Amide Proton Resonances Research Articles

1H NMR characterization of chitin tetrasaccharide in binary H2O:DMSO solution: Evidence for anomeric end-effect propagation.

Chitin oligosaccharides, composed of homogeneous β(1 → 4)-linked N-acetyl-D-glucosamine (GlcNAc) sequences, is a well-known elicitor of plant immune defense, and also occur as structural elements of chitosan and nodulation (Nod) factor. Detailed microstructure characterization is required for understanding the function mode of these bioactive molecules. Herein, experimental conditions for detection and elucidation of the 1H NMR resonances of amide groups in chitin oligosaccharides are presented. The binary mixture of 70% H2O: 30% DMSO‑d6 was found to be the optimal solvent for amide proton measurements in homogeneous GlcNAc sequences, facilitating differentiation of the local chemical microenvironments of all four amide groups of the chitin tetrasaccharide. Experimental evidence that anomeric end-effect triggers amide proton resonance differentiation at the adjacent residue has potential to provide important insights into the solution structure of chitin and other amino sugars containing GlcNAc sequences.

Using High Pressure NMR to Study Folding Cooperativity and Kinetics of Protein L9

The ribosomal protein L9 is comprised of two globular domains, the N-terminal (NTL9) and C-terminal (CTL9) domains, connected by a rigid linker. The folding properties of the two domains have been investigated extensively with a variety of denaturants, including urea, GdnHCl, temperature, and pH. Both NTL9 and CTL9 have exhibited cooperative folding in a two-state manner. Pressure perturbation, as a relatively new denaturing factor, has been proved to unfold proteins locally by targeting their packing defects, and to slow protein folding due to the positive activation volume of folding. Conventional NMR spectroscopy is too slow to characterize the folding transition state of most single domain proteins. When combined with NMR spectroscopy, which provides residue-specific resolution, high pressure (HP) could reveal sequence based information regarding protein folding cooperativity and conformational dynamics. Hence we applied HP-NMR to NTL9, CTL9 and their variants, seeking to gain more insights on their folding mechanism. To study their folding equilibria, 1H-15N HSQC spectra were recorded as a function of pressure, and the intensity of each amide proton resonance was analyzed as a function of pressure. We combined HP with 15N ZZ-exchange NMR experiments to determine residue-specific folding and unfolding rate constants. Our thermodynamic and kinetic results reveal that NTL9 folding is very close to two-state, while small deviations from two-state behavior were observed for CTL9. The volumetric properties of these domains indicate that most of the solvent excluded voids in the hydrophobic cores of the folded structures are formed in their respective transition states.

Chemical exchange saturation transfer of the cervical spinal cord at 7 T.

High-magnetic-field (7 T) chemical exchange saturation transfer (CEST) MRI provides information on the tissue biochemical environment. Multiple sclerosis (MS) affects the entire central nervous system, including the spinal cord. Optimal CEST saturation parameters found via simulation were implemented for CEST MRI in 10 healthy controls and 10 patients with MS, and the results were examined using traditional asymmetry analysis and a Lorentzian fitting method. In addition, T1 - and T2 *-weighted images were acquired for lesion localization and the transmitted B1 (+) field was evaluated to guide imaging parameters. Distinct spectral features for all tissue types studied were found both up- and downfield from the water resonance. The z spectra in healthy subjects had the expected z spectral shape with CEST effects apparent from 2.0 to 4.5 ppm. The z spectra from patients with MS demonstrated deviations from this expected normal shape, indicating this method's sensitivity to known pathology as well as to tissues appearing normal on conventional MRI. Examination of the calculated CESTasym revealed increased asymmetry around the amide proton resonance (Δω = 3.5 ppm), but it was apparent that this measure is complicated by detail in the CEST spectrum upfield from water, which is expected to result from the nuclear Overhauser effect. The z spectra upfield (negative ppm range) were also distinct between healthy and diseased tissue, and could not be ignored, particularly when considering the conventional asymmetry analysis used to quantify the CEST effect. For all frequencies greater than +1 ppm, the Lorentzian differences (and z spectra) for lesions and normal-appearing white matter were distinct from those for healthy white matter. The increased frequency separation and signal-to-noise ratio, in concert with prolonged T1 at 7 T, resulted in signal enhancements necessary to detect subtle tissue changes not possible at lower field strengths. This study presents CEST imaging metrics that may be sensitive to the extensive and temporally varying biochemical neuropathology of MS in the spinal cord. Copyright © 2016 John Wiley & Sons, Ltd.

