Hydrogen Exchange Rates Research Articles

Structure–function relationships of the NMDA receptor antagonist peptide, conantokin-R

Conantokin-R (con-R) is a γ-carboxyglutamate-containing 27-residue neuroactive peptide present in the venom of Conus radiatus, and acts as a non-competitive antagonist of the N-methyl- D-aspartate (NMDA) receptor. This peptide features a single disulfide bond, a type of structural element found in most classes of conotoxins, but not in other conantokins. The NMDA receptor antagonist activity of chemically synthesized con-R was determined through an assay involving inhibition of the spermine-enhanced binding of the NMDA receptor channel blocker, [ 3H]MK-801, to rat brain membranes, and yielded an IC 50 of 93 nM. This value represents a 2–5 times better potency than con-G or con-T, the other two characterized conantokins. Circular dichroism (CD) analysis of the metal-free form of con-R is indicative of a low α-helical content. There is an increase in α-helicity upon the addition of divalent cations, such as Ca 2+, Mg 2+, or Zn 2+. Isothermal titration calorimetry experiments showed one detectable Mg 2+ binding site with a K d of 6.5 μM, and two binding sites for Zn 2+, with K d values of 150 nM and 170 μM. Residue-specific information of the conformational state of con-R was obtained by two-dimensional 1H-NMR. Analyses of the α-proton chemical shifts, NOE patterns, and hydrogen exchange rates of the peptide indicated an α-helical conformation for residues 1–19. Synthetic con-R-derived peptide variants, containing deletions of 7 and 10 amino acid residues from the carboxy-terminus of the wild-type peptide, displayed unaltered cation binding and NMDA receptor antagonist properties. The α-helical secondary structures of the two truncation peptides were more stable than full-length con-R, as evidenced by CD measurements and reduced backbone hydrogen exchange rates. These results provide experimental evidence that the structural elements common to the three conantokins thus far identified are the primary determinants for receptor function and cation binding/secondary structure stability.

A Designed Four- α-Helix Bundle That Binds the Volatile General Anesthetic Halothane with High Affinity

The structural features of volatile anesthetic binding sites on proteins are being examined with the use of a defined model system consisting of a four- α-helix bundle scaffold with a hydrophobic core. Previous work has suggested that introducing a cavity into the hydrophobic core improves anesthetic binding affinity. The more polarizable methionine side chain was substituted for a leucine, in an attempt to enhance the dispersion forces between the ligand and the protein. The resulting bundle variant has an improved affinity ( K d = 0.20 ± 0.01 mM) for halothane binding, compared with the leucine-containing bundle ( K d = 0.69 ± 0.06 mM). Photoaffinity labeling with 14C-halothane reveals preferential labeling of the W15 residue in both peptides, supporting the view that fluorescence quenching by bound anesthetic reports both the binding energetics and the location of the ligand in the hydrophobic core. The rates of amide hydrogen exchange were similar for the two bundles, suggesting that differences in binding affinity were not due to changes in protein stability. Binding of halothane to both four- α-helix bundle proteins stabilized the native folded conformations. Molecular dynamics simulations of the bundles illustrate the existence of the hydrophobic core, containing both W15 residues. These results suggest that in addition to packing defects, enhanced dispersion forces may be important in providing higher affinity anesthetic binding sites. Alternatively, the effect of the methionine substitution on halothane binding energetics may reflect either improved access to the binding site or allosteric optimization of the dimensions of the binding pocket. Finally, preferential stabilization of folded protein conformations may represent a fundamental mechanism of inhaled anesthetic action.

Amide proton hydrogen exchange rates for sperm whale myoglobin obtained from 15N-1H NMR spectra.

The hydrogen exchange behavior of exchangeable protons in proteins can provide important information for understanding the principles of protein structure and function. The positions and exchange rates of the slowly-exchanging amide protons in sperm whale myoglobin have been mapped using 15N-1H NMR spectroscopy. The slowest-exchanging amide protons are those that are hydrogen bonded in the longest helices, including members of the B, E, and H helices. Significant protection factors were observed also in the A, C, and G helices, and for a few residues in the D and F helices. Knowledge of the identity of slowly-exchanging amide protons forms the basis for the extensive quench-flow kinetic folding experiments that have been performed for myoglobin, and gives insights into the tertiary interactions and dynamics in the protein.

