High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. I. The hydrogen bonded protons of the "charge relay" system

High resolution proton nuclear magnetic resonance has been used to observe protons at the active site of chymotrypsin A_δ and at the same region of chymotrypsinogen A. A single resonance with the intensity of one proton is located in the low field region of the nuclear magnetic resonance spectrum. This resonance is observed in H₂O solutions but not in ²H₂O. On going from low to high pH the resonance titrates upfield 3 parts per million in both proteins and has a pK of 7.5. The titration can be prevented by alkylating His57 with either of two active site directed chloromethyl ketones. Using these data the proton resonance has been assigned to a proton in a hydrogen bond between His57 and Asp102. Further confirmation of this assignment lies in the observation of a similar resonance in this same low field region of the nuclear magnetic resonance spectrum of trypsin, trypsinogen, subtilisin BPN′ and α-lytic protease all of which have the Asp-His-Ser triad at their active sites.This proton resonance in chymotrypsin A_δ was used as a probe to monitor the charge state of the active site upon formation of a stable acyl-enzyme analogue N²(N-acetylalanyl)-N¹benzoylcarbazoyl-chymotrypsin A_δ. In this derivative the His-Asp proton resonance titrates from the same low pH end point as in the native enzyme, ?18 parts per million, to a new high pH end point of ?14.4 parts per million (versus ?15.0 parts per million in the native enzyme). The difference of 0.6 parts per million in the high pH end points between the native and acyl enzyme is interpreted as supporting the suggestion that a hydrogen bond exists between Ser195 and His57 in the native enzyme and zymogen.We conclude from these studies that the charge relay system from Asp102 across His57 to Ser195 is intact in chymotrypsin A_δ and chymotrypsinogen A, and that, in the native enzyme, it slightly polarizes Ser195.     

Formation of intermolecular and intramolecular hydrogen bonds in histidine-binding protein J of Salmonella typhimurium upon binding L-histidine. A proton nuclear magnetic resonance study

Histidine-binding protein J of Salmonella typhimurium has been chosen as a model system for a proton nuclear magnetic resonance spectroscopic investigation of binding protein-ligand interaction. This interaction is involved in the recognition step of the osmotic shock-sensitive active transport systems. When J protein binds L-histidine, four new, low-field, exchangeable proton resonances appear in the region +7 to +12 parts per million downfield from the water proton resonance (or +11.7 to +16.7 parts per million downfield from the methyl proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate). Due to their chemical shift range and other properties, they indicate the formation of both intra- and intermolecular hydrogen bonds. Experiments with 15N-labeled compounds confirm this conclusion. The specificity of the hydrogen-bond formation is demonstrated by observing the effects of substrate analogs, temperature, pH, and mutations on the exchangeable proton resonances. Proton-proton nuclear Overhauser effect measurements suggest that two of these exchangeable proton resonances (at +7.2 and +10.6 parts per million from H2O) are most likely from intramolecular hydrogen-bonded protons, while the other two (at +7.1 and +9.5 parts per million from H2O) are intermolecular hydrogen bonds. Our finding of L-histidine-induced hydrogen-bond formation in histidine-binding protein J in the solution state is an excellent demonstration of the production of specific conformational changes in a periplasmic binding protein upon binding of ligand.     

High resolution nuclear magnetic resonance study of the histidine--aspartate hydrogen bond in chymotrypsin and chymotrypsinogen

A high resolution proton nuclear magnetic resonance study of chymotrypsin A_δ and Chymotrypsinogen A in water has shown a single resonance at very low magnetic fields (− 18 to − 15 p.p.m. relative to dimethyl-silapentane-sulfonate). From its pH dependence (pK = 7·2) and response to chemical modification the resonance has been assigned to the hydrogen-bonded proton between His-57 and Asp-102.     

