Hydrogen-deuterium Exchange Rates Research Articles

Relevance of Protein Thin Films Prepared for Attenuated Total Reflection Fourier Transform Infrared Spectroscopy: Significance of the pH

Amide hydrogen–deuterium exchange rates were recorded on thin lysozyme films by attenuated total reflection Fourier transform infrared spectroscopy as a function of the pH of the solution from which the films were prepared. We describe the appropriate spectral corrections for atmospheric water absorption and for lateral side-chain contribution in the amide I/amide II region required to correctly evaluate the exchange rate from the amide II integrated intensity decrease. We show that the pH dependence of the exchange rate in our experimental conditions (0.4 g water/g protein) parallels the solution behavior. This observation confirms that the technique can be used to monitor pH-sensitive effects and allows exchange curves to be made which include most of the protein amide protons.

The pH dependence of hydrogen-deuterium exchange in trp repressor: the exchange rate of amide protons in proteins reflects tertiary interactions, not only secondary structure.

The pH dependence of amide proton exchange rates have been measured for trp-repressor. One class of protons exchanges too fast to be measured in these experiments. Among the protons that have measurable hydrogen-deuterium exchange rates, two additional classes may be distinguished. The second class of protons are in elements of secondary structure that are mostly on the surface of the protein, and exchange linearly with increasing base concentration (log kex versus pH). The third class of amide protons is characterized by much higher protection against exchange at higher pH. These protons are located in the core of the protein, in helices B and C. The exchange rate in the core region does not increase linearly with pH, but rather goes through a minimum around pH 6. The mechanism of exchange for the slowly exchanging core protons is interpreted in terms of the two-process model of Hilton and Woodward (1979, Biochemistry 18:5834-5841), i.e., exchange through both a local mechanism that does not require unfolding of the protein, and a mechanism involving global unfolding of the protein. The increase in exchange rates at low pH is attributed to a partial unfolding of the repressor. It is concluded that the formation of secondary structure alone is insufficient to account for the high protection factors seen in the core of native proteins at higher pH, and that tertiary interactions are essential to stabilize the structure.

Effects of replacement of Lys25 with Gln on the conformation of ribonuclease T1: sequence-specific 1H NMR resonance assignments of Gln25 ribonuclease T1 by two-dimensional NMR spectroscopy.

Two isozymes of ribonuclease (RNase) T1 exist in nature, i.e. Gln25 RNase T1 and Lys25 RNase T1. Gln25 RNase T1 is less stable than Lys25 RNase T1, although the enzymatic activity is not distinguishable between these two isozymes. To elucidate the effects of the replacement of Lys25 with Gln on the conformation and microenvironments of RNase T1 in detail, two-dimensional NMR spectra were measured, sequence-specific 1H NMR resonance assignments of Gln25 RNase T1 were performed, and then the determined parameters and microenvironments of Gln25 RNase T1 were compared with those of Lys25 isozyme [Hoffmann, E. and Rüterjans, H. (1988) Eur. J. Biochem. 177, 539-560]. The main chain protons were assigned for 101 out of the total of 104 amino acid residues. Secondary structure elements were identified from analysis of characteristic NOE patterns, interstrand NOE connectivities, and hydrogen-deuterium exchange rates of main chain amide protons. The results indicated that Gln25 RNase T1 contains a single alpha-helix and seven beta-strands. The secondary structure of Gln25 RNase T1 is, thus, essentially the same as that of Lys25 RNase T1. On the other hand, comparison of the conformation-dependent shifts of Gln25 RNase T1 with these of Lys25 RNase T1 showed that the replacement of Lys25 with Gln has significant effects on the C-terminal part of the alpha-helix region and the base-binding site. These results may indicate that the base-binding site is relatively flexible in the RNase T1 molecule. Among the residues of the C-terminal part of the alpha-helix region, the protons of Asp29 were most affected in terms of their chemical shifts, which may indicate that the side chain carboxylate anion of Asp29 is the counterpart of the electrostatic interaction of Lys25 in Lys25 RNase T1. The Gln25 of Gln25 RNase T1 may have little or no interaction with Asp29, and this may be the reason why Gln25 RNase T1 is less stable than the Lys25 isozyme.

Stability of alpha-helices in a molten globule state of cytochrome c by hydrogen-deuterium exchange and two-dimensional NMR spectroscopy.

