Nitrotyrosine chelation of nuclear magnetic resonance shift probes in proteins: application to bovine pancreatic trypsin inhibitor

The interactions of Pr(III) and Eu(III) with specifically nitrated derivatives of the basic bovine pancreatic trypsin inhibitor have been studied using optical spectroscopy and nuclear magnetic resonance (NMR) at 250 and 270 MHz. Stability constants for proton and metal binding to nitrotyrosines 10 and 21 determined optically are in good agreement with those from NMR. Observations of the Eu(III)-induced NMR shifts of the ring protons of nitrotyrosine 21 allowed calibration of the magnetic interactions for this binding site. The Pr(III)-induced shifts for several resolved nonexchangeable backbone proton resonances were compared with calculated shifts using the known x-ray structure. With several simplifying assumptions, the Pr(III)-induced shifts were used to assign one alpha-CH and five NH protons to compatible sets of backbone positions which are consistent with the known pH dependence and resistance to exchange with solvent D2O. Some of the more general aspects of lanthanide-induced shifts are discussed with reference to their use in proteins. Due to the complexities of the analysis of the shift data, the most straightforward use of this technique is in conjunction with the relaxation probe Gd(III) for measurement of intramolecular distances.     

Complete tyrosine assignments in the high-field 1H nuclear magnetic resonance spectrum of bovine pancreatic trypsin inhibitor selectively reduced and carboxamidomethylated at cystine 14-38.

The low-field portions of the 250-MHz 1H nuclear magnetic resonance spectra of native and chemically modified basic pancreatic trypsin inhibitor have been studied as a function of pH over the range pH 5-13. In derivatives selectively reduced and carboxamidomethylated at cystine 14-38, resonances associated with 15 of the 16 protons of the aromatic rings of the four tyrosines of the inhibitor have been located and assigned to specific tyrosyl residues. Titrations of pH yielded pK's for tyrosines 10, 21, 23, and 35 in the modified inhibitor of 9.9, 10.6, 11.6, and 11.0, respectively. Resonances associated with the three nitrotyrosine 10 protons of the mononitrated derivative and the six nitrotyrosine 10 and 21 protons of the dinitrated derivative have been similarly located, assigned, and titrated, yielding pK's for nitrotyrosines 10 and 21 of 6.5 and 6.4, respectively. Previously reported results for derivatives with cystine 14-38 intact have been revised on the basis of new data. Comparison of these revised results with the new data for derivatives with modified cystine 14-38 reveals no changes in pK's for any tyrosine or nitrotyrosing ring and no changes in chemical shift for resonances of nitrotyrosine 21 or tyrosines 21 and 23. However, modification of cystine 14-38 causes significant changes in chemical shifts of resonances of the nearby nitrotyrosine 10 and tyrosine 10 and 35 rings. Tyrosine 35 remains relatively immobile, rotating less than 1600 times/s at 25 degrees C for pH's in the range 5-13.     

NMR studies of the backbone protons and secondary structure of pentapeptide and heptapeptide substrates bound to bovine heart protein kinase

The conformations of enzyme-bound pentapeptide (Arg-Arg-Ala-Ser-Leu) and heptapeptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) substrates of protein kinase have been studied by NMR in quaternary complexes of the type (Formula: see text). Paramagnetic effects of Mn2+ bound at the inhibitory site of the catalytic subunit on the longitudinal relaxation rates of backbone Ca protons, as well as on side-chain protons of the bound pentapeptide and heptapeptide substrates, have been used to determine Mn2+ to proton distances which range from 8.2 to 12.4 A. A combination of the paramagnetic probe-T1 method with the Redfield 2-1-4-1-2 pulse sequence for suppression of the water signal has been used to measure distances from Mn2+ to all of the backbone amide (NH) protons of the bound pentapeptide and heptapeptide substrates, which range from 6.8 to 11.1 A. Paramagnetic effects on the transverse relaxation rates yield rate constants for peptide exchange, indicating that the complexes studied by NMR dissociate rapidly enough to participate in catalysis. Model-building studies based on the Mn2+-proton distances, as well as on previously determined distances from Cr3+-AMPPCP to side-chain protons [Granot, J., Mildvan, A.S., Bramson, H. N., & Kaiser, E. T. (1981) Biochemistry 20, 602], rule out alpha-helical, beta-sheet, beta-bulge, and all possible beta-turn conformations within the bound pentapeptide and heptapeptide substrates. The distances are fit only by extended coil conformations for the bound peptide substrates with a minor difference between the pentapeptides and heptapeptides in the phi torsional angle at Arg3C alpha and in psi at Arg2C alpha. An extended coil conformation, which minimizes the number of interactions within the substrate, would facilitate enzyme-substrate interaction and could thereby contribute to the specificity of protein kinase.     