Hydrogen-exchange stabilities of RNase T1 and variants with buried and solvent-exposed Ala --> Gly mutations in the helix.

Hydrogen-exchange rates were measured for RNase T1 and three variants with Ala --> Gly substitutions at a solvent-exposed (residue 21) and a buried (residue 23) position in the helix: A21G, G23A, and A21G + G23A. These results were used to measure the stabilities of the proteins. The hydrogen-exchange stabilities (DeltaG(HX)) for the most stable residues in each variant agree with the equilibrium conformational stability measured by urea denaturation (DeltaG(U)), if the effects of D(2)O and proline isomerization are included [Huyghues-Despointes, B. M. P., Scholtz, J. M., and Pace, C. N. (1999) Nat. Struct. Biol. 6, 210-212]. These residues also show similar changes in DeltaG(HX) upon Ala --> Gly mutations (DeltaDeltaG(HX)) as compared to equilibrium measurements (DeltaDeltaG(U)), indicating that the most stable residues are exchanging from the globally unfolded ensemble. Alanine is stabilizing compared to glycine by 1 kcal/mol at a solvent-exposed site 21 as seen by other methods for the RNase T1 protein and peptide helix [Myers, J. K., Pace, C. N., and Scholtz, J. M. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 3833-2837], while it is destabilizing at the buried site 23 by the same amount. For the A21G variant, only local NMR chemical shift perturbations are observed compared to RNase T1. For the G23A variant, large chemical shift changes are seen throughout the sequence, although X-ray crystal structures of the variant and RNase T1 are nearly superimposable. Ala --> Gly mutations in the helix of RNase T1 at both helical positions alter the native-state hydrogen-exchange stabilities of residues throughout the sequence.

Secondary structures and structural fluctuation in a dimeric protein, Streptomyces subtilisin inhibitor.

Based on the nuclear magnetic resonance assignments of a dimeric protein, Streptomyces subtilisin inhibitor (SSI), microscopic details of secondary structures in solution have been elucidated. The chemical shift index of C(alpha) signals, together with information on the hydrogen exchange rates of the backbone amide protons, were used to identify secondary structures. The locations of these secondary structures were found to be different in some critical points from those determined earlier by X-ray crystallography of the crystal. Notably, the beta3 strand is completely missing and the alpha2 helix is extended toward the C-terminus. Furthermore, hydrogen exchange experiments of individual peptide NH protons under strongly folding conditions revealed mechanisms of global and local structural fluctuation within the dimeric structure. It has been suggested that the global fluctuation of the monomeric unit occurs without affecting the accompanying monomer, in contrast to the equilibrium thermal unfolding, which is cooperative. Higher protection against hydrogen exchange for residues in part of the beta4 strand implies that this region might serve as a folding core.

Secondary structure of a calcium binding protein (CaBP) from Entamoeba histolytica

A calcium binding protein from Entamoeba histolytica, ( EhCaBP, M r∼15 kDa) is the causative agent for amoebiosis and has a very low sequence homology (∼30%) with other known CaBPs. Almost complete sequence specific resonance assignments for 1H, 13C and 15N spins in EhCaBP were obtained using double and triple resonance NMR experiments. Qualitative interpretation of the nuclear Overhauser enhancements, chemical shift indices and of hydrogen exchange rates threw valuable light upon the secondary structure of this protein. CaBP is found to have two globular domains each of which consists of two pairs of helix-loop-helix motifs. Though this protein has a very small sequence homology with calmodulins, the topological arrangement of the α-helices and β-strands in EhCaBP resemble them.

HYDROGEN BONDS REVEALED BY NMR

Hydrogen bonds-molecular interactions involving the sharing of a hydrogen atom by a weakly acidic donor atom and a weakly basic acceptor atom-are important structural features, especially in biological molecules. Hydrogen bonds are crucial elements of the three-dimensional conformations that permit proteins and nucleic acids to carry out their biological functions in an effective manner. However, experimental tools for detecting in a direct and unambiguous manner the conventional weak hydrogen bonds found in many biological macromolecules haven't been available until recently. Researchers have clearly known that hydrogen bonds were present in macromolecules. But existence of the bonds was generally inferred from the spatial proximity of donor and acceptor groups in X-ray and nuclear magnetic resonance (NMR) structures or from other indirect evidence, such as reduced hydrogen-exchange rates with solvent or changes in chemical shifts of protons in putative hydrogen bonds. In biological macromolecules, one ...