Proton nuclear magnetic resonance studies on glutamine-binding protein from Escherichia coli. Formation of intermolecular and intramolecular hydrogen bonds upon ligand binding

Proton nuclear magnetic resonance studies have revealed several structural and dynamic properties of the glutamine-binding protein of Escherichia coli. When this protein binds L-glutamine, six low-field, exchangeable proton resonances appear in the region from +5.5 to +10 parts per million downfield from water (or +10.2 to +14.7 parts per million downfield from the methyl proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate). This suggests that the binding of L-glutamine induces specific conformational changes in the protein molecule, involving the formation of intermolecular and intramolecular hydrogen bonds between the glutamine-binding protein and L-glutamine, and within the protein molecule. The oxygen atom of the gamma-carbonyl group of L-glutamine is likely to be involved in the formation of an intermolecular hydrogen bond between the ligand and the binding protein. We have shown that at least one phenylalanine and one methyl-containing residue are spatially close to this intermolecular hydrogen-bonded proton. The intermolecular and intramolecular hydrogen-bonded protons of the ligand-protein complex undergo solvent exchange. The local conformations around these intermolecular and intramolecular hydrogen bonds are quite stable when subjected to pH and temperature variations. From these results, the utility of proton nuclear magnetic resonance spectroscopy for investigating such binding proteins has been shown, and a picture of the ligand-binding process can be drawn.     

Assignments of the paramagnetically shifted heme methyl nuclear magnetic resonance peaks of cyanometmyoglobin by selective deuteration

Three of the four paramagnetically shifted heme methyl nuclear magnetic resonance peaks of cyanometmyoglobin could be assigned by comparing the proton nuclear magnetic resonance spectra of myoglobins reconstituted from selectively deuterated hemes. These spectra indicate that the fourth methyl nuclear magnetic resonance peak has to be looked for outside the region ?9 to ?43 parts per million.     

Nitrogen-15 NMR spectroscopy of the catalytic-triad histidine of a serine protease in peptide boronic acid inhibitor complexes

15N NMR spectroscopy was used to examine the active-site histidyl residue of alpha-lytic protease in peptide boronic acid inhibitor complexes. Two distinct types of complexes were observed: (1) Boronic acids that are analogues of substrates form complexes in which the active-site imidazole ring is protonated and both imidazole N-H protons are strongly hydrogen bonded. With the better inhibitors of the class this arrangement is stable over the pH range 4.0-10.5. The results are consistent with a putative tetrahedral intermediate like complex involving a negatively charged, tetrahedral boron atom covalently bonded to O gamma of the active-site serine. (2) Boronic acids that are not substrate analogues form complexes in which N epsilon 2 of the active-site histidine is covalently bonded to the boron atom of the inhibitor. The proton bound to N delta 1 of the histidine in these histidine-boronate adducts remains strongly hydrogen bonded, presumably to the active-site aspartate. Benzeneboronic acid, which falls in this category, forms an adduct with histidine. In both types of complexes the N-H protons of His-57 exchange unusually slowly as evidenced by the room temperature visibility of the low-field 1H resonances and the 15N-H spin couplings. These results, coupled with the kinetic data of the preceding paper [Kettner, C. A., Bone, R., Agard, D. A., & Bachovchin, W. W. (1988) Biochemistry (preceding paper in this issue)], indicate that occupancy of the specificity subsites may be required to fully form the transition-state binding site. The significance of these findings for understanding inhibitor binding and the catalytic mechanism of serine proteases is discussed.     

Ring-current shifts in the 300 MHz nuclear magnetic resonance spectra of six purified transfer RNA molecules

We present the 300 MHz high-resolution proton nuclear magnetic resonance spectra of the ring NH hydrogen-bonded protons of six purified tRNAs. Good agreement was obtained between the observed spectra and those computed on the assumption of the suitable cloverleaf models. In the computation it is assumed that the hydrogen-bonded ring NH in each type of base pair has an intrinsic position with respect to 2,2-dimethyl-2-silapentane-5-sulfonate, i.e. in A·U it is at ?14·8 parts per million, in G·C at ?13·7 parts per million and in A·Ψat ?13·5 parts per million. The shifts of these resonances from these positions are calculated by including ring current fields from the nearest neighbors. The agreement is very good, adding support to our earlier findings that there is no evidence for additional Watson-Crick base pairs detected beyond those in the cloverleaf. In general, resolved resonances are fitted by the computed spectra to within ±0·2 part per million showing that there is no need for any additional physical mechanism to explain the nuclear magnetic resonance positions. Hence, the nuclear magnetic resonance spectra can be interpreted in terms of the structure of their neighbors and in a few important cases this has been particularly valuable in understanding the structure beyond the end of a helical region. In the tRNA^Glu_E.coli′ for example, the positions of the resonances in A·U no. 7 and A·U no. 49 at the interior ends of the acceptor and -T-Ψ-C- stems, respectively, strongly suggest that these two stems are in a continuous helix. Other structural effects at the ends of the helical regions are also suggested by the nuclear magnetic resonance spectra.     