Fluorescent probes for measuring the binding constants and distances between the metal ions bound to Escherichia coli glutamine synthetase.

TNS, 2-p-toluidinylnaphthalene-6-sulfonate, has been used as a fluorescent probe to determine the binding constants of metal ions to the two binding sites of Escherichia coli glutamine synthetase (GS). TNS fluorescence is enhanced dramatically when bound to proteins due to its high quantum yield resulting from its interactions with hydrophobic regions in proteins. The fluorescence energy transfer from a hydrophobic tryptophan residue of GS to TNS has been detected as an excitation band centered at 280 nm. Therefore, TNS is believed to be bound to a hydrophobic site on the GS surface other than the active site and is located near a hydrophobic Trp residue of GS. GS binds lanthanide ions [Ln(III)] more tightly than either Mn(II) or Mg(II), and the binding constants of several lanthanide ions were determined to be in the range (2.1-4.6) x 10(10) and (1.4-3.0) x 10(8) M-1 to the two metal binding sites of GS, respectively. The intermetal distances between the two metal binding sites of GS were also determined by measuring the efficiencies of energy transfer from Tb(III) to other Ln(III) ions. The intermetal distances of Tb(III)-Ho(III) and Tb(III)-Nd(III) were 7.9 and 6.8 A, respectively.     

Complete tyrosine assignments in the high field 1H nuclear magnetic resonance spectrum of the bovine pancreatic trypsin inhibitor.

The low-field portions of the 250-MHz 1H nuclear magnetic resonance (NMR) specra of native and chemically modified bovine basic pancreatic trypsin inhibitor (BPTI) have been studied as a function of pH over the range pH 5-13. Resonances associated with the 16 protons of the aromatic rings of the four BPTI tyrosines have been located and assigned to specific tyrosyl residues. Titrations of pH yielded pK's for tyrosines-10, -21, -23, and -35 of 10.4, 11.0, 11.7, and 11.1, respectively. The resonances associated with the nitrotyrosine-10 protons of mononitrated BPTI and the nitrotyrosine-10 and -21 protons of dinitrated BPTI have been similarly located, assigned and titrated yielding pK's for nitrotyrosine-10 and -21 of 6.6 and 6.4, respectively. The high-field NMR spectrum indicates that the aromatic ring of tyrosine-35 rotates less than 160 times per second at 25 degrees for pH's in the range 5-9.     

Interaction of aromatic donor molecules with manganese(III) reconstituted horseradish peroxidase: proton nuclear magnetic resonance and optical difference spectroscopic studies

The interaction of aromatic donor molecules with manganese(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by optical difference spectroscopy and relaxation rate measurements of 1H resonances of aromatic donor molecules (at 500 MHz). pH dependence of substrate proton resonance line-widths indicated that the binding was facilitated by protonation of an amino acid residue (with a pKa of 6.1), which is presumably distal histidine. Dissociation constants were evaluated from both optical difference spectroscopy and 1H-NMR relaxation measurements (pH 6.1). The dissociation constants of aromatic donor molecules were not affected by the presence of excess of I-, CN- and SCN-. From competitive binding studies it was shown that all these aromatic donor molecules bind to Mn(III)HRP at the same site, which is different from the binding site of I-, CN- and SCN-. Comparison of the dissociation constants between the different substrates suggests that hydrogen bonding of the donors with distal histidyl amino acid and hydrophobic interaction between the donors and active site contribute significantly towards the associating forces. Free energy, entropy and enthalpy changes associated with the Mn(III)HRP-substrate equilibrium have been evaluated. These thermodynamic parameters were found to be all negative. Distances of the substrate protons from the paramagnetic manganese ion of Mn(III)HRP were found to be in the range of 7.7 to 9.4 A. The Kd values, the thermodynamic parameters and the distances of the bound aromatic donor protons from metal center in the case of Mn(III)HRP were found to be very similar as in the case of native Fe(III)HRP.     

