Hydrogen Bond Equilibrium Constants of Some Unusual Nucleotide Base Pairs

Abstract

Approximate hydrogen bond association constants were determined for base pairs formed by an adenine derivative and a number of unusual pyrimidine bases. A series is found in which the H-bond strength in the base-pairs varies. In certain cases the H-bond equilibrium constant is larger than in the adenine-thymine pair. Inosine derivatives seem to have a non-negligible chance of replacing guanosine in the guanosine-cytosine pair. Infrared, near-infrared (overtone) and NMR spectra were used to determine the equilibrium constants.     

Observation of H-bond mediated 3hJH2H3 coupling constants across Watson-Crick AU base pairs in RNA

^3hJ_H2H3trans-hydrogen bond scalar coupling constants have been observed for the first time in Watson-Crick AU base pairs in uniformly ¹⁵N-labeled RNA oligonucleotides using a new ^2hJ_NN-HNN-E. COSY experiment. The experiment utilizes adenosine H2 (AH2) for original polarization and detection, while employing ^2hJ_NNcouplings for coherence transfer across the hydrogen bonds (H-bonds). The H3 protons of uracil bases are unperturbed throughout the experiment so that these protons appear as passive spins in E. COSY patterns. ^3hJ_H2H3coupling constants can therefore be accurately measured in the acquisition dimension from the displacement of the E. COSY multiplet components, which are separated by the relatively large ¹J_H3N3coupling constants in the indirect dimension of the two-dimensional experiment. The ^3hJ_H2H3scalar coupling constants determined for AU base pairs in the two RNA hairpins examined here have been found to be positive and range in magnitude up to 1.8 Hz. Using a molecular fragment representation of an AU base pair, density functional theory/finite field perturbation theory (DFT/FPT) methods have been applied to attempt to predict the relative contributions of H-bond length and angular geometry to the magnitude of ^3hJ_H2H3coupling constants. Although the DFT/FPT calculations did not reproduce the full range of magnitude observed experimentally for the ^3hJ_H2H3coupling constants, the calculations do predict the correct sign and general trends in variation in size of these coupling constants. The calculations suggest that the magnitude of the coupling constants depends largely on H-bond length, but can also vary with differences in base pair geometry. The dependency of the ^3hJ_H2H3coupling constant on H-bond strength and geometry makes it a new probe for defining base pairs in NMR studies of nucleic acids.     

Observation of the closing of individual hydrogen bonds during TFE-induced helix formation in a peptide

Helix formation of an S-peptide analog, comprising the first 20 residues of Ribonuclease A and two additional N-terminal residues, was studied by measuring hydrogen bond (H-bond) (h3)J(NC') scalar couplings as a function of 2,2,2-trifluoroethanol (TFE) concentration. The (h3)J(NC') couplings give direct evidence for the closing of individual backbone N-H***O = C H-bonds during the TFE-induced formation of secondary structure. Whereas no (h3)J(NC') correlations could be detected without TFE, alpha-helical (i,i +4) H-bond correlations were observed for the amides of residues A5 to M15 in the presence of TFE. The analysis of individual coupling constants indicates that alpha-helix formation starts at the center of the S-peptide around residue E11 and proceeds gradually from there to both peptide ends as the TFE concentration is increased. At 60% to 90% TFE, well-formed alpha-helical H-bonds were observed for the amides hydrogens of residues K9 to Q13, whereas H-bonds of residues T5 to A8, H14, and M15 are affected by fraying. No intramolecular backbone H-bonds are present at and beyond the putative helix stop signal D16. As the (h3)J(NC') constants represent ensemble averages and the dependence of (h3)J(NC') on H-bond lengths is very steep, the size of the individual (h3)J(NC') coupling constants can be used as a measure for the population of a closed H-bond. These individual populations are in agreement with results derived from the Lifson-Roig theory for coil-to-helix transitions. The present work shows that the closing of individual H-bonds during TFE-induced helix formation can be monitored by changes in the size of H-bond scalar couplings.     

Geometrical correlations useful for design of sequence-specific DNA narrow groove binding ligands

Isohelical geometry of sequence-specific DNA narrow groove binding ligands was analyzed in terms of H-bond donor/acceptor complementarity between the base pair atoms facing into the narrow groove and the corresponding H-bond donating atoms regularly disposed along the ligand molecule. Spatial correlations found in analytical form were applied to analysis of naturally occurring and hypothetical drug molecule structures. For the case of B-like isohelices the permitted values of the distance L0 between each two neighboring H-bond donating atoms of the ligand as well as the bending angle tau 0 of the line subsequently connecting these atoms were estimated as follows: L0 congruent to (5.0 +/- 0.4) A; tau 0 congruent to (26 +/- 2) degrees.     

