms compared with wild-type, indicating a change in the conformation of the mutants. Three regions demonstrated significant differences in deuterium incorporation: the first 33 residues of the N terminus and residues 243-255 (both previously implicated in receptor interaction), and residues 271-299. The results suggest that subtle differences in conformation are responsible for the significant difference in biological activity displayed by pre-activated arrestin mutants and that similar changes occur in the process of arrestin binding to the receptor.     

The prohormone proenkephalin possesses differential conformational features of subdomains revealed by rapid H-D exchange mass spectrometry

Active enkephalin and related peptide hormones or neurotransmitters are generated by proteolytic processing of inactive prohormone precursors. Little is known about the relative accessibilities of prohormone cleavage sites and conformations of subdomains that undergo proteolytic processing. Therefore, this study investigated the conformational features of the prohormone proenkephalin (PE) by rapid hydrogen-deuterium exchange mass spectrometry (DXMS). DXMS analyzes rates of hydrogen exchange of the polypeptide backbone of PE with deuterium from D(2) O (heavy water) by mass spectrometry, accomplished at sub-second and multisecond time periods. Results showed differential accessibilities of cleavage sites and adjacent subdomains of PE to the aqueous environment. Importantly, protease cleavage sites of PE with greater relative accessibilities correspond to sites most readily cleaved by processing proteases to generate active peptide neurotransmitters. For comparison, peptides derived from PE (by pepsin digestion) displayed greater accessibility to the solvent environment, illustrated by their higher rates of H-D exchange compared to that of intact PE protein. The more limited H-D exchange accessibilities of PE protein, compared to peptides derived from PE, indicate that PE possesses tertiary conformation. These results demonstrate that differential tertiary conformations of PE subdomains undergo ordered proteolytic processing to generate active enkephalin peptides for cell-cell communication in the nervous and endocrine systems.