Amide proton exchange rates of oxidized and reduced Saccharomyces cerevisiae iso-1-cytochrome c.

Proton NMR spectroscopy was used to determine the rate constant, kobs, for exchange of labile protons in both oxidized (Fe(III)) and reduced (Fe(II)) iso-1-cytochrome c. We find that slowly exchanging backbone amide protons tend to lack solvent-accessible surface area, possess backbone hydrogen bonds, and are present in regions of regular secondary structure as well as in omega-loops. Furthermore, there is no correlation between kobs and the distance from a backbone amide nitrogen to the nearest solvent-accessible atom. These observations are consistent with the local unfolding model. Comparisons of the free energy change for denaturation, delta Gd, at 298 K to the free energy change for local unfolding, delta Gop, at 298 K for the oxidized protein suggest that certain conformations possessing higher free energy than the denatured state are detected at equilibrium. Reduction of the protein results in a general increase in delta Gop. Comparisons of delta Gd to delta Gop for the reduced protein show that the most open states of the reduced protein possess more structure than its chemically denatured form. This persistent structure in high-energy conformations of the reduced form appears to involve the axially coordinated heme.     

Proteins from the prokaryotic nucleoid. High-resolution 1H NMR spectroscopic study of Escherichia coli DNA-binding proteins NS1 and NS2

The 1H-NMR spectra of the two Escherichia coli basic, low-Mr (approximately equal to 9000) DNA-binding proteins NS1 and NS2 and of their native complex NS were studied at 400 MHz and a number of resonances and resonance peaks were assigned. As in the case of some eukaryotic histones, the presence of a large number of high-field perturbed Phe resonances, several shielded and deshielded methyl resonances and backbone NH protons quite inaccessible to the solvent clearly indicate the existence of extensive tertiary and, even more so, quaternary structures involving hydrophobic interactions. These structures are lost upon heating, but readily reform upon cooling. Spectral differences between NS1, NS2 and NS and the greater thermal stability of NS indicate that molecules of the heterologous subunits (NS1 and NS2) aggregate (dimerize) preferentially in comparison to the self-aggregation of the homologous subunits. Unlike those of the eukaryotic histones, the tertiary and quaternary structures of NS are insensitive to extensive variations of the ionic strength.     

Hydrogen exchange kinetics of surface peptide amides in bovine pancreatic trypsin inhibitor

The acid and base catalytic rate constants, kH, obs and kOH, obs and the pH at the minimum rate, pHmin, of 25 rapidly exchanging protons in bovine pancreatic trypsin inhibitor have been determined. Here we report the labeling procedure giving 1H nuclear magnetic resonance spectral resolution of seven additional rapidly exchanging NH protons and the pH dependence of their chemical shifts. Values of kH,obs kOH,obs and pHmin are given for Ala16, Gly28 and Arg53 NH groups, the only backbone amide protons with static accessibility of more than zero in the crystal structure not previously reports, and for Gly56 NH, buried at the C terminus of an alpha-helix. All four protons reported here have pH min greater than or equal to 3. Conclusions of the previous study predict that peptide protons with pHmin higher than those of model compounds have greater static accessibility of the peptide O than of the peptide N atom. The locations in the crystal structure of the four NH groups whose exchange rates are reported here are in qualitative agreement with these predictions. The ionic strength dependence of Ala16 at pH 5.5 shows a sharp increase in the exchange rate with decreasing salt concentration, as expected for base-catalyzed exchange in a positive electrostatic field.     