Intramolecular hydrogen bonding as a determinant of the inhibitory potency of N-unsubstituted imidazole derivatives towards mammalian hemoproteins

Enzymes involved in the mammalian microsomal metabolism of drugs are, in numerous cases, inhibited by compounds bearing an imidazolyl scaffold. However, the inhibition potency is highly dependent upon the accessibility of the imidazolyl nitrogen lone pair. In order to highlight some structural parameters of inhibitors that control this phenomenon, a series of compounds containing a nitrogen unsubstituted imidazolyl moiety with varying degrees of nitrogen lone pair accessibility was tested on human and rat hepatic cytochromes P450 and microperoxidase 8, an enzymatically active peptide derived from cytochrome c. In each case, we have shown that the accessibility of the imidazole lone pair determined the extent of inhibition. Nitrogen accessibility was tuned not only by varying the steric hindrance in the vicinity of the imidazolyl ring but also by modifying its surrounding hydrogen bonding network. Compounds in which there exists intramolecular hydrogen bonding between the imidazole moiety and an H-bond acceptor, such as an appropriately positioned amide carbonyl group, demonstrated enhanced inhibitory effects. Conversely, imidazole moieties which are in proximity to H-bond donors, such as an amide NH group, displayed reduced potency. This trend was observed in cyclo-peptide derivatives in which the intramolecular H-bond network was adjusted through the modification of the stereochemistry of a dehydrohistidine residue. It was observed that (Z)-isomers weakly bind heme, whereas (E)-isomers demonstrated higher degrees of metal binding. Therefore, enzymatic inhibition of heme-containing proteins by compounds bearing a dehydrohistidine motif seems to be closely related to its stereochemistry and hydrogen binding propensity. At neutral pH, these differences in binding affinities can be confidently attributed to the ambident H-bond properties of imidazole nitrogen atoms. This structure-activity relationship may be of use for the design of novel imidazolyl compounds as new P450 inhibitors or drug candidates.     

CH...O hydrogen bonds at protein-protein interfaces

For the first time, a statistical potential has been developed to quantitatively describe the CH.O hydrogen bonding interaction at the protein-protein interface. The calculated energies of the CH.O pair interaction show a favorable valley at approximately 3.3 A, exhibiting a feature typical of an H-bond and similar to the ab initio quantum calculation result (Scheiner, S., Kar, T., and Gu, Y. (2001) J. Biol. Chem. 276, 9832-9837). The potentials have been applied to a set of 469 protein-protein complexes to calculate the contribution of different types of interactions to each protein complex: the average energy contribution of a conventional H-bond is approximately 30%; that of a CH.O H-bond is 17%; and that of a hydrophobic interaction is 50%. In some protein-protein complexes, the contribution of the CH.O H-bond can reach as high as approximately 40-50%, indicating the importance of the CH.O H-bond at the protein interface. At the interfaces of these complexes, C(alpha)H.O H-bonds frequently occur between adjacent strands in both parallel and antiparallel orientations, having the obvious structural motif of bifurcated H-bonds. Our study suggests that the weak CH.O H-bond makes an important contribution to the association and stability of protein complexes and needs more attention in protein-protein interaction studies.     

DNA-sequence recognition by CAP: role of the adenine N6 atom of base pair 6 of the DNA site.

Two similar, but not identical, models have been proposed for the amino acid-base pair contacts in the CAP-DNA complex ('Model I,' Weber, I. and Steitz, T., Proc. Natl. Acad. Sci. USA, 81, 3973-3977, 1984; 'Model II,' Ebright, et al., Proc. Natl. Acad. Sci. USA, 81, 7274-7278, 1984). One difference between the two models involves Glu181 of CAP. Model I predicts that Glu181 of CAP makes two specificity determining contacts: one H-bond with the cytosine N4 atom of G:C at base pair 7 of the DNA half site, and one H-bond with the adenine N6 atom of T:A at base pair 6 of the DNA half site. In contrast, Model II predicts that Glu181 makes only one specificity determining contact: one H-bond with the cytosine N4 atom of G:C at base pair 7 of the DNA half site. In the present work, we show that replacement of T:A at base pair 6 of the DNA half site by T:N6-methyl-adenine has no, or almost no, effect on the binding of CAP. We conclude, contrary to Model I, that Glu181 of CAP makes no contact with the adenine N6 atom of base pair 6 of the DNA half site.     