To distinguish between intrinsically stable helices and those stabilized by the protein three-dimensional structure, we report the hydrogen-deuterium exchange (H2H) rates of 29 amide protons of ferricytochrome c in a molten globule state (at 35 degrees C, pH 2.0 with 0.5 M KCl), monitored by 2D-NMR. The results of the H2H-exchange experiments have been analyzed to calculate the protection factors. The helices were not uniformly stable and amide protons of residues belonging to the N and C-terminal helices had high protection factors. The protection factors of amide protons involved in the 50's helix were low, and could not be determined quantitatively. In the 60's helix we found two amide protons with protection factors comparable to those of the N and C-terminal helices. These results, compared with previously reported intrinsic helicities of peptide fragments, indicate that the relative helicities of isolated fragments are not directly reflected in the stability of the helices in the molten globule state, even though this state has no rigid tertiary structure. This suggests that loose interactions between helices are present in the molten globule state of cytochrome c, and that they are essential for keeping the helicity of the helical segments. The loose tertiary interactions discussed here differ from the usual tertiary interaction found in the native state in that the specific interdigitization between side-chains is absent.

Influence of Murchison or Allende minerals on hydrogen-deuterium exchange of amino acids

Deuterium-enriched amino acids occur in the Murchison carbonaceous chrondrite. This meteorite underwent a period of aqueous alteration with isotopically light water. With the objective of setting limits on the conditions of aqueous alteration, the exchange of the carbon-bonded hydrogen atoms of amino acids with D 2O has been studied from 295 to 380 K as a function of time and meteorite/heavy water ratio. The amount of Murchison or Allende dust present has a significant effect on the rate and amount of hydrogen-deuterium exchange observed. At elevated temperatures, the a-hydrogens of all the amino acids studied were found to exchange with deuterium. In glycine and aspartic acid, this process resulted in total exchange of the carbon-bonded hydrogen. A completely deuterated isotopomer of alanine was produced in significant quantities only when the rock/water ratio was greater than 0.5. No exchange of carbonbonded hydrogens was observed in the case of amino acids which do not possess an α-hydrogen atom. The rates of H/D exchange for amino acids observed here did not correspond to deuterium enrichment of the amino acids in the Murchison meteorite. These results suggest that H/D exchange with water had a negligible effect on the observed deuterium enrichment of amino acids found in Murchison and that the temperature at which the amino acids were exposed to liquid water was close to 273 K.

Stability of α-helices in a molten globule state of cytochrome c by hydrogen-deuterium exchange and two-dimensional NMR spectroscopy

To distinguish between intrinsically stable helices and those stabilized by the protein three-dimensional structure, we report the hydrogen-deuterium exchange (H2H) rates of 29 amide protons of ferricytochrome c in a molten globule state (at 35°C, pH 2.0 with 0.5 M KCl), monitored by 2D-NMR. The results of the H2H-exchange experiments have been analyzed to calculate the protection factors. The helices were not uniformly stable and amide protons of residues belonging to the N and C-terminal helices had high protection factors. The protection factors of amide protons involved in the 50's helix were low, and could not be determined quantitatively. In the 60's helix we found two amide protons with protection factors comparable to those of the N and C-terminal helices. These results, compared with previously reported intrinsic helicities of peptide fragments, indicate that the relative helicities of isolated fragments are not directly reflected in the stability of the helices in the molten globule state, even though this state has no rigid tertiary structure. This suggests that loose interactions between helices are present in the molten globule state of cytochrome c, and that they are essential for keeping the helicity of the helical segments. The loose tertiary interactions discussed here differ from the usual tertiary interaction found in the native state in that the specific interdigitization between side-chains is absent.

Role of the Domain-Domain Interaction in the Construction of the Antigen Combining Site: A Comparative Study by 1H- 15N Shift Correlation NMR Spectroscopy of the Fv and Fab Fragments of Anti-dansyl Mouse Monoclonal Antibody

A comparative NMR structural study of anti-dansyl Fv and Fab fragments is reported. Both of these antigen binding fragments have been prepared using antibodies that originate from the identical anti-dansyl switch variant cell lines. It has been confirmed that the Fv and Fab fragments possess the identical binding property. The antigen binding fragment analogs labeled with 15 N of the main chain amide group of the aromatic residues (His, Phe, Trp, and Tyr) were used. The chemical shift and hydrogen-deuterium exchange rate of the amide protons are compared for the Fv and Fab fragments. On the basis of the NMR data obtained, we have concluded that (1) the structural change induced in the V H domain upon antigen binding significantly affects the dynamical structure of the V L domain and (2) the existence of the constant regions affects the fluctuation of the V L domain, increasing the thermal stability of the variable region.