1H nuclear magnetic resonance double resonance study of oxytocin in aqueous solution.

Peptide NH resonances in the 250 MHZ 1H nuclear magnetic resonance (NMR) spectrum of oxytocin in H2O were assigned to specific amino acid residues by the "underwater decoupling" technique (i.e., decoupling from corresponding CalphaH resonances, which are buried beneath the intense water peak). These experiments confirm previous assignments of A. I. Brewster an V. J. Hruby ((1973), Proc. Natl. Acad. Sci. U.S.A. 70, 3806) and A. F. Bradbury et al. ((1974), FEBS Lett. 42, 179). Three methods of assigning NH resonances of peptides--solvent titration, underwater decoupling, and isotopic labeling--are compared. As the solvet composition is gradually changed from dimethyl sulfoxide to H2O, oxytocin undergoes a conformational change at 70-90 mol % of H2O. Exposure to solvent of specific hydrogens of oxytocin in H2O was studied by monitoring intensity changes of solute resonances when the solvent peak was saturated. Positive nuclear Overhauser effects (NOE's) of 14 +/- 5 were observed for the Tyr ortho CH and meta CH resonances, respectively. Comparative studies with deamino-oxytocin indicate that these effects result predominantly from intermolecular dipoledipole interaction between aromatic side chain CH protons and protons of the solvent. The NOE's therefore indicate intimate contact between water and the aromatic CH hydrogens of the Tyr side chain. The extent of saturation transferred by proton exchange between water and NH group varies with Ph in a manner which appears to reflect the acid-base catalysis of the protolysis reaction. There is no indication that any NH protons are substantially shiedled from the solvent.     

Study of the spatial structure of a cyclic analog of bradykinin in solution by two-dimensional NMR spectroscopy

H NMR resonances of [cyclo (9----18) Lys1, Gly6]bradykinin (CBK) in (CD3)2SO and H2O solution have been assigned by combined analysis of two-dimensional COSY and NOESY spectra. The presence of two slowly interchangeable conformers of CBK in (CD3)2SO is established, the minor conformer not exceeding 15% in the population. The minor conformer is absent from the aqueous solution, chemical shifts of the CBK and bradykinin NH and C alpha H protons differ insignificantly. The major CBK conformer contains at least two X-Pro trans-peptide groups and three amide protons NH Phe5, NH Arg9 and N zeta H Lys1 protected from solvent. A system of cross-peaks from the NOESY spectra of CBK in (CD3)2SO has been analysed and the maximum distance between backbone protons and neighbouring amino acid residues evaluated. The experimental data agree well with the assumed type II beta-bend in the sequence Pro2-Pro3-Gly4-Phe5. Spatial structure models for the backbone fragment 6-9 of CBK containing two intramolecular hydrogen bonds that involve the NH Arg9 and N zeta H Lys1 protons and the carbonyl groups of Phe5 and Gly4 are proposed.     

A joint 2D NMR and theoretical investigation of Ca2+ binding loops III and IV of calmodulin

A 2D NMR NOESY spectrum of integral CaM in water(148 residues) reveals a series of downfield-shifted crosspeaks stemming from the NH protons of the Ca2(+)-binding loops III and IV. Their attribution, with the help of already assigned proton resonances of isolated tryptic fragments, was complemented by means of energy-minimizations on the Ca2+ complexes of loops III and IV. From these calculations, a set of two alternative, related conformations was obtained for each loop. The first type of conformation provides a coordination pattern for Ca2+ that is similar to that found in loop EF of parvalbumin. The computed interproton distances in both loops are fully compatible with the inferences from the sets of NOESY cross-peaks. Evidence is also provided for interloop interactions.     