Solution structures of purine base analogues 9-deazaguanine and 9-deazahypoxanthine

Deaza analogues of nucleobases are potential drugs against infectious diseases caused by parasites. A caveat is that apart from binding their target parasite enzymes, they also bind and inhibit enzymes of the host. In order to design derivatives of deaza analogues which specifically bind target enzymes, knowledge of their molecular structure, protonation state, and predominant tautomers at physiological conditions is essential. We have employed resonance Raman spectroscopy at an excitation wavelength of 260 nm, to decipher solution structure of 9-deazaguanine (9DAG) and 9-deazahypoxanthine (9DAH). These are analogues of guanine and hypoxanthine, respectively, and have been exploited to study static complexes of nucleobase binding enzymes. Such enzymes are known to perturb pK_a of their ligands, and thus, we also determined solution structures of these analogues at two, acidic and alkaline, pH. Structure of each possible protonation state and tautomer was computed using density functional theoretical calculations. Species at various pHs were identified based on isotopic shifts in experimental wavenumbers and by comparing these shifts with corresponding computed isotopic shifts. Our results show that at physiological pH, N1 of pyrimidine ring in 9DAG and 9DAH bears a proton. At lower pH, N3 is place of protonation, and at higher pH, deprotonation occurs at N1 position. The proton at N7 of purine ring remains intact even at pH 12.5. We have further compared these results with naturally occurring nucleotides. Our results identify key vibrational modes which can report on hydrogen bonding interactions, protonation and deprotonation in purine rings upon binding to the active site of enzymes.     

Kinetic properties of the binding of alpha-lytic protease to peptide boronic acids

The kinetic parameters for peptide boronic acids in their interaction with alpha-lytic protease were determined and found to be similar to those of other serine proteases [Kettner, C., & Shenvi, A. B. (1984) J. Biol. Chem. 259, 15106-15114]. alpha-Lytic protease hydrolyzes substrates with either alanine or valine in the P1 site and has a preference for substrate with a P1 alanine. The most effective inhibitors are tri- and tetrapeptide analogues that have a -boroVal-OH residue in the P1 site. At pH 7.5, MeOSuc-Ala-Ala-Pro-boroVal-OH has a Ki of 6.4 nM and Boc-Ala-Pro-boroVal-OH has a Ki of 0.35 nM. Ac-boroVal-OH and Ac-Pro-boroVal-OH are 220,000- and 500-fold less effective, respectively, than the tetrapeptide analogue. The kinetic properties of the tri- and tetrapeptide analogues are consistent with the mechanism for slow-binding inhibition, E + I in equilibrium EI in equilibrium EI*, while the less effective inhibitors are simple competitive inhibitors. MeO-Suc-Ala-Ala-Pro-boroAla-OH is a simple competitive inhibitor with a Ki of 67 nM at pH 7.5. Other peptide boronic acids, which are analogues of nonsubstrates, are less effective than substrate analogues but still are effective competitive inhibitors. For example, MeOSuc-Ala-Ala-Pro-boroPhe-OH has a Ki of 0.54 microM although substrates with a phenylalanine in the P1 position are not hydrolyzed. Binding for boronic acid analogues of both substrate and nonsubstrate analogues is pH dependent with higher affinity near pH 7.5. Similar binding properties have been observed for pancreatic elastase. Both enzymes have almost identical requirements for an extended peptide inhibitor sequence in order to exhibit highly effective binding and slow-binding characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Easily polarizable proton transfer hydrogen bonds between the side chains of histidine and the carboxylic acid groups of glutamic and aspartic acid residues in proteins

The nature of the hydrogen bonds formed between glutamic acid and histidine residues between aspartic acid and histidine residues is studied by i.r. spectroscopy. These studies were performed with $\text{(}\text{l}\text{-Glu}\text{)}{}_{\mathrm{n}}\text{+(}\text{l}\text{-His}\text{)}{}_{\mathrm{n}}$ and with associates of monomeric Glu + His and Asp + His systems in solutions whereby these amino acids had protected α-amino and α-carboxylic groups. It is shown that the OH …N?^?O?H⁺N bonds are easily polarizable proton transfer hydrogen bonds. The residence time of the proton at the His is a little larger in the case of the Asp + His than in the case of the Glu + His systems. Polar environments shift this proton transfer equilibrium in favour of the proton limiting structure ^?O?H⁺N, and less polar ones in favour of the structure OH?N. These results demonstrate that the large proton polarizability of the hydrogen bonded system in the active centre of chymotrypsin is responsible for the charge shift caused by the substrate, and thus for the increase in reactivity of the serine residue and the catalytic activity of the enzyme.     

