Electron Transfer Dissociation Reveals Changes in the Cleavage Frequencies of Backbone Bonds Distant to Amide-to-Ester Substitutions in Polypeptides

Interrogation of electron transfer dissociation (ETD) mass spectra of peptide amide-to-ester backbone bond substituted analogues (depsipeptides) reveals substantial differences in the entire backbone cleavage frequencies. It is suggested that the point permutation of backbone bonds leads to changes in the predominant ion structures by removal/weakening of specific hydrogen bonding. ETD responds to these changes by redistributing the cleavage frequencies of the peptide backbone bonds. In comparison, no distinction between depsi-/peptide was observed using collision-activated dissociation, which is consistent with a general unfolding and elimination of structural information of these ions. These results should encourage further exploration of depsipeptides for gas-phase structural characterization.     

Modeling competitive interactions in proteins: vibrational spectroscopy of M+(n-methylacetamide)1(H2O)n=0-3, M=Na and K, in the 3 microm region

To properly understand the preferred structures and biological properties of proteins, it is important to understand how they are influenced by their immediate environment. Competitive intrapeptide, peptide...water, ion...water, and ion...peptide interactions, such as hydrogen bonding, play a key role in determining the structures, properties, and functionality of proteins. The primary types of hydrogen bonding involving proteins are intramolecular amide...amide (N-H...O=C) and intermolecular amide...water (O-H...O=C and H-O...H-N). n-Methylacetamide (NMA) is a convenient model for investigating these competitive interactions. An analysis of the IR photodissociation (IRPD) spectra of M+(n-methylacetamide)1(H2O)n=0-3 (M=Na and K) in the O-H and N-H spectral regions is presented. Ab initio calculations (MP2/cc-pVDZ) are used as a guide in identifying both the type and location of hydrogen bonds present. In larger clusters, where several structural isomers may be present in the molecular beam, ab initio calculations are also used to suggest assignments for the observed spectral features. The results presented offer insight to the nature of ion...NMA interactions in an aqueous environment and reveal how different ion...ligand pairwise interactions direct the extent of water...water and water...NMA hydrogen bonding observed.     

PA6/CaCl_2复合物的络合机理研究

通过负离子淤浆聚合制备了高相对黏度的聚酰胺6(PA6)粉末料,将其加入氯化钙甲酸溶液中制备不同的PA6/CaCl2单位链节摩尔比络合溶液.采用X射线光电子能谱(XPS)、红外分析方法研究了PA6/CaCl2复合物的络合机理.结果表明,复合物中钙原子与聚酰胺6分子链上的羰基氧原子发生配位作用,破坏了PA6本身的氢键,释放出自由NH,而氯离子则与NH形成氢键.同时通过电导率测试推测氯化钙与酰胺键之间的配合形式为四配位或者六配位.     

Dissociation of disulfide‐intact somatostatin ions: the roles of ion type and dissociation method

The dissociation chemistry of somatostatin‐14 was examined using various tandem mass spectrometry techniques including low‐energy beam‐type and ion trap collision‐induced dissociation (CID) of protonated and deprotonated forms of the peptide, CID of peptide‐gold complexes, and electron transfer dissociation (ETD) of cations. Most of the sequence of somatostatin‐14 is present within a loop defined by the disulfide linkage between Cys‐3 and Cys‐14. The generation of readily interpretable sequence‐related ions from within the loop requires the cleavage of at least one of the bonds of the disulfide linkage and the cleavage of one polypeptide backbone bond. CID of the protonated forms of somatostatin did not appear to give rise to an appreciable degree of dissociation of the disulfide linkage. Sequential fragmentation via multiple alternative pathways tended to generate very complex spectra. CID of the anions proceeded through CH₂? S cleavages extensively but relatively few structurally diagnostic ions were generated. The incorporation of Au(I) into the molecule via ion/ion reactions followed by CID gave rise to many structurally relevant dissociation products, particularly for the [M+Au+H]²⁺ species. The products were generated by a combination of S? S bond cleavage and amide bond cleavage. ETD of the [M+3H]³⁺ ion generated rich sequence information, as did CID of the electron transfer products that did not fragment directly upon electron transfer. The electron transfer results suggest that both the S? S bond and an N? C_α bond can be cleaved following a single electron transfer reaction. Copyright © 2009 John Wiley & Sons, Ltd.     