Amide hydrogen exchange of the central B-chain helix within the T- and R-states of insulin hexamers

Comparative analysis of the 1H-NMR spectra of human insulin shows that in the presence of the allosteric ligand, phenol, the tertiary structure of the protein is altered as evidenced by the decreased rate of amide hydrogen-deuterium exchange. In particular, exchange of amide protons in residues of the B-chain helix (B 9-B 20) are significantly affected suggesting either a stabilization of this helix or a reduction in the solvent accessibility of the helix in the R-state. This paper exemplifies the exchange rates of two amides (Val B18 and Tyr B16) from this helix which decrease by approximately 400-fold as a result of this ligand induced conformational transition.

Conformational analysis of an antitumour cyclic pentapeptide, astin B, from Aster tataricus)

Conformational analysis of an antitumour cyclic pentapeptide, astin B, isolated from Aster tataricus, was conducted by 2D-NMR techniques, temperature effects on NH protons, rate of hydrogen-deuterium exchange, vicinal NH-CαH coupling constants, and NOE experiments. The methods of molecular mechanics and restrained molecular dynamics calculations were applied to understand the energetic preferences of various conformations of astin B. Distances involving in three intramolecular hydrogen bonds and the NOE correlations were used for the refinements AMBER program. These results indicated that the conformation in the solution state was, on the whole, homologous to that observed in the solid state. Conformational difference between astin B, showing a cis configuration in a proline amide bond, and cyclochlorotine from Penicillium islandicum, showing all trans amide configurations, was also discussed.

Nuclear magnetic resonance study and secondary structure determination of the antibiotic peptide, aibellin.

Aibellin is a 20-residue peptide antibiotic that has been isolated from the fungus Verticimonosporium ellipticum. Sequence-specific assignment of the 1H- and 13C-NMR signals of aibellin in a methanol solution was achieved by using the two-dimensional NMR technique. Furthermore, its secondary structure was characterized by circular dichroism (CD) and NOESY spectra. The observed NOEs, 3JNHC alpha H coupling constants and amide hydrogen-deuterium (H-D) exchange rates show that the peptide consisted of two alpha-helices and a bent structure around a Pro-14 residue.

Identification of the barstar binding site of barnase by NMR spectroscopy and hydrogen-deuterium exchange

The extracellular ribonuclease from Bacillus amyloliquifaciens, barnase, forms a tightly-bound one-to-one complex with its intracellular inhibitor barstar. The barstar binding site on barnase was characterised by comparing the differences in the chemical shift and hydrogen-deuterium exchange rates between free and bound barnase. Chemical shift assignments of barnase in the complex with barstar were determined from 3D NOESY-HMQC and TOCSY-HMQC spectra of a complex that had been prepared with uniformly 15N-labelled barnase and unlabelled barstar. Hydrogen exchange rates were obtained from an analysis of a series of [ 15N]HMQC spectra of a sample prepared in the same manner exchanged into D 2O. The largest changes in either chemical shift or hydrogen-deuterium exchange rate are observed for residues located in the active-site and substrate binding loops indicating that barstar inhibits barnase activity by sterically blocking the active site.

Identification of the adsorbing site of lysozyme onto the hydroxyapatite surface using hydrogen exchange and 1H NMR.

The lysozyme-hydroxyapatite interaction was studied by measuring individual hydrogen-deuterium (H-D) exchange rates of amide protons. The H-D exchange reaction was initiated by transferring the lysozyme adsorbed on hydroxyapatite powder from H2O into D2O. After various H-D exchange time periods (pH 7.0, 25 degrees C), the complex was dissociated and the remaining hydrogen label was determined by 2D NMR analysis. The H-D exchange rate of amide protons of residues 9, 11, 13, and 83 was slowed in the hydroxyapatite-lysozyme complex compared with free lysozyme. Residues 9, 11 and 13 positioned at the back of the active site would be the location of the binding site.

Sequence and solution conformation of the 20-residue peptaibols, saturnisporins SA II and SA IV.