A calorimetric investigation of the binding of indole and phenylethane boronic acid to chymotrypsin

The heat of formation of the chymotrypsin-phenylethane boronic acid complex has been observed calorimetrically from pH 4 to 8 at 25 degrees C and is found to be pH-dependent, changing from near -6 kcal/mol at pH 4 to -13 kcal/mol at pH 8. The heat of formation of the chymotrypsin-indole complex is a nearly constant -6 kcal/mol over most of the same pH range. alpha-Chymotrypsin has been purified by pH gradient elution from an immobilized lima bean inhibitor column. Solutions of the enzyme up to 400 microM, prepared in this manner, have a zero heat of dilution from pH 5 to 8 in 0.1 M KCl, with or without added 0.05 M Tris, N-(tris[hydroxy-methyl]methyl-2-amino) ethanesulfonic acid, 4-morpholineethanesulfonic acid, or acetate buffers. Binding of phenylethane boronic acid causes a pH-dependent decrease in proton binding to chymotrypsin; the decrease in proton binding evoked by formation of the indole complex is much less, with a much smaller pH dependence. The calorimetric and proton-binding results are applied to a model for boronic acid binding (Hanai, K. (1976) J. Biochem. (Tokyo) 79, 107-116). We conclude that the thermodynamics of formation of the trigonal boronic acid complex are quite similar to those for the formation of the noncovalent complex formed by indole and related ligands. The trigonal-tetrahedral tautomerism in the boronic acid-chymotrypsin complex is characterized by thermodynamic changes similar to those accompanying the binding of virtual substrates to chymotrypsin.     

Tautomerism,acid-base equilibria,and H-bonding of the six histidines in subtilisin BPN' by NMR

We have determined by (15)N, (1)H, and (13)C NMR, the chemical behavior of the six histidines in subtilisin BPN' and their PMSF and peptide boronic acid complexes in aqueous solution as a function of pH in the range of from 5 to 11, and have assigned every (15)N, (1)H, C(epsilon 1), and C(delta2) resonance of all His side chains in resting enzyme. Four of the six histidine residues (17, 39, 67, and 226) are neutrally charged and do not titrate. One histidine (238), located on the protein surface, titrates with pK(a) = 7.30 +/- 0.03 at 25 degrees C, having rapid proton exchange, but restricted mobility. The active site histidine (64) in mutant N155A titrates with a pK(a) value of 7.9 +/- 0.3 and sluggish proton exchange behavior, as shown by two-site exchange computer lineshape simulation. His 64 in resting enzyme contains an extremely high C(epsilon 1)-H proton chemical shift of 9.30 parts per million (ppm) owing to a conserved C(epsilon 1)-H(.)O=C H-bond from the active site imidazole to a backbone carbonyl group, which is found in all known serine proteases representing all four superfamilies. Only His 226, and His 64 at high pH, exist as the rare N(delta1)-H tautomer, exhibiting (13)C(delta1) chemical shifts approximately 9 ppm higher than those for N(epsilon 2)-H tautomers. His 64 in the PMSF complex, unlike that in the resting enzyme, is highly mobile in its low pH form, as shown by (15)N-(1)H NOE effects, and titrates with rapid proton exchange kinetics linked to a pK(a) value of 7.47 +/- 0.02.     

Proton relay system in the active site of maltodextrinphosphorylase via hydrogen bonds with large proton polarizability: an FT-IR difference spectroscopy study