An Easy Access to Organic Salt‐Based Stimuli‐Responsive and Multifunctional Supramolecular Hydrogels

By exploiting orthogonal hydrogen bonding involving supramolecular synthons and hydrophobic/hydrophilic interactions, a new series of simple organic salt based hydrogelators derived from pyrene butyric acid and its β‐alanine amide derivative, and various primary amines has been achieved. The hydrogels were characterised by microscopy, table‐top rheology and dynamic rheology. FTIR, variable‐temperature ¹H NMR and emission spectroscopy established the role of various supramolecular interactions such as hydrogen bonding and π–π stacking in hydrogelation. Single‐crystal X‐ray diffraction (SXRD) studies supported the conclusion that orthogonal hydrogen bonding involving amide–amide and primary ammonium monocarboxylate (PAM) synthons indeed played a crucial role in hydrogelation. The hydrogels were found to be stimuli‐responsive and were capable of sensing ammonia and adsorbing water‐soluble dye (methylene blue). All the hydrogelators were biocompatible (MTT assay in RAW 264.7 cells), indicating their suitability for use in drug delivery.     

Peptide and protein quantification using iTRAQ with electron transfer dissociation

Electron transfer dissociation (ETD) has become increasingly used in proteomic analyses due to its complementarity to collision-activated dissociation (CAD) and its ability to sequence peptides with post-translation modifications (PTMs). It was previously unknown, however, whether ETD would be compatible with a commonly employed quantification technique, isobaric tags for relative and absolute quantification (iTRAQ), since the fragmentation mechanisms and pathways of ETD differ significantly from CAD. We demonstrate here that ETD of iTRAQ labeled peptides produces c- and z -type fragment ions as well as reporter ions that are unique from those produced by CAD. Exact molecular formulas of product ions were determined by ETD fragmentation of iTRAQ-labeled synthetic peptides followed by high mass accuracy orbitrap mass analysis. These experiments revealed that ETD cleavage of the N-C(alpha) bond of the iTRAQ tag results in fragment ions that could be used for quantification. Synthetic peptide work demonstrates that these fragment ions provide up to three channels of quantification and that the quality is similar to that provided by beam-type CAD. Protein standards were used to evaluate peptide and protein quantification of iTRAQ labeling in conjunction with ETD, beam-type CAD, and pulsed Q dissociation (PQD) on a hybrid ion trap-orbitrap mass spectrometer. For reporter ion intensities above a certain threshold all three strategies provided reliable peptide quantification (average error < 10%). Approximately 36%, 8%, and 16% of scans identified fall below this threshold for ETD, HCD, and PQD, respectively. At the protein level, average errors were 2.3%, 1.7%, and 3.6% for ETD, HCD, and PQD, respectively.     

Theoretical and experimental investigation of the energetics of cis-trans proline isomerization in peptide models

The energetics of cis-trans proline isomerization in small peptide models have been investigated using the hybrid density functional theory method B3LYP with a 6-31+G* basis set. The molecules studied are models for the phospho-Ser/Thr-Pro substrate for Pin-1, a peptidyl-prolyl isomerase (PPIase) involved in cell division. Pin-1 requires phosphorylation of a Ser or Thr residue adjacent to a Pro residue in the substrate and catalyzes cis-trans isomerization about the proline amide bond. The dihedral angle that would correspond to the reaction coordinate for isomerization of the omega peptide bond was investigated for several small models. Relaxed potential energy scans for this dihedral angle in N-methylacetamide, 1, N,N-dimethylacetamide, 2, acetylpyrrolidine, 3 and acetylproline, 4, were carried out in 20 degrees steps using the B3LYP/6-31+G* level of theory. In addition, similar scans were carried out for 1-4 protonated on the acetylamide carbonyl oxygen. Optimized structures for 1-4 protonated on the amide nitrogen were also obtained at B3LYP/6-31+G*. Relative proton affinities were determined for each site at various angles along the reaction coordinate for isomerization. The relative proton affinities were anchored to experimental gas phase proton affinities, which were taken from the literature for 1 and 2, or determined in an electrospray ionization-quadrupole ion trap instrument using the extended kinetic method for 3 and 4. Proton affinities of 925 +/- 10 and 911 +/- 12 kJ/mol were determined for 3 and 4, respectively. These studies suggest that the nitrogen atom in these amides becomes the most basic site in the molecule at a dihedral angle of ca. 130 degrees . In addition, the nitrogen atoms in 2-4 are predicted to attain basicities in the range 920-950 kJ/mol, making them basic enough to be the preferred site for hydrogen bonding in the Pin-1 active site, in support of the proposed mechanism for PPIases.     