Proton-detected 3D 15N/1H/1H isotropic/anisotropic/isotropic chemical shift correlation solid-state NMR at 70 kHz MAS

Chemical shift anisotropy (CSA) tensors offer a wealth of information for structural and dynamics studies of a variety of chemical and biological systems. In particular, CSA of amide protons can provide piercing insights into hydrogen-bonding interactions that vary with the backbone conformation of a protein and dynamics. However, the narrow span of amide proton resonances makes it very difficult to measure 1H CSAs of proteins even by using the recently proposed 2D 1H/1H anisotropic/isotropic chemical shift (CSA/CS) correlation technique. Such difficulties due to overlapping proton resonances can in general be overcome by utilizing the broad span of isotropic chemical shifts of low-gamma nuclei like 15N. In this context, we demonstrate a proton-detected 3D 15N/1H/1H CS/CSA/CS correlation experiment at fast MAS frequency (70kHz) to measure 1H CSA values of unresolved amide protons of N-acetyl-15N-l-valyl-15N-l-leucine (NAVL).

Protein stabilizer, NDSB-195, enhances the dynamics of the β4 -α2 loop of ubiquitin.

Non-detergent sulfobetaines (NDSBs) are a new group of small, synthetic protein stabilizers, which have advantages over classical compatible osmolytes, such as polyol, amines, and amino acids: they do not increase solution viscosity, unlike polyols, and they are zwitterionic at all pH ranges, unlike amines and amino acids. NDSBs also facilitate the crystallization and refolding of proteins. The mechanism whereby NDSBs exhibit such activities, however, remains elusive. To gain insight into this mechanism, we studied, using nuclear magnetic resonance (NMR), the effects of dimethylethylammonium propane sulfonate (NDSB-195) on the dynamics of ubiquitin, on which a wealth of information has been accumulated. By analyzing the line width of amide proton resonances and the transverse relaxation rates of nitrogen atoms, we found that NDSB-195 enhances the microsecond-millisecond dynamics of a β4 -α2 loop of ubiquitin. Although those compounds that enhance protein dynamics are generally considered to destabilize protein molecules, NDSB-195 enhanced the stability of ubiquitin against guanidium chloride denaturation. Thus, the simultaneous enhancement of stability and flexibility by a single compound can be attained.

On the potential of hyperpolarized water in biomolecular NMR studies.

A main obstacle arising when using ex situ hyperpolarization to increase the sensitivity of biomolecular NMR is the fast relaxation that macromolecular spins undergo upon being transferred from the polarizer to the spectrometer, where their observation takes place. To cope with this limitation, the present study explores the use of hyperpolarized water as a means to enhance the sensitivity of nuclei in biomolecules. Methods to achieve proton polarizations in excess of 5% in water transferred into the NMR spectrometer were devised, as were methods enabling this polarization to last for up to 30 s. Upon dissolving amino acids and polypeptides sited at the spectrometer into such hyperpolarized water, a substantial enhancement of certain biomolecular amide and amine proton resonances was observed. This exchange-driven (1)H enhancement was further passed on to side-chain and to backbone nitrogens, owing to spontaneous one-bond Overhauser processes. (15)N signal enhancements >500 over 11.7 T thermal counterparts could thus be imparted in a kinetic process that enabled multiscan signal averaging. Besides potential bioanalytical uses, this approach opens interesting possibilities in the monitoring of dynamic biomolecular processes, including solvent accessibility and exchange process.

Backbone 1H, 13C, 15N NMR assignments of yeast OMP synthase in unliganded form and in complex with orotidine 5′-monophosphate

The type I phosphoribosyltransferase OMP synthase (EC 2.4.2.10) is involved in de novo synthesis of pyrimidine nucleotides forming the UMP precursor orotidine 5'-monophosphate (OMP). The homodimeric enzyme has a Rossman α/β core topped by a base-enclosing "hood" domain and a flexible domain-swapped catalytic loop. High-resolution X-ray structures of the homologous Salmonella typhimurium and yeast enzymes show that a general compacting of the core as well as movement of the hood and a major disorder-to-order transition of the loop occur upon binding of ligands MgPRPP and orotate. Here we present backbone NMR assignments for the unliganded yeast enzyme (49 kDa) and its complex with product OMP. We were able to assign 212-213 of the 225 non-proline backbone (15)N and amide proton resonances. Significant difference in chemical shifts of the amide cross peaks occur in regions of the structure that undergo movement upon ligand occupancy in the S. typhimurium enzyme.