1H NMR sequential assignments and secondary structure analysis of human fibrinogen gamma-chain C-terminal residues 385-411

The human fibrinogen gamma-chain, C-terminal fragment, residues 385-411, i.e., KIIPFNRLTIGEGQQHHLGGAKQAGDV, contains two biologically important functional domains: (1) fibrinogen gamma-chain polymerization center and (2) platelet receptor recognition domain. This peptide was isolated from cyanogen bromide degraded human fibrinogen and was investigated by 1H NMR (500 MHz) spectroscopy. Sequence-specific assignments of NMR resonances were obtained for backbone and side-chain protons via analysis of 2D NMR COSY, double quantum filtered COSY, HOHAHA, and NOESY spectra. The N-terminal segment from residues 385-403 seems to adopt a relatively fixed solution conformation. Strong sequential alpha CH-NH NOESY connectivities and a continuous run of NH-NH NOESY connectivities and several long-lived backbone NH protons strongly suggest the presence of multiple-turn or helix-like structure for residues 390 to about 402. The conformation of residues 403-411 seems to be much less constrained as evidenced by the presence of weaker and sequential alpha CH-NH NOEs, the absence of sequential NH-NH NOEs, and the lack of longer lived amides. Chemical shifts of resonances from backbone and side-chain protons of the C-terminal dodecapeptide, residues 400-411, differ significantly from those of the parent chain, suggesting that some preferred C-terminal conformation does exist.     

Application of peptide-mediated ring current shifts to the study of neurophysin-peptide interactions: a partial model of the neurophysin-peptide complex

Perdeuteriated peptides were synthesized that are capable of binding to the hormone binding site of neurophysin but that differ in the position of aromatic residues. The binding of these peptides to bovine neurophysin I and its des-1-8 derivative was studied by proton nuclear magnetic resonance spectroscopy in order to identify protein residues near the binding site through the observation of differential ring current effects on assignable protein resonances. Phenylalanine in position 3 of bound peptides was shown to induce significant ring current shifts in several resonances assignable to the 1-8 sequence, including those of Leu-3 and/or Leu-5, but was without effect on Tyr-49 ring protons. The magnitude of these shifts was dependent on the identity of peptide residue 1. By contrast, the sole demonstrable direct effect of an aromatic residue in position 1 was a downfield shift in Tyr-49 ring protons. Study of peptide binding to des-1-8-neurophysin demonstrated similar conformations of native and des-1-8 complexes except for the environment of Tyr-49, confirmed the peptide-induced ring current shift assignments in native neurophysin, and indicated an effect of binding on Thr-9. These observations are integrated with other results to provide a partial model of neurophysin-peptide complexes that places the ring of Tyr-49 at a distance 5-10 A from residue 1 of bound peptide and that places both the 1-8 sequence and the protein backbone region containing Tyr-49 proximal to each other and to peptide residue 3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the unfolding of ribonuclease a by a pulsed hydrogen exchange study: evidence for competing pathways for unfolding

The unfolding of ribonuclease A was studied in 5.2 M guanidine hydrochloride at pH 8 and 10 degrees C using multiple optical probes, native-state hydrogen exchange (HX), and pulse labeling by hydrogen exchange. First, native-state HX studies were used to demonstrate that the protein exists in two slowly interconverting forms under equilibrium native conditions: a predominant exchange-incompetent N form and an alternative ensemble of conformations, N(I), in which some amide hydrogens are fully exposed to exchange. Pulsed HX studies indicated that, during unfolding, the rates of exposure to exchange with solvent protons were similar for all backbone NH probe protons. It is shown that two parallel routes of unfolding are available to the predominant N conformation as soon as it encounters strong unfolding conditions. A fraction of molecules appears to rapidly form N(I) on one route. On the other route an exchange-incompetent intermediate state ensemble, I(U)(2), is formed. The kinetics of unfolding measured by far-UV circular dichroism (CD) were faster than those measured by near-UV CD and intrinsic tyrosine fluorescence of the protein. The logarithms of the rate constants of the unfolding reaction measured by all three optical probes also showed a nonlinear dependence on GdnHCl concentration. All of the data suggest that N(I) and I(U)(2) are nativelike in their secondary and tertiary structures. While N(I) unfolds directly to the fully exchange-competent unfolded state (U), I(U)(2) forms another intermediate I(U)(3) which then unfolds to U. I(U)(3) is devoid of all native alpha-helical secondary structure and has only 30% of the tertiary interactions still intact. Since the rates of global unfolding measured by near-UV CD and fluorescence agree well with the rates of exposure determined for all of the backbone NH probe protons, it appears that the rate-limiting step for the unfolding of RNase A is the dissolution of the entire native tertiary structure and penetration of water into the hydrophobic core.     