Maltodextrinphosphorylase (MDP) was studied in the pH range 5.4–8.4 by Fourier transform infrared (FT-IR) spectroscopy. The pK _a value of the cofactor pyridoxalphosphate (PLP) was found between 6.5 and 7.0, which closely resembles the second pK _a of free PLP. FT-IR difference spectra of the binary complex of MDP+α-d-glucose-1-methylenephosphonate (Glc-1-MeP) minus native MDP were taken at pH 6.9. Following binary complex formation, two Lys residues, tentatively assigned to the active site residues Lys533 and Lys539, became deprotonated, and PLP as well as a carboxyl group, most likely of Glu637, protonated. A system of hydrogen bonds which shows large proton polarizability due to collective proton tunneling was observed connecting Lys533, PLP, and Glc-1-MeP. A comparison with model systems shows, furthermore, that this hydrogen bonded chain is highly sensitive to local electrical fields and specific interactions, respectively. In the binary complex the proton limiting structure with by far the highest probability is the one in which Glc-1-MeP is singly protonated. In a second hydrogen bonded chain the proton of Lys539 is shifted to Glu637. In the binary complex the proton remains located at Glu637. In the ternary complex composed of phosphorylase, glucose-1-phosphate (Glc-1-P), and the nonreducing end of a polysaccharide chain (primer), a second proton may be shifted to the phosphate group of Glc-1-P. In the doubly protonated phosphate group the loss of mesomeric stabilization of the phosphate ester makes the C₁–O₁ bond of Glc-1-P susceptible to bond cleavage. The arising glucosyl carbonium ion will be a substrate for nucleophilic attack by the nonreducing terminal glucose residue of the polysaccharide chain. Received: 15 June 1997 / Revised version: 15 October 1998 / Accepted: 15 October 1998     

Studies on the conformation and interactions of elastin: nuclear magnetic resonance of the polyhexapeptide.

Synthesis, proton magnetic resonance and carbon-13 magnetic resonance characterizations, including complete assignments, are reported for the polyhexapeptide of elastin, HCO-Val(Ala1-Pro2-Gly3-Val4-Gly5-Val6)18-OMe. Temperature dependence of peptide NH chemical shifts and solvent dependence of peptide C-O chemical shifts have been determined in several solvents and have been interpreted in terms of four hydrogen bonded rings for each repeat of the polyhexapeptide. The more stable hydrogen bonded ring is a beta-turn involving Ala1C-O--HN-Val4. More dynamic hydrogen bonds are an 11-atom hydrogen bonded ring Gly3NH--O-C Gly5, a 7-atom hydrogen bonded ring (a gamma-turn) Gly3 C-O--NH-Gly5, and a 23-atom hydrogen bonded ring Val6inH--O-C Val6(i+1). This set of hydrogen bonds results in a right-handed beta-spiral structure with slightly more than two repeats (approximately 2.2) per turn of spiral. The beta-spiral structure is briefly discussed relative to data on the elastic fiber.     

Assignment of the histidine 12-threonine 45 hydrogen-bonded proton in the NMR spectrum of ribonuclease A in H2O.

Described herein are proton nmr experiments on chemically modified derivatives of ribonuclease A designed to elucidate the origin of an exchangeable resonance, assigned previously to a histidine ring N proton that titrates between 11 to 13 ppm with a pK_a of 6.1 in H₂O solution. Histidines 48 and 105, which are distant from the active site, are eliminated as candidates for this resonance from inhibitor binding studies on the enzyme in acetate–water solutions. This exchangeable resonance titrates with modified pK_a's and constant area over the above pH range in His-119-N₁-carboxymethylated-RNase A and des-(121–124)-RNase A, thus eliminating the imidazole N₃ proton in the His 119-Asp 121 hydrogen bond. In His-12-N₁-carboxymethylated-RNase A, this resonance is also observable, but broadens on raising the pH above 7 and at elevated temperatures above neutrality. It exhibits a pH-independent chemical shift characteristic of the protonated state of histidine. On the basis of these findings, this exchangeable resonance, designated a, is assigned to the imidazole N₁ proton of His 12, which is hydrogen-bonded to the carbonyl oxygen of Thr 45 in the crystal.     

The influence of hydrogen bonds on electron transfer rate in photosynthetic RCs

Hydrogen bonds formed between photosynthetic reaction centers (RCs) and their cofactors were shown to affect the efficacy of electron transfer. The mechanism of such influence is determined by sensitivity of hydrogen bonds to electron density rearrangements, which alter hydrogen bonds potential energy surface. Quantum chemistry calculations were carried out on a system consisting of a primary quinone Q_A, non-heme Fe²⁺ ion and neighboring residues_. The primary quinone forms two hydrogen bonds with its environment, one of which was shown to be highly sensitive to the Q_A state. In the case of the reduced primary quinone two stable hydrogen bond proton positions were shown to exist on [Q_A-His^M219] hydrogen bond line, while there is only one stable proton position in the case of the oxidized primary quinone. Taking into account this fact and also the ability of proton to transfer between potential energy wells along a hydrogen bond, theoretical study of temperature dependence of hydrogen bond polarization was carried out. Current theory was successfully applied to interpret dark P⁺/Q_A⁻ recombination rate temperature dependence.     