Intramolecular acid-catalyzed amide isomerization in aqueous solution

[reaction: see text] We report for the first time that stoichiometric and even catalytic quantities of weak acids in aqueous solution can very efficiently catalyze amide isomerization in a carefully designed system in which a proton donor is situated so that intramolecular hydrogen bonding to the amide nitrogen is highly favored. Our results provide the first experimental verification that hydrogen bond donation to the amide nitrogen by charged proton donors may play a very significant role in the enzymatic catalysis of amide isomerization.     

UV raman examination of alpha-helical peptide water hydrogen bonding

UV resonance Raman spectra (UVRS) of an alpha-helical, 21 residue, mainly Ala peptide (AP) in the dehydrated solid state were compared to those in aqueous solution at different temperatures. The UVRS amide band frequencies of a dehydrated solid alpha-helix peptide show frequency shifts compared to those in aqueous solution due to the loss of amide backbone hydrogen bonding to water; the amide II and amide III bands of the solid alpha-helix downshift, while the amide I band upshifts. The shifts are identical in direction but smaller than those that occur for alpha-helices in aqueous solution as the temperature increases; water hydrogen bonding strengths decrease as the temperature increases. The UV Raman amide band frequency shifts can be used to monitor alpha-helix hydrogen bonding.     

On performing simultaneous electron transfer dissociation and collision-induced dissociation on multiply protonated peptides in a linear ion trap

We propose a tandem mass spectrometry method that combines electron-transfer dissociation (ETD) with simultaneous collision-induced dissociation (CID), termed ETD/CID. This technique can provide more complete sequence coverage of peptide ions, especially those at lower charge states. A selected precursor ion is isolated and subjected to ETD. At the same time, a residual precursor ion is subjected to activation via CID. The specific residual precursor ion selected for activation will depend upon the charge state and m/z of the ETD precursor ion. Residual precursor ions, which include unreacted precursor ions and charge-reduced precursor ions (either by electron-transfer or proton transfer), are often abundant remainders in ETD-only reactions. Preliminary results demonstrate that during an ETD/CID experiment, b, y, c, and z-type ions can be produced in a single experiment and displayed in a single mass spectrum. While some peptides, especially doubly protonated ones, do not fragment well by ETD, ETD/CID alleviates this problem by acting in at least one of three ways: (1) the number of ETD fragment ions are enhanced by CID of residual precursor ions, (2) both ETD and CID-derived fragments are produced, or (3) predominantly CID-derived fragments are produced with little or no improvement in ETD-derived fragment ions. Two interesting scenarios are presented that display the flexibility of the ETD/CID method. For example, smaller peptides that show little response to ETD are fragmented preferentially by CID during the ETD/CID experiment. Conversely, larger peptides with higher charge states are fragmented primarily via ETD. Hence, ETD/CID appears to rely upon the fundamental reactivity of the analyte cations to provide the best fragmentation without implementing any additional logic or MS/MS experiments. In addition to the ETD/CID experiments, we describe a novel dual source interface for providing front-end ETD capabilities on a linear ion trap mass spectrometer.     