Atomic dissection of the hydrogen bond network for transition-state analogue binding to purine nucleoside phosphorylase

Immucillin-H (ImmH) and immucillin-G (ImmG) were previously reported as transition-state analogues for bovine purine nucleoside phosphorylase (PNP) and are the most powerful inhibitors reported for the enzyme (K(i) = 23 and 30 pM). Sixteen new immucillins are used to probe the atomic interactions that cause tight binding for bovine PNP. Eight analogues of ImmH are identified with equilibrium dissociation constants of 1 nM or below. A novel crystal structure of bovine PNP-ImmG-PO(4) is described. Crystal structures of ImmH and ImmG bound to bovine PNP indicate that nearly every H-bond donor/acceptor site on the inhibitor is fully engaged in favorable H-bond partners. Chemical modification of the immucillins is used to quantitate the energetics for each contact at the catalytic site. Conversion of the 6-carbonyl oxygen to a 6-amino group (ImmH to ImmA) increases the dissociation constant from 23 pM to 2.6 million pM. Conversion of the 4'-imino group to a 4'-oxygen (ImmH to 9-deazainosine) increases the dissociation constant from 23 pM to 2.0 million pM. Substituents that induce small pK(a) changes at N-7 demonstrate modest loss of affinity. Thus, 8-F or 8-CH(3)-substitutions decrease affinity less than 10-fold. But a change in the deazapurine ring to convert N-7 from a H-bond donor to a H-bond acceptor (ImmH to 4-aza-3-deaza-ImmH) decreases affinity by >10(7). Introduction of a methylene bridge between 9-deazahypoxanthine and the iminoribitol (9-(1'-CH(2))-ImmH) increased the distance between leaving and oxacarbenium groups and increased K(i) to 91 000 pM. Catalytic site energetics for 20 substitutions in the transition-state analogue are analyzed in this approach. Disruption of the H-bond pattern that defines the transition-state ensemble leads to a large decrease in binding affinity. Changes in a single H-bond contact site cause up to 10.1 kcal/mol loss of binding energy, requiring a cooperative H-bond pattern in binding the transition-state analogues. Groups involved in leaving group activation and ribooxacarbenium ion stabilization are central to the H-bond network that provides transition-state stabilization and tight binding of the immucillins.     

Structure of the 2-aminopurine-cytosine base pair formed in the polymerase active site of the RB69 Y567A-DNA polymerase

The adenine base analogue 2-aminopurine (2AP) is a potent base substitution mutagen in prokaryotes because of its enhanceed ability to form a mutagenic base pair with an incoming dCTP. Despite more than 50 years of research, the structure of the 2AP-C base pair remains unclear. We report the structure of the 2AP-dCTP base pair formed within the polymerase active site of the RB69 Y567A-DNA polymerase. A modified wobble 2AP-C base pair was detected with one H-bond between N1 of 2AP and a proton from the C4 amino group of cytosine and an apparent bifurcated H-bond between a proton on the 2-amino group of 2-aminopurine and the ring N3 and O2 atoms of cytosine. Interestingly, a primer-terminal region rich in AT base pairs, compared to GC base pairs, facilitated dCTP binding opposite template 2AP. We propose that the increased flexibility of the nucleotide binding pocket formed in the Y567A-DNA polymerase and increased "breathing" at the primer-terminal junction of A+T-rich DNA facilitate dCTP binding opposite template 2AP. Thus, interactions between DNA polymerase residues with a dynamic primer-terminal junction play a role in determining base selectivity within the polymerase active site of RB69 DNA polymerase.     

Heat transitions in DNA molecule. The life-time of excited h-b-1 hydrogen bond in paired guanine-cytosine bases

A theory of radiationless transitions of protons of the hydrogen bonds between the paired DNA bases is constructed. The lifetime of excited h-b-1 bond in guanine-cytosine pair is found to be 4.6 x 10(-9) sec. The dependence of frequencies, amplitudes and normal coordinates of heavy atoms (N, O) on the state of H-bond (ground or excited) are taken into account.     