Hydrogen exchange kinetics of proteins in denaturants: a generalized two-process model

The recent progress in measurements on the amide hydrogen exchange (HX) in proteins under varying denaturing conditions, both at equilibrium and in transient relaxation, necessitates the development of a unifying theory which quantitatively relates the HX rates to the conformational energetics of the proteins. We present here a comprehensive kinetic model for the site-specific HX of proteins under varying solvent denaturing conditions based on the two-state protein folding model. The generalized two-process model considers both conformational fluctuations and residual protections, respectively, within the folded and unfolded states of a protein, as well as a global kinetic folding-unfolding transition between the two states. The global transition can be either rapid or slow, depending on the solvent condition for the protein. This novel model is applicable to the traditional equilibrium HX measurements in both EX2 and EX1 regimes, and also the recently introduced transient pulse-labeling HX experiments. A set of simple analytical equations is provided for quantitative interpretation of experimental data. The model emphasizes the use of full time-course of bi-exponential HX kinetics, rather than fitting time-course data to single rate constants, to obtain quantitative information about fluctuating conformers within the folded and unfolded states of proteins. This HX kinetic model naturally unfolds into a simple two-state and two-stage kinetic interpretation for protein folding. It suggests that the various observed intermediates of a protein can be interpreted as dominant isomers of either the folded or the unfolded state under different solvent conditions. This simple, minimalist’s view of protein folding is consistent with various recent experimental observations on folding kinetics by HX.

Fractionation factors and activation energies for exchange of the low barrier hydrogen bonding proton in peptidyl trifluoromethyl ketone complexes of chymotrypsin.

NMR investigations have been carried out of complexes between bovine chymotrypsin Aalpha and a series of four peptidyl trifluoromethyl ketones, listed here in order of increasing affinity for chymotrypsin: N-Acetyl-L-Phe-CF3, N-Acetyl-Gly-L-Phe-CF3, N-Acetyl-L-Val-L-Phe-CF3, and N-Acetyl-L-Leu-L-Phe-CF3. The D/H fractionation factors (phi) for the hydrogen in the H-bond between His 57 and Asp 102 (His 57-Hdelta1) in these four complexes at 5 degreesC were in the range phi = 0.32-0.43, expected for a low-barrier hydrogen bond. For this series of complexes, measurements also were made of the chemical shifts of His 57-Hepsilon1 (delta2,2-dimethylsilapentane-5-sulfonic acid 8.97-9. 18), the exchange rate of the His 57-Hdelta1 proton with bulk water protons (284-12.4 s-1), and the activation enthalpies for this hydrogen exchange (14.7-19.4 kcal.mol-1). It was found that the previously noted correlations between the inhibition constants (Ki 170-1.2 microM) and the chemical shifts of His 57-Hdelta1 (delta2, 2-dimethylsilapentane-5-sulfonic acid 18.61-18.95) for this series of peptidyl trifluoromethyl ketones with chymotrypsin [Lin, J., Cassidy, C. S. & Frey, P. A. (1998) Biochemistry 37, 11940-11948] could be extended to include the fractionation factors, hydrogen exchange rates, and hydrogen exchange activation enthalpies. The results support the proposal of low barrier hydrogen bond-facilitated general base catalysis in the addition of Ser 195 to the peptidyl carbonyl group of substrates in the mechanism of chymotrypsin-catalyzed peptide hydrolysis. Trends in the enthalpies for hydrogen exchange and the fractionation factors are consistent with a strong, double-minimum or single-well potential hydrogen bond in the strongest complexes. The lifetimes of His 57-Hdelta1, which is solvent shielded in these complexes, track the strength of the hydrogen bond. Because these lifetimes are orders of magnitude shorter than those of the complexes themselves, the enzyme must have a pathway for hydrogen exchange at this site that is independent of dissociation of the complexes.