Beam-type collisional activation of polypeptide cations that survive ion/ion electron transfer

Doubly protonated peptides that undergo an electron transfer reaction without dissociation in a linear ion trap can be subjected to beam-type collisional activation upon transfer from the linear ion trap into an adjacent mass analyzer, as demonstrated here with a hybrid triple quadrupole/linear ion trap system. The activation can be promoted by use of a DC offset difference between the ion trap used for reaction and the ion trap into which the products are injected of 12-16 V, which gives rise to energetic collisions between the transferred ions and the collision/bath gas employed in the linear ion trap used for ion/ion reactions. Such a process can be executed routinely on hybrid linear ion trap/triple quadrupole tandem mass spectrometers and is demonstrated here with several model peptides as well as a few dozen tryptic peptides. Collisional activation of the peptide precursor ions that survive electron transfer frequently provides structural information that is absent from the precursor ions that fragment spontaneously upon electron transfer. The degree to which additional structural information is obtained by collisional activation of the surviving singly charged peptide ions depends upon peptide size. Little or no additional structural information is obtained from small peptides (<8 residues) due to the high electron transfer dissociation (ETD) efficiencies noted for these peptides as well as the extensive sequence information that tends to be forthcoming from ETD of such species. Collisional activation of the surviving electron transfer products provided greatest benefit for peptides of 8-15 residues.     

Strategies for generating peptide radical cations via ion/ion reactions

Several approaches for the generation of peptide radical cations using ion/ion reactions coupled with either collision induced dissociation (CID) or ultraviolet photo dissociation (UVPD) are described here. Ion/ion reactions are used to generate electrostatic or covalent complexes comprised of a peptide and a radical reagent. The radical site of the reagent can be generated multiple ways. Reagents containing a carbon–iodine (C―I) bond are subjected to UVPD with 266‐nm photons, which selectively cleaves the C―I bond homolytically. Alternatively, reagents containing azo functionalities are collisionally activated to yield radical sites on either side of the azo group. Both of these methods generate an initial radical site on the reagent, which then abstracts a hydrogen from the peptide while the peptide and reagent are held together by either electrostatic interactions or a covalent linkage. These methods are demonstrated via ion/ion reactions between the model peptide RARARAA (doubly protonated) and various distonic anionic radical reagents. The radical site abstracts a hydrogen atom from the peptide, while the charge site abstracts a proton. The net result is the conversion of a doubly protonated peptide to a peptide radical cation. The peptide radical cations have been fragmented via CID and the resulting product ion mass spectra are compared to the control CID spectrum of the singly protonated, even‐electron species. This work is then extended to bradykinin, a more broadly studied peptide, for comparison with other radical peptide generation methods. The work presented here provides novel methods for generating peptide radical cations in the gas phase through ion/ion reaction complexes that do not require modification of the peptide in solution or generation of non‐covalent complexes in the electrospray process. Copyright © 2015 John Wiley & Sons, Ltd.     

Multipurpose Dissociation Cell for Enhanced ETD of Intact Protein Species

We describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. We partitioned the collision cell into a multi-section rf ion storage and transfer device to enable injection and simultaneous separate storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell end lens electrodes enables charge-sign independent trapping for ion–ion reactions. The approximately 2-fold higher quadrupole field frequency of this cell relative to that of the A-QLT enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell’s longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z analysis of the ETD product ions. This extends the intra-scan dynamic range by increasing the maximum number of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, we demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS analysis of intact protein species as compared to the standard ETD implementation.

Application of electron transfer dissociation mass spectrometry in analyses of non-enzymatically glycated peptides

Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the context of development of diabetic complications. The fragmentation behavior of glycated peptides produced from reaction of D-glucose with lysine residues was investigated by electron transfer dissociation (ETD) and collision-induced dissociation (CID) tandem mass spectrometry. It was found that high abundance ions corresponding to various degrees of neutral water losses, as well as furylium ion production, dominate the CID spectra, and that the sequence-informative b and y ions were rarely observed when Amadori-modified peptides were fragmented. Contrary to what was observed under CID conditions, ions corresponding to neutral losses of water or furylium ion production were not observed in the ETD spectra. Instead, abundant and almost complete series of c- and z-type ions were observed regardless of whether the modification site was located in the middle of the sequence or close to the N-terminus, greatly facilitating the peptide sequencing. This study strongly suggests that ETD is a better technique for proteomic studies of non-enzymatically glycated peptides and proteins.     