Interaction of nucleic acid bases with water molecules and formation of mismatched nucleotide pairs

The possibility of the inclusion of water molecules in the formation of mismatched nucleotide pairs was considered in relation to the mechanisms of point errors in template directed biosynthesis. Calculations of the intermolecular interaction energy for systems containing two bases and one water molecule were carried out by the method of atom-atom potential functions. There exist energy minima for each base pair, corresponding to a single N--H...O or N--H...N H-bond between the bases and H-bonding of the water molecule with both bases. The relative positions of glycosyl bonds in some of these minima are closer to those for Watson--Crick pairs, than the positions of minima for these pairs without water. For other minima, the H-bond formation between the water molecule and the two bases additionally stabilizes the relative base position in wobble-pairs with two H-bonds between the bases. The base and water positions in energy minima are compared with the positions in some pairs proposed on the basis of NMR and X-ray data for double helical oligonucleotides.     

Conformational effects on the electron-transfer efficiency in peptide foldamers based on alpha,alpha-disubstituted glycyl residues

Peptide foldamers based on alpha,alpha-disubstituted glycyl residues were synthesized and chemically characterized to investigate the effects of the electric field generated by a 3(10)-helix on the rate of intramolecular photoinduced electron-transfer reactions. To this end, two new octapeptides having identical sequences were suitably side-chain functionalized with the same electron-transfer donor-acceptor pair, but inverting the position of the pair along the main chain. The electron-transfer rate constants, measured by time-resolved spectroscopy techniques (nanosecond transient absorption and time-resolved fluorescence), indicated that, in the case of the 3(10)-helix, the electrostatic effect is significant, but smaller than that obtained for alpha-helical peptides. This finding can be likely ascribed to the distortion of the H-bond network with respect to the helical axis taking place in the former secondary structure. Overall, these results could have implications on electron-transfer phenomena in model and biomembranes facilitated by peptaibiotics.     

Effects of mutations and truncations on the kinetic behavior of IIAGlc, a phosphocarrier and regulatory protein of the phosphoenolpyruvate phosphotransferase system of Escherichia coli

IIAGlc, a component of the glucose-specific phosphoenolpyruvate:phosphotransferase system (PTS) of Escherichia coli, is important in regulating carbohydrate metabolism. In Glc uptake, the phosphotransfer sequence is: phosphoenolpyruvate --> Enzyme I --> HPr --> IIAGlc --> IICBGlc --> Glc. (HPr is the first phosphocarrier protein of the PTS.) We previously reported two classes of IIAGlc mutations that substantially decrease the P-transfer rate constants to/from IIAGlc. A mutant of His75 which adjoins the active site (His90), (H75Q), was 0.5% as active as wild-type IIAGlc in the reversible P-transfer to HPr. Two possible explanations were offered for this result: (a) the imidazole ring of His75 is required for charge delocalization and (b) H75Q disrupts the hydrogen bond network: Thr73, His75, phospho-His90. The present studies directly test the H-bond network hypothesis. Thr73 was replaced by Ser, Ala, or Val to eliminate the network. Because the rate constants for phosphotransfer to/from HPr were largely unaffected, we conclude that the H-bond network hypothesis is not correct. In the second class of mutants, proteolytic truncation of seven residues of the IIAGlc N terminus caused a 20-fold reduction in phosphotransfer to membrane-bound IICBGlc from Salmonella typhimurium. Here, we report the phosphotransfer rates between two genetically constructed N-terminal truncations of IIAGlc (Delta7 and Delta16) and the proteins IICBGlc and IIBGlc (the soluble cytoplasmic domain of IICBGlc). The truncations did not significantly affect reversible P-transfer to IIBGlc but substantially decreased the rate constants to IICBGlc in E. coli and S. typhimurium membranes. The results support the hypothesis (Wang, G., Peterkofsky, A., and Clore, G. M. (2000) J. Biol. Chem. 275, 39811-39814) that the N-terminal 18-residue domain "docks" IIAGlc to the lipid bilayer of membranes containing IICBGlc.     