Energetic components of the allosteric machinery in hemoglobin measured by hydrogen exchange

A hydrogen exchange (HX) functional labeling method was used to study allosterically active segments in human hemoglobin (Hb) at the α-chain N terminus and the β-chain C terminus. Allosterically important interactions that contact these segments were removed one or more at a time by mutation (Hbs Cowtown, Bunbury, Barcelona, Kariya), proteolysis (desArg141α, desHis146β), chemical modification (N-ethylsuccinimidyl-Cys93β), and the withdrawal of extrinsic effectors (phosphate groups, chloride). The effects of each modification on HX rate at the local and the remote position were measured in the deoxy Hb T-state and translated into change in structural free energy at each position.The removal of individual salt links destabilizes local structure by 0.4 to 0.75 kcal/mol (pH 7.4, 0°C, 0.35 M ionic strength) and often produces cross-subunit effects while hemoglobin remains in the T-state. In doubly modified hemoglobins, different changes that break the same links produce identical destabilization, changes that are structurally independent show energetic additivity, and changes that intersect show energetic overlap. For the overall T-state to R-state transition and for some but not all modifications within the T-state, the summed loss in stabilization free energy measured at the two chain termini matches the total loss in allosteric free energy measured by global methods. These observations illustrate the importance of evaluating the detailed energetics and the modes of energy transfer that define the allosteric machinery.

An isoleucine zipper peptide forms a native-like triple stranded coiled coil in solution.

Recent studies in the field of de novo protein design have focused on the construction of native-like structures. Here we describe the design and characterization of an isoleucine zipper peptide intended to form a parallel triple-stranded coiled coil. To obtain the native-like structural uniqueness, the hydrophobic interface of the peptide consists of beta-branched Ile residues for complementary side chain packing. The peptide forms a stable triple-stranded coiled coil, as determined by circular dichroism and sedimentation equilibrium analyses. A fluorescence quenching assay after the incorporation of acridine revealed a parallel orientation of the peptides. The structural uniqueness of the coiled coil was confirmed by proton-deuterium amide hydrogen exchange and hydrophobic dye binding. The peptide contains amide protons with hydrogen exchange rates that are approximately an order of magnitude slower than those expected if the exchange occurred via global unfolding. A hydrophobic dye does not bind to the peptide. These results strongly suggest that the peptide folds into a well-packed structure that is very similar to the native state of a natural protein. Thus, Ile residues in the hydrophobic interface can improve the side chain packing, which can impart native-like structural uniqueness to the designed coiled coil.

Hydrogen exchange reactions in supercritical media monitored by in situ NMR

Well established, simple NMR techniques are applied to the in situ study of hydrogen exchange reactions in supercritical aqueous solutions and the supercritical alcohols methanol and ethanol. In the case of the rapidly exchanging hydroxyl protons of alcohols and water, changes in the exchange rate are evidenced by the collapse of chemical shift differences (in solutions) and the collapse of J-multiplets. We show that the hydrogen exchange rate can be adjusted by changing pressure (density) at a constant supercritical temperature. Higher pressure at constant temperature increases the exchange rate, indicating that hydrogen exchange at supercritical conditions proceeds via charged intermediates. The pure alcohols at temperatures above 200°C exhibit exchange of hydroxyl protons at a rate of 5 s −1 already at low, gas-like densities. However, the hydrogen exchange rate in the pure and aqueous alcohols is found to be very sensitive to the presence of impurities. The much slower deuteration of aliphatic and aromatic protons in 4-ethylphenol by supercritical D 2O was monitored in situ from the time dependence of the resonance intensities. Suitable pressure–temperature conditions are identified for the purpose of selective deuteration of 4-ethylphenol. The corresponding reaction rates are reported.

Stabilizing Effect of a Multiple Salt Bridge in a Prenucleated Peptide

The free energy contribution of salt bridges to protein stability remains a controversial issue. The role of single salt bridges has been investigated in protein and peptide studies; there is no reported value of a multiple salt bridge employing peptide models. We have designed a de novo peptide model to evaluate the strength of a multiple salt bridge. Measurement of the rate of hydrogen exchange (HX) of the backbone NH groups in α-helical peptides allows determination of the absolute free energy of individual interactions that participate in helix stabilization. We apply this procedure here to evaluate the stabilizing contribution of a multiple salt bridge involving Glu3, Asp4, and Arg7 in an 11-mer peptide. The peptide is prenucleated by a lactam bridge connecting two side chains spaced four residues apart. Measurement of the HX rates of each NH group in this peptide as a function of pH, together with the pH independent rate of a Gln3 and Asn4 analogue, reveals a substantial favorable contribution to th...