Saturnisporins SA II and SA IV are the major components of the 20-residue peptaibol mixture isolated from a culture of the fungus Trichoderma saturnisporum. These peptides exhibit antibiotic activity against Staphylococcus aureus. Their sequences were derived from unequivocal methodology implying the combined use of positive ion FAB mass spectrometry and NMR: the majority of the sequences result from mass spectrometry fragmentations and the location of isomeric residues arises either from analysis of ROESY cross-peaks between contiguous amide protons or from heteronuclear 2J or 3J 1H-13C couplings detected in long-range 1H-13C COSY experiments. The sequence specific 1H and 13C NMR assignments are described. Saturnisporins SA II and SA IV exhibit similar secondary structures, as deduced from their ROESY patterns and 3JNHC alpha H coupling constant values, together with amide hydrogen-deuterium exchange rates and temperature coefficients of amide and carbonyl groups. An overall alpha-helical structure is proposed, maintaining two regions of distortion to this regular structure; i) the N-terminal part, which contains 3(10) and mixed alpha-3(10) turns, and ii) the Aib10-Val15 region, including a Pro residue which accommodates a bend stabilized by two 3(10) H-bonds.

Helical structure and orientation of melittin in dispersed phospholipid membranes from amide exchange analysis in situ.

A trapping method combined with high-resolution nuclear magnetic resonance spectroscopy is described for the measurement of hydrogen-deuterium exchange rates for individual amides of polypeptides bound to fully hydrated, dispersed phospholipid bilayers. Exchange rates were measured for 22 of the 24 amide hydrogens of bee venom melittin bound to bilayers composed of egg phosphatidylcholine/phosphatidylserine (88:12, mol/mol) dispersed in 20 mM sodium acetate, pH 4.0. Amides of residues 5-11 and 16-22 had exchange rates suppressed by between 30- and 1000-fold, and the rate suppression exhibited a helical periodicity with amides on the hydrophobic helix face up to 20-fold more stable than those on the hydrophilic face of the helix. These results demonstrate that under the conditions studied melittin adopts a helical conformation with stable helical hydrogen bonds extending to residue 22 and that the helix is oriented with the hydrophobic face directed toward the membrane interior.

Dynamical structure of the antibody combining site as studied by 1H-15N shift correlation NMR spectroscopy.

The Fv fragment, which is a smallest antigen-binding unit of immunoglobulin, has been used for a 1H-15N shift correlation NMR study of the dynamical structure of the antibody combining site. Fv has been prepared by clostripain digestion of a mouse anti-dansyl IgG2a monoclonal antibody that lacks the entire CH1 domain. We have previously reported that of the six hypervariable regions, three each from the heavy chain (H1, H2, and H3) and the light chain (L1, L2, and L3), H3 is primarily responsible for the antigen binding in the anti-dansyl Fv fragment. The backbone amide nitrogens of all non-proline amino acid residues in H3 have been multiply labeled with 15N. [15N]T2 relaxation times and hydrogen-deuterium exchange rates of the amide groups of the main chain were measured in the absence and presence of epsilon-dansyl-L-lysine (DNS-Lys). It has been shown that (1) in the absence of DNS-Lys H3 displays a significant degree of internal motion and (2) antigen binding induces a significant change in the dynamical structure of H3.

A reexamination of the stopped-flow measured hydrogen-deuterium exchange rates in nucleic acid double helices

Using the deuteration labelling method in conjunction with an improved stopped-flow instrument, we have reexamined the proton exchange process in poly(dA-dT) · poly(dA-dT). A single proton exchange class is found with a rate constant at 20°C of 0.4 s −1. This exchange rate is independent of buffer concentration. From the comparison of these results with those obtained in a former study where two rates were measured, it is concluded that the present deuteration signal corresponds to the exchange of the amino protons, whereas the imino proton exchange is not detected.

Solution structure and dynamics of epidermal growth factor and transforming growth factor alpha.

Circular dichroism (CD) and Fourier transform infrared spectroscopic studies have shown that the secondary structure of transforming growth factor alpha (TGF-alpha) is very similar to that of epidermal growth factor (EGF). The infrared spectra revealed a minor difference between the two proteins, in particular in the beta-sheet structure. A large difference was observed with CD between the two proteins in the apparent conformation each adopts when the disulfide bonds are reduced. Reduced TGF-alpha showed a distinct alpha-helical conformation only at a high trifluoroethanol concentration, whereas reduced EGF assumed an alpha-helical conformation in the absence of trifluoroethanol. This indicates that these two proteins adopt different secondary structures in the absence of disulfide bonds, although they assume similar folding structures in their presence. These data suggest that the disulfide bonds to a large degree dictate the conformation of these two proteins. Additionally, differences in the dynamic behavior between EGF and TGF-alpha were also observed. Infrared experiments showed that the hydrogen-deuterium exchange rate is much higher for TGF-alpha than for EGF, indicating that TGF-alpha is a more flexible molecule. The rate of reduction of the disulfide bonds by dithiothreitol was also faster for TGF-alpha. Therefore, it can be concluded that although EGF and TGF-alpha have a similar overall conformation, TGF-alpha is a more flexible molecule than EGF.