Conformational analysis of biologically active thyroliberin analogs by two-dimensional NMR spectroscopy]

Preferable conformations of thyrotropin-releasing hormone (TRH, Glp-His-Pro-NH2) and its analogues Glp-Glu(R)-Pro-NH2 (R = NHCH(CH3)CH2Ar), Glp-Gln-Abu-NH2, Dho-Gln-Abu-NH2 in DMSO solution are determined using two-dimensional 1H NMR spectroscopy (delta-J-correlated, COSY and NOESY). Torsion angles psi i and chi i for every amino acid were calculated on the basis of the spin-spin coupling constants 3JNH-C alpha H and 3JC alpha H-C beta H values. The NOESY data were used for selecting the peptide conformations realized in solution. Distances between protons interacting by the dipole mechanism (d-contacts) were calculated using NOE values. These experiments allow one to estimate the torsion angles psi (between C alpha H-CO). TRH has an intramolecular H-bond between NH2-protons and His carbonyl with the torsion angles omega 3 = 180 degrees and psi 3 = 0 degrees. It is formation of this H-bond that apparently promotes the domination of the trans configuration of the His-Pro peptide bond. An intramolecular NH2-C alpha CO (Glp) H-bonding is revealed in other investigated compounds. It is known that a similar conformation of the TRH is realized in the course of its interaction with receptor.     

诺氟沙星与牛血清白蛋白相互作用的研究

用荧光光谱法研究诺氟沙星与牛血清白蛋白之间的结合作用,确定了诺氟沙星与牛血清白蛋白的荧光猝灭机制为静态猝灭。通过测定和计算不同温度下该结合反应的结合常数和结合位点数,并根据热力学方程求得了结合反应的热力学参数,讨论了两者问的主要作用力类型是范德华力和氢键。同时采用同步荧光技术考察了诺氟沙星对BSA构象的影响。并从荧光寿命进一步证明诺氟沙星与牛血清白蛋白的荧光猝灭机制为静态猝灭。     

Thermodynamics and kinetics for base-pair opening in the P1 duplex of the Tetrahymena group I ribozyme

The thermodynamics and kinetics for base-pair opening of the P1 duplex of the Tetrahymena group I ribozyme were studied by NMR hydrogen exchange experiments. The apparent equilibrium constants for base pair opening were measured for most of the imino protons in the P1 duplex using the base catalysts NH3, HPO4(2-) or TRIS. These equilibrium constants were also measured for several modified P1 duplexes, and the C-2.G23 base pair was the most stable base pair in all the duplexes. The conserved U-1*G22 base pair is required for activity of the ribozyme and the data here show that this wobble base pair destabilizes neighboring base pairs on only one side of the wobble. A 2'-OMe modification on the U-3 residue stabilized its own base pair but had little effect on the neighboring base pairs. Three base pairs, U-1*G22, C-2*G23 and A2*U21 showed unusual equilibrium constants for opening and possible implications of the opening thermodynamics of these base pairs on the undocking rates of the P1 helix with catalytic core are discussed.     

Presence versus absence of hydrogen bond donor Tyr-39 influences interactions of cationic trypsin and mesotrypsin with protein protease inhibitors

Mesotrypsin displays unusual resistance to inhibition by polypeptide trypsin inhibitors and cleaves some such inhibitors as substrates, despite a high degree of conservation with other mammalian trypsins. Substitution of Arg for the generally conserved Gly-193 has been implicated as a critical determinant of the unusual behavior of mesotrypsin toward protein protease inhibitors. Another relatively conserved residue near the trypsin active site, Tyr-39, is substituted by Ser-39 in mesotrypsin. Tyr-39, but not Ser-39, forms a hydrogen bond with the main chain amide nitrogen of the P₄′ residue of a bound protease inhibitor. To investigate the role of the Tyr-39 H-bond in trypsin-inhibitor interactions, we reciprocally mutated position 39 in mesotrypsin and human cationic trypsin to Tyr-39 and Ser-39, respectively. We assessed inhibition constants and cleavage rates of canonical protease inhibitors bovine pancreatic trypsin inhibitor (BPTI) and the amyloid precursor protein Kunitz protease inhibitor domain by mesotrypsin and cationic trypsin variants, finding that the presence of Ser-39 relative to Tyr-39 results in a 4- to 13-fold poorer binding affinity and a 2- to 18-fold increase in cleavage rate. We also report the crystal structure of the mesotrypsin-S39Y•BPTI complex, in which we observe an H-bond between Tyr-39 OH and BPTI Ile-19 N. Our results indicate that the presence of Ser-39 in mesotrypsin, and corresponding absence of a single H-bond to the inhibitor backbone, makes a small but significant functional contribution to the resistance of mesotrypsin to inhibition and the ability of mesotrypsin to proteolyze inhibitors.     