A Simple Model for Understanding the Origin of the Amide Proton Transfer MRI Signal in Tissue

Amide proton transfer (APT) imaging is a new molecular MRI technique that gives contrast at the cellular protein level. To better understand the origin of the APT signal in tissue, fresh and cooked hen eggs (n = 4) were imaged at 4.7 Tesla. The APT effect was quantified using the asymmetry in the magnetization transfer ratio (MTR(asym)) at the composite amide proton resonance frequency (3.5 ppm from the water resonance). The measured APT signals were significantly higher in the fresh egg white (20.1% ± 0.9%) than in the fresh egg yolk (-1.4% ± 1.1%; P < 0.001), and in the cooked egg white (2.8% ± 0.7%; P < 0.001), all of which have similar absolute protein contents. The data support the notion that the APT effect observed in vivo is associated with mobile proteins in tissue, such as those in the cytoplasm.

A simplified recipe for assigning amide NMR signals using combinatorial 14N amino acid inverse-labeling

Assignment of backbone amide proton resonances is one of the most time-consuming stages of any protein NMR study when the protein samples behave non-ideally. A robust and convenient NMR procedure for analyzing spectra of marginal-to-low quality is helpful for high-throughput structure determination. The 14N selective- and inverse-labeling method is a candidate solution. Here, we present a simplified protocol for assigning protein backbone amide NMR signals. When 14N inversely labeled residues are present in a protein, their backbone NH cross peaks vanish from the protein's 1H-(15)N HSQC spectrum, and thus, their chemical shifts can be readily identified by a process of elimination. Some metabolically related amino acids, for example, Ile, Leu, and Val, cannot be individually incorporated but can be inversely labeled together. We optimized and simplified the protocol and M9-based medium formula for the 14N selective- and inverse-labeling method without any additives. Our approach should be cost-effective, because the method could be additively applied stepwise, even when the proteins of interest were found to be non-ideal.

Structure of the p53 Transactivation Domain in Complex with the Nuclear Receptor Coactivator Binding Domain of CREB Binding Protein

The activity and stability of the tumor suppressor p53 are regulated by interactions with key cellular proteins such as MDM2 and CBP/p300. The transactivation domain (TAD) of p53 contains two subdomains (AD1 and AD2) and interacts directly with the N-terminal domain of MDM2 and with several domains of CBP/p300. Here we report the NMR structure of the full-length p53 TAD in complex with the nuclear coactivator binding domain (NCBD) of CBP. Both the p53 TAD and NCBD are intrinsically disordered and fold synergistically upon binding, as evidenced by the observed increase in helicity and increased level of dispersion of the amide proton resonances. The p53 TAD folds to form a pair of helices (denoted Pα1 and Pα2), which extend from Phe19 to Leu25 and from Pro47 to Trp53, respectively. In the complex, the NCBD forms a bundle of three helices (Cα1, residues 2066-2075; Cα2, residues 2081-2092; and Cα3, residues 2095-2105) with a hydrophobic groove into which p53 helices Pα1 and Pα2 dock. The polypeptide chain between the p53 helices remains flexible and makes no detectable intermolecular contacts with the NCBD. Complex formation is driven largely by hydrophobic contacts that form a stable intermolecular hydrophobic core. A salt bridge between D49 of p53 and R2105 of NCBD may contribute to the binding specificity. The structure provides the first insights into simultaneous binding of the AD1 and AD2 motifs to a target protein.

Detection of Amide and Aromatic Proton Resonances of Human Brain Metabolites Using Localized Correlated Spectroscopy Combined with Two Different Water Suppression Schemes.