Stopped‐flow NMR measurement of hydrogen exchange rates in reduced horse cytochrome c under strongly destabilizing conditions

Stopped‐flow NMR measurement of hydrogen exchange rates in reduced horse cytochrome c under strongly destabilizing conditions

Stopped-flow NMR measurement of hydrogen exchange rates in reduced horse cytochromec under strongly destabilizing conditions

A procedure to measure exchange rates of fast exchanging protein amide hydrogens by time-resolved NMR spectroscopy following in situ initiation of the reaction by diluting a native protein solution into an exchanging deuterated buffer is described. The method has been used to measure exchange rates of a small set of amide hydrogens of reduced cytochrome c, maintained in a strictly anaerobic atmosphere, in the presence of an otherwise inaccessible range of guanidinium deuterochloride concentrations. The results for the measured protons indicate that hydrogen exchange in the unfolding transition region of cytochrome c reach the EX2 limit, but emphasize the difficulty in interpretation of the exchange mechanism in protein hydrogen exchange studies. Comparison of free energies of structure opening for the measured hydrogens with the global unfolding free energy monitored by far-UV CD measurements has indicated the presence of at least one partially unfolded equilibrium species of reduced cytochrome c. The results provide the first report of measurement of free energy of opening of structure to exchange in the 0-2-kcal/mol range.

Potentiometric and NMR spectroscopic study of protonations and amide hydrogen exchange rates of DTPA-bis(butylamide), DTPA-bis(glucamide), and their lanthanide(III) complexes

The macroscopic and microscopic protonations of DTPA-GlucA 2 (DTPA-bis(glucamide)) have been investigated using potentiometry, 1H and 13C NMR. The protonation behavior appears to be similar to that of the corresponding bis(alkylamides), showing that it is not affected by the presence of the polyhydroxy side chains. Consideration of the various possible protonation pathways leads to the conclusion that the differences in basicity of the amino functions in DTPA and DTPA-bis(amides) result in different protonation sequences of these ligands, which is reflected in the macroscopic protonation constants. The significance for the design of DTPA-based contrast agents for MRI is discussed. Exchange rates of the amide NH of this compound and that of DTPA-bis(butylamide) (DTPA-BuA 2) were determined via longitudinal relaxation rate measurements of the amide 1H resonances in H 2OD 2O (9:1) as solvent. The reaction is strongly base catalyzed and the rate increases substantially upon coordination of the DTPA-bis(amide) by La(III).

TIMP-1 contact sites and perturbations of stromelysin 1 mapped by NMR and a paramagnetic surface probe.

Surfaces of the 173 residue catalytic domain of human matrix metalloproteinase 3 (MMP-3(DeltaC)) affected by binding of the N-terminal, 126 residue inhibitory domain of human TIMP-1 (N-TIMP-1) have been investigated using an amide-directed, NMR-based approach. The interface was mapped by a novel method that compares amide proton line broadening by paramagnetic Gd-EDTA in the presence and absence of the binding partner. The results are consistent with the X-ray model of the complex of MMP-3(DeltaC) with TIMP-1 (Gomis-Rüth et al. (1997) Nature 389, 77-81). Residues Tyr155, Asn162, Val163, Leu164, His166, Ala167, Ala169, and Phe210 of MMP-3(DeltaC) are protected from broadening by the Gd-EDTA probe by binding to N-TIMP-1. N-TIMP-1-induced exposure of backbone amides of Asp238, Asn240, Gly241, and Ser244 of helix C of MMP-3(DeltaC) to Gd-EDTA confirms that the displacement of the N-terminus of MMP-3(DeltaC) occurs not only in the crystal but also in solution. These results validate comparative paramagnetic surface probing as a means of mapping protein-protein interfaces. Novel N-TIMP-1-dependent changes in hydrogen bonding near the active site of MMP-3(DeltaC) are reported. N-TIMP-1 binding causes the amide of Tyr223 of MMP-3(DeltaC) bound by N-TIMP-1 to exchange with water rapidly, implying a lack of the hydrogen bond observed in the crystal structure. The backbone amide proton of Asn162 becomes protected from rapid exchange upon forming a complex with N-TIMP-1 and could form a hydrogen bond to N-TIMP-1. N-TIMP-1 binding dramatically increases the rate of amide hydrogen exchange of Asp177 of the fifth beta strand of MMP-3(DeltaC), disrupting its otherwise stable hydrogen bond.