Periodicity of amide proton exchange rates in a coiled-coil leucine zipper peptide.

The two-stranded coiled-coil motif, which includes leucine zippers, is a simple protein structure that is well suited for studies of helix-helix interactions. The interaction between helices in a coiled coil involves packing of "knobs" into "holes", as predicted by Crick in 1953 and confirmed recently by X-ray crystallography for the GCN4 leucine zipper [O'Shea, E.K., Klemm, J.D., Kim, P.S., & Alber, T. (1991) Science 254, 539]. A striking periodicity, extending over six helical turns, is observed in the rates of hydrogen-deuterium exchange for amide protons in a peptide corresponding to the leucine zipper of GCN4. Protons at the hydrophobic interface show the most protection from exchange. The NMR chemical shifts of amide protons in the helices also show a pronounced periodicity which predicts a short H-bond followed by a long H-bond every seven residues. This variation was anticipated in 1953 by Pauling and is sufficient to give rise to a local left-handed superhelical twist characteristic of coiled coils. The amide protons that lie at the base of the "hole" in the "knobs-into-holes" packing show slow amide proton exchange rates and are predicted to have short H-bond lengths. These results suggest that tertiary interactions can lead to highly localized, but substantial, differences in stability and dynamics within a secondary structure element and emphasize the dominant nature of packing interactions in determining protein structure.

Structural description of acid-denatured cytochrome c by hydrogen exchange and 2D NMR

Hydrogen exchange and two-dimensional nuclear magnetic resonance (2D NMR) techniques were used to characterize the structure of oxidized horse cytochrome c at acid pH and high ionic strength. Under these conditions, cytochrome c is known to assume a globular conformation (A state) with properties resembling those of the molten globule state described for other proteins. In order to measure the rate of hydrogen-deuterium exchange for individual backbone amide protons in the A state, samples of oxidized cytochrome c were incubated at 20 degrees C in D2O buffer (pD 2.2, 1.5 M NaCl) for time periods ranging from 2 min to 500 h. The exchange reaction was then quenched by transferring the protein to native conditions (pD 5.3). The extent of exchange for 44 amide protons trapped in the refolded protein was measured by 2D NMR spectroscopy. The results show that this approach can provide detailed information on H-bonded secondary and tertiary structure in partially folded equilibrium forms of a protein. All of the slowly exchanging amide protons in the three major helices of native cytochrome c are strongly protected from exchange at acid pH, indicating that the A state contains native-like elements of helical secondary structure. By contrast, a number of amide protons involved in irregular tertiary H-bonds of the native structure (Gly37, Arg38, Gln42, Ile57, Lys79, and Met80) are only marginally protected in the A state, indicating that these H-bonds are unstable or absent. The H-exchange results suggest that the major helices of cytochrome c and their common hydrophobic domain are largely preserved in the globular acidic form while the loop region of the native structure is flexible and partly disordered.

Role of arginine 67 in the stabilization of chymotrypsin inhibitor 2: examination of amide proton exchange rates and denaturation thermodynamics of an engineered protein

We have examined the contribution to protein stability of an interaction involving a charged hydrogen bond from an arginyl side chain (Arg67) in the serine proteinase inhibitor chymotrypsin inhibitor 2 (CI-2), by replacing this side chain with an alanyl residue by protein engineering. Using nuclear magnetic resonance spectroscopy (NMR), we have examined the effect of this mutation on the hydrogen-deuterium exchange rates of several backbone amide protons in the native and engineered proteins at 50 degrees C. These exchange rates provide a localized probe at multiple discrete sites throughout the protein and from comparison of native and mutant exchange rates allow calculation of the difference in free energy of exchange (delta delta Gex) resulting from the mutation. The results show that for the majority of amides observed this mutation results in delta delta Gex of ca. 1.7 kcal mol-1 over the whole CI-2 molecule. However, for two relatively exposed amide protons the exchange rates are found to be far less perturbed, implying that local unfolding mechanisms predominate for these protons. Direct measurement of the stability of both proteins to denaturation by guanidinum hydrochloride shows that the interaction contributes 1.4 kcal mol-1 to the stability of the molecule. This value is comparable to those obtained from the NMR exchange measurements and indicates that the exchange processes reflect the differences in stability between the native and mutant proteins.(ABSTRACT TRUNCATED AT 250 WORDS)