Nuclear magnetic resonance study of exchangeable protons in ferrocytochrome c

Five well-resolved exchangeable proton resonances have been observed in horse ferrocytochrome c in the low-field region between ?10.0 and ?12.0 ppm. A resonance at ?11.6 ppm is assigned to an amino proton of His 26 on the basis of its magnetic field position, pH dependence and its correlation with histidine at this position in eight species. A resonance at ?10.9 ppm observed in horse and donkey ferrocytochrome c is assigned to a hydrogen-bonded ?-amino proton of Lys 60. This resonance shifts upfield with increasing salt and decreasing pH, with the shifts increasing as one goes from chloride to bromide to iodide. With less assurance, a resonance at ?10.6 ppm is assigned to the indole amino of Trp 59 and a resonance at ?10.3 ppm to the amino proton of the His 18. These resonances have been used to study the binding of small anions to ferrocytochrome c, the results of which show that inorganic phosphate, ADP and ATP all bind in the immediate vicinity of His 26, and that the inorganic phosphate has the greatest effect upon the imidazole amino exchange time.     

The presence of a histidine-aspartic acid pair in the active site of 2-hydroxyacid dehydrogenases. X-ray refinement of cytoplasmic malate dehydrogenase

The structure of cytoplasmic malate dehydrogenase has been partially refined by crystallographic least squares methods. Using x-ray phases based on the refined coordinates, analysis of the resultant electron density maps has led to a new model of cytoplasmic malate dehydrogenase and a tentative "x-ray sequence." The two crystallographically independent subunits comprising the dimeric enzyme are nearly identical in structure and are related to each other by roughly 2-fold rotational symmetry. The best fit of the molecular structure of cytoplasmic malate dehydrogenase to that of lactate dehydrogenase has been obtained by least squares methods. The active sites of these two enzymes contain similarly oriented His-Asp pairs linked by a hydrogen bond which may function as a proton relay system during catalysis. This pair could also provide an explanation for the relatively stronger binding by cytoplasmic malate dehydrogenase and lactate dehydrogenase of NADH versus NAD. Similar His-Asp pairs have been observed in the serine proteases, thermolysin, and phospholipase A2, and the His-Asp pair may play a similar functional role in all of these enzymes.     

Proton NMR and Optical Spectroscopic Studies on the DNA Triplex Formed by d-A-(G-A)7-G and d-C-(T-C)7-T

Abstract

Triplex and duplex formation of two deoxyribohexadecamers d-A-(G-A)₇-G (a) and d-C-(T-C)₇-T (b) have been studied by UV, CD, fluorescence, and proton NMR spectroscopy. Optical studies of a and b at dilute concentrations (μM range) yielded results similar to those seen for polymers of the same sequence, indicating that these hexadecamers have properties similar to the polymers in regard to triplex formation. The CD spectra of concentrated NMR samples (mM range) are similar to those observed at optical concentrations at both low and high pH, making possible a correlation between CD and NMR studies. In NMR spectra, two imido NH-N hydrogen bonded resonance envelopes at 12.6 and 13.7 ppm indicate that only the duplex conformation is present at pH > 7.7. Four new NH-N hydrogen-bonded resonance envelopes at 12.7, 13.5, 14.2. and 14.9 ppm are observed under acidic conditions (pH 5.6) and the two original NH-N resonances gradually disappear as the pH is lowered. Assignment of these four peaks to Watson-Crick G · C, Hoogsteen T · A. Watson-Crick A · T. and Hoogsteen C⁺ · G hydrogen-bonded imidos, respectively, confirm the formation of triple-stranded DNA NMR results also show that triplex is more stable than duplex at the same salt condition and that triplex melts to single strands directly without going through a duplex intermediate. However, in the melting studies, a structural change within the triple-stranded complex is evident at temperatures significantly below the major helix-to-coil transition. These studies demonstrate the feasibility of using NMR spectroscopy and oligonucleotide model compounds a and b for the study of DNA triplex formation.