The purpose of the study was to demonstrate the J-coupling connectivity network between the amide, aliphatic, and aromatic proton resonances of metabolites in human brain using two-dimensional (2D) localized correlated spectroscopy (L-COSY). Two different global water suppression techniques were combined with L-COSY, one before and another after localizing the volume of interest (VOI). Phantom solutions containing several cerebral metabolites at physiological concentrations were evaluated initially for sequence optimization. Nine healthy volunteers were scanned using a 3T whole body MRI scanner. The VOI for 2D L-COSY was placed in the right occipital white/gray matter region. The 2D cross and diagonal peak volumes were measured for several metabolites such as N-acetyl aspartate (NAA), creatine (Cr), free choline (Ch), glutamate/glutamine (Glx), aspartate (Asp), myo-inositol (mI), GABA, glutathione (GSH), phosphocholine (PCh), phosphoethanolamine (PE), tyrosine (Tyr), lactate (Lac), macromolecules (MM) and homocarnosine (Car). Using the pre-water suppression technique with L-COSY, the above mentioned metabolites were clearly identifiable and the relative ratios of metabolites were calculated. In addition to detecting multitude of aliphatic resonances in the high field region, we have demonstrated that the amide and aromatic resonances can also be detected using 2D L-COSY by pre water suppression more reliably than the post-water suppression.

Molecular Cloning and NMR Characterization of the Nonreceptor Tyrosine Kinase PTK6 SH3-SH2-Linker Domain

Human protein tyrosine kinase-6 (PTK6) is a member of the non-receptor protein tyrosine kinase family and it is found in two-thirds of all breast tumors. Very recently, we proposed that the SH3 domain of PTK6 interacts with the linker region (Linker) between the SH2 and kinase domains, proving that the interaction between SH3 domain and Linker plays an important role in auto-inhibition mechanism. Residues from 1 to 191 corresponding region of SH3-SH2-Linker (SH32L) of PTK6 was cloned into the pET32a expression vector with Tobbaco etch virus (TEV) protease enzyme site by sequence homology and 3D structural model. The purified PTK6-SH32L was determined as a monomer conformation in solution. The amide proton resonances in the <TEX>$^{15}N-^{1}H$</TEX> 2D-HSQC spectrum suggest that PTK6-SH32L possesses disordered structural region of the flexible/unstructured linker region. In addition, the backbone amide proton chemical shifts of the SH3 domain in the PTK6-SH32L differ from that of the independent domain, indicating that intra-molecular interaction between SH3 and Linker in the PTK6-SH32L is present.

Magnetization transfer magic-angle-spinning z-spectroscopy of excised tissues

NMR experiments devised to aid in analyses of tissues include magnetization transfer (MT), which can highlight the signals of biological macromolecules through cross-relaxation and/or chemical exchange processes with the bulk 1H water resonance, and high-resolution magic-angle-spinning (HRMAS) methods, akin to those used in solid-state NMR to introduce additional spectral resolution via the averaging of spin anisotropies. This paper explores the result of combining these methodologies, and reports on MT “z-spectroscopy” between water and cell components in excised tissues under a variety of HRMAS conditions. Main features arising from the resulting 1H “MTMAS” experiments include strong spinning sideband manifolds centered at the liquid water shift, high-resolution isotropic features coinciding with aliphatic and amide proton resonances, and a second sideband manifold arising as spinning speeds are increased. Interpretations are given for the origin of these various features, including simulations shedding further light onto the nature of MT NMR signals observed for tissue samples. Concurrently, histological examinations are reported validating the limits of HRMAS NMR procedures to the analysis of tissue samples preserved in a number of different ways.

A Sequential Assignment Procedure for Proteins that have Intermediate Line Widths in MAS NMR Spectra: Amyloid Fibrils of Human CA150.WW2

The second WW domain (WW2) of CA150, a human transcriptional activator, forms amyloid fibrils in vitro under physiological conditions. Based on experimental constraints from MAS NMR spectroscopy experiments, alanine scanning and electron microscopy, a structural model of CA150.WW2 amyloid fibrils was calculated earlier. Here, the assignment strategy is presented and suggested as a general approach for proteins that show intermediate line width. The (13)C,(13)C correlation experiments were recorded on fully or partially (13)C-labelled fibrils. The earlier (13)C assignment (26 residues) was extended to 34 of the 40 residues by direct (13)C-excitation experiments by using a deuterated sample that showed strongly improved line width. A 3D HNC-TEDOR (transferred-echo double-resonance) experiment with deuterated CA150.WW2 fibrils yielded 14 amide nitrogen and proton resonance assignments. The obtained chemical shifts were compared with the chemical shifts determined with the natively folded WW domain. TALOS (Torsion angle likelihood obtained from shift and sequence similarity) predictions confirmed that, under physiological conditions, the fibrillar form of CA150.WW2 adopts a significantly different beta structure than the native WW-domain fold.