Hypervalent radical formation probed by electron transfer dissociation of zwitterionic tryptophan and tryptophan‐containing dipeptides complexed with Ca2+ and 18‐crown‐6 in the gas phase

The relationship between peptide structure and electron transfer dissociation (ETD) is important for structural analysis by mass spectrometry. In the present study, the formation, structure and reactivity of the reaction intermediate in the ETD process were examined using a quadrupole ion trap mass spectrometer equipped with an electrospray ionization source. ETD product ions of zwitterionic tryptophan (Trp) and Trp‐containing dipeptides (Trp‐Gly and Gly‐Trp) were detected without reionization using non‐covalent analyte complexes with Ca²⁺ and 18‐crown‐6 (18C6). In the collision‐induced dissociation, NH₃ loss was the main dissociation pathway, and loss related to the dissociation of the carboxyl group was not observed. This indicated that Trp and its dipeptides on Ca²⁺(18C6) adopted a zwitterionic structure with an NH₃⁺ group and bonded to Ca²⁺(18C6) through the COO^? group. Hydrogen atom loss observed in the ETD spectra indicated that intermolecular electron transfer from a molecular anion to the NH₃⁺ group formed a hypervalent ammonium radical, R‐NH₃, as a reaction intermediate, which was unstable and dissociated rapidly through N–H bond cleavage. In addition, N–C_α bond cleavage forming the z₁ ion was observed in the ETD spectra of Trp‐GlyCa²⁺(18C6) and Gly‐TrpCa²⁺(18C6). This dissociation was induced by transfer of a hydrogen atom in the cluster formed via an N–H bond cleavage of the hypervalent ammonium radical and was in competition with the hydrogen atom loss. The results showed that a hypervalent radical intermediate, forming a delocalized hydrogen atom, contributes to the backbone cleavages of peptides in ETD. Copyright © 2015 John Wiley & Sons, Ltd.     

Investigating the effect of hydrogen bonding environments in amide cleavage reactions at zinc(II) complexes with intramolecular amide oxygen co-ordination

Amide oxygen co-ordination to a zinc(II) ion around a hydrogen bonding microenvironment is a common structural/functional feature of metalloproteases. We report two strategies to position hydrogen bonding groups in the proximity of a zinc(II)-bound amide oxygen, and we investigate their effect on the stability of the amide group. Polydentate tripodal ligands (6-R1-2-pyridylmethyl)-R2 (R1= NHCOtBu, R2= N(CH2-py-6-X)2 X = H L1, X = NH2, H L2, X = NH2 L3) form [(L)Zn]2+ cations (L =L1, 1; L2, 2; L3, 3) with intramolecular amide oxygen co-ordination (1-3), and intramolecular N-H...O=C(amide) hydrogen bonding (2, 3) rigidly fixed by the ligand framework. 1-3 undergo cleavage of the tert-butyl amide upon addition of Me4NOH.5H2O (1 equiv.) in methanol at 50(1) degrees C. Under these conditions the half-life, t(1/2), of the amide bond is 0.4 h for 1, 9 h for 2 and 320 h for 3. Mononuclear zinc(II) complexes of (6-NHCOtBu-2-pyridylmethyl)-R2(R2= N(CH2CH2)2S) L4 and chelating N2 ligands without hydrogen bonding groups (1,10-phenanthroline L5, 2-(aminomethyl)pyridine L6) as control compounds, and with an amino hydrogen bonding group (6-amino-2-(aminomethyl)pyridine L7) have been synthesised. Amide cleavage is in this case faster at the zinc(II) complex with the amino hydrogen bonding group. Thus, hydrogen bonding environments can both accelerate and slow down amide bond cleavage reactions at zinc(II) sites. Importantly, the magnitude of the effect exerted by the hydrogen bonding environments was found to be significant; 800-fold rate difference. This result highlights the importance of hydrogen bonding environments around metal centres in amide cleavage reactions, which may be relevant to the chemistry of natural metalloproteases and applicable to the design of more efficient artificial protein cleaving agents.     