Local dynamics and stability of apocytochrome b562 examined by hydrogen exchange.

Cytochrome b562 is a heme-binding protein consisting of four helices folded into a classic helix bundle motif. Though retaining much of the topology of the holoprotein, apocytochrome b562 displays physical features commonly associated with so-called protein molten globules. Here, the stability and dynamics of this "structured" molten globule are probed by examination of the dependence of its hydrogen exchange behavior upon the presence of a chemical denaturant. Compared to other systems studied in this manner, apocytochrome b562 displays a limited dynamic range of hydrogen exchange rates and the analysis required the development of a quantitative approach. The protein is found to have three regions of subglobal cooperative stability. The most stable region, or core, is composed of the central two helices of the bundle, with the N- and C-terminal helices being of independent and lower stability. The dependence of the global unfolding free energy upon denaturant concentration indicates the applicability of a binding model and explains the observed difference between global unfolding free energies obtained by the linear extrapolation method and those obtained by calorimetry and hydrogen exchange. These observations place a significant restraint upon the type of folding pathway that is operative for this protein and suggest that that the N- and C-terminal helices fold and unfold independently of the core of the molecule.

Effect of osmolytes on the exchange rates of backbone amide protons in proteins.

Osmolytes are small organic solutes produced by the cells of all organisms (except halobacteria) in high stress situations (e.g. extremes of salt concentration, high temperature, etc.) to stabilize their macromolecules and so conserve biological activity. They do not interact with the macromolecule directly but act by altering the solvent properties in the cellular environment, and so their presence indirectly modifies the stability of proteins. In this paper we examine the effect of a model osmolyte, glycine, on the stabilization of two proteins, chymotrypsin inhibitor 2 and horse heart cytochrome c. We have used NMR to monitor the effect of this osmolyte on amide hydrogen exchange rates, which allows a probe at discrete points within the protein structure. Hydrogen exchange rates of specific backbone amide protons provide information about the localized structural fluctuations that expose these amides to solvent and allow exchange to take place. We find that the presence of a high concentration of glycine osmolyte has a profound stabilizing effect on the proteins studied, which is accompanied by a large reduction of the exchange rate constants of most slowly exchanging amide protons. The spectra indicate that this arises without significant changes in the three-dimensional structure. However, the effects on individual amide protons within a single protein were not uniform, and a wide variation in the magnitude of the effects was observed. This ranged from no apparent change in the exchange rate, to decreases in the exchange rate constant by over 2 orders of magnitude. The osmolyte appears to alter a number of different processes that contribute to the observed exchange rates, and no simple generalization allows prediction of the extent of stabilization at any individual location. The results are discussed in light of the available structures of the proteins studied.

Molten globule unfolding monitored by hydrogen exchange in urea.

The molten globule state of Escherichia coli ribonuclease H1 was studied by hydrogen exchange in order to understand how the energetics of individual regions react to the presence of denaturant. Hydrogen exchange rates were monitored (1) directly by NMR spectroscopy and (2) indirectly by quenching the exchange process and returning to the native state for NMR detection. Direct hydrogen exchange on the molten globule state demonstrated that the observed protons exchange 3-100-fold more slowly than in an unfolded peptide. The quenched hydrogen exchange experiments were modeled after the recently developed "native state hydrogen exchange" experiment and were carried out as a function of urea concentration. The free energy of hydrogen exchange varied linearly with denaturant concentration for all 29 measurable protons, suggesting that each amide's exchange behavior can be modeled with only one type of opening transition. The free energy of unfolding measured by hydrogen exchange and corresponding m values varied for each residue implying a noncooperative molten globule structure. These results are in contrast to similar exchange experiments on native proteins which generally display more than one type of exchange behavior. The single type of exchange seen in the molten globule is probably due to its larger conformational freedom and noncooperative nature.