Crystal Structure of a Prolactin Receptor Antagonist Bound to the Extracellular Domain of the Prolactin Receptor

The crystal structure of the complex between an N-terminally truncated G129R human prolactin (PRL) variant and the extracellular domain of the human prolactin receptor (PRLR) was determined at 2.5A resolution by x-ray crystallography. This structure represents the first experimental structure reported for a PRL variant bound to its cognate receptor. The binding of PRL variants to the PRLR extracellular domain was furthermore characterized by the solution state techniques, hydrogen exchange mass spectrometry, and NMR spectroscopy. Compared with the binding interface derived from mutagenesis studies, the structural data imply that the definition of PRL binding site 1 should be extended to include residues situated in the N-terminal part of loop 1 and in the C terminus. Comparison of the structure of the receptor-bound PRL variant with the structure reported for the unbound form of a similar analogue ( Jomain, J. B., Tallet, E., Broutin, I., Hoos, S., van Agthoven, J., Ducruix, A., Kelly, P. A., Kragelund, B. B., England, P., and Goffin, V. (2007) J. Biol. Chem. 282, 33118-33131 ) demonstrates that receptor-induced changes in the backbone of the four-helix bundle are subtle, whereas large scale rearrangements and structuring occur in the flexible N-terminal part of loop 1. Hydrogen exchange mass spectrometry data imply that the dynamics of the four-helix bundle in solution generally become stabilized upon receptor interaction at binding site 1.

Spontaneous Assembly of Photosynthetic Supramolecular Complexes as Mediated by the Intrinsically Unstructured Protein CP12

CP12 is a protein of 8.7 kDa that contributes to Calvin cycle regulation by acting as a scaffold element in the formation of a supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) in photosynthetic organisms. NMR studies of recombinant CP12 (isoform 2) of Arabidopsis thaliana show that CP12-2 is poorly structured. CP12-2 is monomeric in solution and contains four cysteines, which can form two intramolecular disulfides with midpoint redox potentials of -326 and -352 mV, respectively, at pH 7.9. Site-specific mutants indicate that the C-terminal disulfide is involved in the interaction between CP12-2 and GAPDH (isoform A(4)), whereas the N-terminal disulfide is involved in the interaction between this binary complex and PRK. In the presence of NAD, oxidized CP12-2 interacts with A(4)-GAPDH (K(D) = 0.18 microm) to form a binary complex of 170 kDa with (A(4)-GAPDH)-(CP12-2)(2) stoichiometry, as determined by isothermal titration calorimetry and multiangle light scattering analysis. PRK is a dimer and by interacting with this binary complex (K(D) = 0.17 microm) leads to a 498-kDa ternary complex constituted by two binary complexes and two PRK dimers, i.e. ((A(4)-GAPDH)-(CP12-2)(2)-(PRK))(2). Thermodynamic parameters indicate that assembly of both binary and ternary complexes is exoergonic although penalized by a decrease in entropy that suggests an induced folding of CP12-2 upon binding to partner proteins. The redox dependence of events leading to supramolecular complexes is consistent with a role of CP12 in coordinating the reversible inactivation of chloroplast enzymes A(4)-GAPDH and PRK during darkness in photosynthetic tissues.

Mechanism of Constitutive Phosphoinositide 3-Kinase Activation by Oncogenic Mutants of the p85 Regulatory Subunit