Neutral loss of water from the b ions with histidine at the C-terminus and formation of the c ions involving lysine side chains

Neutral loss of water from the amide bond induced by the His side chain has been reported. The proposed fragmentation pathway is a retro-Ritter reaction catalyzed by the imidazole nitrogen. In our MS/MS study of the neuropeptide GAHKNYLRFamide, we observed that the neutral loss of water from the b(3) ion is abundant. The b(3) ion has a His residue at the C-terminus. As reported previously, in the b ions with His at the C-terminus, the imidazole residue is connected to the carbonyl carbon to form a five-membered ring. Therefore, it is unlikely that the neutral loss of water from the b(3) ion is catalyzed by the imidazole nitrogen. Through MS2 and MS3 studies of a synthetic peptide standard AGHKLL and its chemically labeled and isotope-encoded forms, we discovered that the water loss from the b(3) ion involves the carbonyl group of His, the hydrogen connected to the alpha-carbon of Gly, and the amide hydrogen of His. We also discovered the formation of an unusual c(x) ion in peptides with a Lys or Arg residue at the (x + 1) position of the peptide.     

Electron transfer dissociation of peptide anions

Ion/ion reactions of multiply deprotonated peptide anions with xenon radical cations result in electron abstraction to generate charge-reduced peptide anions containing a free-radical site. Peptide backbone cleavage then occurs by hydrogen radical abstraction from a backbone amide N to facilitate cleavage of the adjacent C-C bond, thereby producing a- and x-type product ions. Introduction of free-radical sites to multiply charged peptides allows access to new fragmentation pathways that are otherwise too costly (e. g., lowers activation energies). Further, ion/ion chemistry, namely electron transfer reactions, presents a rapid and efficient means of generating odd-electron multiply charged peptides; these reactions can be used for studying gas-phase chemistries and for peptide sequence analysis.     

Negative-ion electron capture dissociation: radical-driven fragmentation of charge-increased gaseous peptide anions

The generation of gaseous polyanions with a Coulomb barrier has attracted attention as exemplified by previous studies of fullerene dianions. However, this phenomenon has not been reported for biological anions. By contrast, electron attachment to multiply charged peptide and protein cations has seen a surge of interest due to the high utility for tandem mass spectrometry (MS/MS). Electron capture dissociation (ECD) and electron transfer dissociation (ETD) involve radical-driven fragmentation of charge-reduced peptide/protein cations to yield N-C(α) backbone bond cleavage, resulting in predictable c'/z(?)-type product ions without loss of labile post-translational modifications (PTMs). However, acidic peptides, e.g., with biologically important PTMs such as phosphorylation and sulfonation, are difficult to multiply charge in positive ion mode and show improved ionization in negative-ion mode. We found that peptide anions ([M - nH](n-), n ≥ 1) can capture electrons within a rather narrow energy range (~3.5-6.5 eV), resulting in charge-increased radical intermediates that undergo dissociation analogous to that in ECD/ETD. Gas-phase zwitterionic structures appear to play an important role in this novel MS/MS technique, negative-ion electron capture dissociation (niECD).     

Novel ion-pair receptors based on hexahomotrioxacalix[3]arene derivatives

A series of novel heteroditopic hexahomotrioxacalix[3]arene triamide receptors capable of binding an anion and cation simultaneously in a cooperative fashion has been prepared. The lower rim functionalized cone-hexahomotrioxacalix[3]arene derivatives cone-5a-5d bearing three amide groups were synthesized from cone-3 by a stepwise reaction. The crystal structures of 5c and 5d and (1)H NMR studies in nonpolar solvents strongly indicate that a number of interesting intramolecular hydrogen bonding interactions exist in these receptors. The binding abilities of these compounds towards n-butylammonium chloride and bromide salts have been investigated using (1)H NMR titration experiments in CDCl(3) solvent. Owing to the 'flattened cone' conformations and intramolecular hydrogen bonding involving the amide NH and neighbouring O atoms in cone-5a-5d, the affinities toward n-Bu(4)NX (X = Cl(-) and Br(-)) were weakened. However, it should be noted that triamides cone-5a-5d show a single selectivity for halide anions in the presence of n-BuNH(3)(+) through intermolecular hydrogen bonding with the amide NH hydrogen atoms in the receptors in CDCl(3) solution. Association constants were calculated from the chemical shift changes of the amide protons.