Characterization of the trans Watson-Crick GU base pair located in the catalytic core of the antigenomic HDV ribozyme

The HDV ribozyme's folding pathway is, by far, the most complex folding pathway elucidated to date for a small ribozyme. It includes 6 different steps that have been shown to occur before the chemical cleavage. It is likely that other steps remain to be discovered. One of the most critical of these unknown steps is the formation of the trans Watson-Crick GU base pair within loop III. The U(23) and G(28) nucleotides that form this base pair are perfectly conserved in all natural variants of the HDV ribozyme, and therefore are considered as being part of the signature of HDV-like ribozymes. Both the formation and the transformation of this base pair have been studied mainly by crystal structure and by molecular dynamic simulations. In order to obtain physical support for the formation of this base pair in solution, a set of experiments, including direct mutagenesis, the site-specific substitution of chemical groups, kinetic studies, chemical probing and magnesium-induced cleavage, were performed with the specific goal of characterizing this trans Watson-Crick GU base pair in an antigenomic HDV ribozyme. Both U(23) and G(28) can be substituted for nucleotides that likely preserve some of the H-bond interactions present before and after the cleavage step. The formation of the more stable trans Watson-Crick base pair is shown to be a post-cleavage event, while a possibly weaker trans Watson-Crick/Hoogsteen interaction seems to form before the cleavage step. The formation of this unusually stable post-cleavage base pair may act as a driving force on the chemical cleavage by favouring the formation of a more stable ground state of the product-ribozyme complex. To our knowledge, this represents the first demonstration of a potential stabilising role of a post-cleavage conformational switch event in a ribozyme-catalyzed reaction.     

Tyrosine hydrogen-bonding and environmental effects in proteins probed by ultraviolet resonance Raman spectroscopy

Ultraviolet resonance Raman spectra with 229-nm excitation are reported for aqueous tyrosine and for ovomucoid third domain proteins from chicken [OMCHI3(-)] and from chachalaca [OMCHA(-)], as well as alpha 1-, alpha 2-, and beta-purothionin. At this excitation wavelength interference from phenylalanine is minimized, and it is possible to determine the frequencies of the Tyr ring modes nu 8a and nu 8b. The nu 8b frequency decreases with the degree of Tyr H-bond donation, reaching a limiting value for deprotonated tyrosine. This spectroscopic indicator of H-bond strength was calibrated by using the model compound p-cresol in H-bond acceptor solutions for which the enthalpy of H-bond formation can be obtained from the literature. With this calibration it is possible to estimate Tyr H-bond enthalpies in proteins for which Tyr is a H-bond donor; values of 13.7, 9.6, and 11.2 kcal/mol were found for OMCHA3(-) and for alpha 1- (or alpha 2-) and beta-purothionin, respectively. The intensity of the 1176-cm-1 nu 9a band of Tyr excited at 229 nm and also the intensity ratio of the Tyr 830/850-cm-1 Fermi doublet excited at 200 nm both correlate strongly with the estimated H-bond enthalpies, but large deviations are seen for the purothionins, reflecting a special environment for the Tyr residue of these proteins, which is believed to be constrained in a hydrophobic pocket. The molar intensity of the strong approximately 1000-cm-1 nu 12 band of phenylalanine in aqueous solution is about half the value observed in most proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantification of H/D Isotope Effects on Protein Hydrogen-bonds by h3JNC′ and 1JNC′ Couplings and Peptide Group 15N and 13C′ Chemical Shifts

The effect of hydrogen/deuterium exchange on protein hydrogen bond coupling constants (h3)J(NC') has been investigated in the small globular protein ubiquitin. The couplings across deuterated or protonated hydrogen bonds were measured by a long-range quantitative HA(CACO)NCO experiment. The analysis is combined with a determination of the H(N)/D(N) isotope effect on the amide group (1)J(NC') couplings and the (15)N and (13)C' chemical shifts. On average, H-bond deuteration exchange weakens (h3)J(NC') and strengthens (1)J(NC') couplings. A correlation is found between the size of the (15)N isotope shift, the (15)N chemical shift, and the (h3)J(NC') coupling constants. The data are consistent with a reduction of donor-acceptor overlap as expected from the classical Ubbelohde effect and the common understanding that H(N)/D(N) exchange leads to a shortening of the N-hydron bond length.