p85/p110 phosphoinositide 3-kinases regulate multiple cell functions and are frequently mutated in human cancer. The p85 regulatory subunit stabilizes and inhibits the p110 catalytic subunit. The minimal fragment of p85 capable of regulating p110 is the N-terminal SH2 domain linked to the coiled-coil iSH2 domain (referred to as p85ni). We have previously proposed that the conformationally rigid iSH2 domain tethers p110 to p85, facilitating regulatory interactions between p110 and the p85 nSH2 domain. In an oncogenic mutant of murine p85, truncation at residue 571 leads to constitutively increased phosphoinositide 3-kinase activity, which has been proposed to result from either loss of an inhibitory Ser-608 autophosphorylation site or altered interactions with cellular regulatory factors. We have examined this mutant (referred to as p65) in vitro and find that p65 binds but does not inhibit p110, leading to constitutive p110 activity. This activated phenotype is observed with recombinant proteins in the absence of cellular factors. Importantly, this effect is also produced by truncating p85ni at residue 571. Thus, the phenotype is not because of loss of the Ser-608 inhibitory autophosphorylation site, which is not present in p85ni. To determine the structural basis for the phenotype of p65, we used a broadly applicable spin label/NMR approach to define the positioning of the nSH2 domain relative to the iSH2 domain. We found that one face of the nSH2 domain packs against the 581-593 region of the iSH2 domain. The loss of this interaction in the truncated p65 would remove the orienting constraints on the nSH2 domain, leading to a loss of p110 regulation by the nSH2. Based on these findings, we propose a general model for oncogenic mutants of p85 and p110 in which disruption of nSH2-p110 regulatory contacts leads to constitutive p110 activity.

Distortion of the three-dimensional structure of the vnd/NK-2 homeodomain bound to DNA induced by an embryonically lethal A35T point mutation.

The three-dimensional solution structure obtained by NMR of the A35T mutant vnd/NK-2 homeodomain bound to the vnd/NK-2 consensus 16 bp DNA sequence was determined. This mutation to threonine from alanine in position 35 in helix II of the vnd/NK-2 homeodomain is associated with early embryonic lethality in Drosophila melanogaster. Although the unbound mutant protein is not structured, in the DNA-bound state it adopts the three-helix fold characteristic of all known homeodomains, but with alterations relative to the structure of the wild-type analogue. These structural modifications occur, and are accompanied by a 50-fold reduction in the DNA binding affinity, even though most of the protein-DNA interactions originally seen for the wild-type homeodomain are found likewise in the threonine analogue. Alterations include torsional angle changes in the loop between helix I and helix II, and in the turn between helix II and helix III, as well as in a distortion of the usual antiparallel orientation of helix I with respect to helix II. The alteration of the position of leucine 40 in the A35T mutant is proposed to explain the observed 1.27 ppm upfield shift of the corresponding amide proton resonance relative to the value observed for the wild-type analogue. A detailed comparison of the structures of the mutant A35T and wild-type vnd/NK-2 homeodomains bound to the cognate DNA is presented. The consequences of the structural alteration of the DNA-bound A35T mutant vnd/NK-2 protein may constitute the basis of the observed early embryonic lethality.

Structural studies on a twin-arginine signal sequence

Translocation of folded proteins across biological membranes can be mediated by the so-called ‘twin-arginine translocation’ (Tat) system. To be translocated, Tat substrates require N-terminal signal sequences which usually contain the eponymous twin-arginine motif. Here we report the first structural analysis of a twin-arginine signal sequence, the signal sequence of the high potential iron-sulfur protein from Allochromatium vinosum. Nuclear magnetic resonance (NMR) analyses of amide proton resonances did not indicate a signal sequence structure. Accordingly, data from H/D exchange matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry showed that the amide protons of the signal sequence exchange rapidly, indicating the absence of secondary structure in the signal sequence up to L29. We conclude that the conserved twin-arginine motif does not form a structure by itself or as a result of intramolecular interactions.

Assignment of amide proton signals by combined evaluation of HN, NN and HNCA MAS-NMR correlation spectra.

In this paper, we present a strategy for the (1)H(N) resonance assignment in solid-state magic-angle spinning (MAS) NMR, using the alpha-spectrin SH3 domain as an example. A novel 3D triple resonance experiment is presented that yields intraresidue H(N)-N-C(alpha) correlations, which was essential for the proton assignment. For the observable residues, 52 out of the 54 amide proton resonances were assigned from 2D ((1)H-(15)N) and 3D ((1)H-(15)N-(13)C) heteronuclear correlation spectra. It is demonstrated that proton-driven spin diffusion (PDSD) experiments recorded with long mixing times (4 s) are helpful for confirming the assignment of the protein backbone (15)N resonances and as an aid in the amide proton assignment.