Epidermal growth factor from the mouse. Physical evidence for a tiered beta-sheet domain: two-dimensional NMR correlated spectroscopy and nuclear Overhauser experiments on backbone amide protons

When H2O-exchanged, lyophilized mouse epidermal growth factor (mEGF) is dissolved in deuterium oxide at low pH (i.e., below approximately 6.0), 13 well-resolved, amide proton resonances are observed in the downfield region of an NMR spectrum (500 MHz). Under the conditions of these experiments, the lifetimes of these amide protons in exchange for deuterons of the deuterium oxide solvent suggest that these amide protons are hydrogen-bonded, backbone amide protons. Several of these amide proton resonances show splittings (i.e., JNH alpha-CH) of approximately 8-10 Hz, indicating that their associated amide protons are in some type of beta-structure. Selective nuclear Overhauser effect (NOE) experiments performed on all amide proton resonances strongly suggest that all 13 of these backbone amide protons are part of a single-tiered beta-sheet structural domain in mEGF. Correlation of 2D NMR correlated spectroscopy data, identifying scaler coupled protons, with NOE data, identifying protons close to the irradiated amide protons, allows tentative assignment of some resonances in the NOE difference spectra to specific amino acid residues. These data allow a partial structural model of the tiered beta-sheet domain in mEGF to be postulated.     

Microsecond to minute dynamics revealed by EX1-type hydrogen exchange at nearly every backbone hydrogen bond in a native protein

A previous comprehensive analysis of the pH dependence of native-state amide hydrogen (NH) exchange in turkey ovomucoid third domain (OMTKY3) yielded apparent opening and closing rate constants (k(op) and k(cl)) at 14 NH groups involved in global conformational changes. This analysis has been extended to 18 additional slowly exchanging NH groups. Quench-flow experiments were performed to monitor NH exchange in native OMTKY3 from neutral to very alkaline pH ( approximately 12) conditions. Above pH 10 the mechanism of exchange switched from one governed by a rapid equilibrium preceding the chemistry of exchange (i.e. EX2 exchange), to one where exchange was limited by the rate of opening (i.e. EX1 exchange). Kinetics of solvent exposure are now known for nearly all backbone NH groups in native OMTKY3, yielding rate constants that span five orders of magnitude, 0.004 to 200 s(-1).     

Conformation of a neurokinin antagonist in solution. 2D NMR and restrained molecular dynamics study.

The hexapeptide [cyclo(Leu1 psi(CH2NH2)Leu2-Gln3-Trp4-Phe5-Gly6)]+1 is a potent antagonist of neurokinin A activity in tissues of hamster urinary bladder. The solution conformation of this cyclic hexapeptide has been characterized by the combined use of two dimensional nuclear magnetic resonance spectroscopy and restrained molecular dynamics. The proton spectrum of the peptide was fully assigned by the sequential assignment procedure. Interproton distances were derived from crosspeak volumes in two dimensional Nuclear Overhauser Effect spectra, and dihedral angles were calculated from appropriate coupling constants. Temperature coefficients of the amide protons were determined. Restrained molecular dynamics simulations were carried out using the backbone interproton distances as constraints. During 210 ps of restrained molecular dynamics the peptide interconverted among three closely related families of conformations. These interconversions occurred at picosecond timescales under the simulation conditions.     

Determination of the relative NH proton lifetimes of the peptide analogue viomycin in aqueous solution by NMR-based diffusion measurement

In aqueous solution, exchanging peptide NH protons experience two environments, that of the peptide itself with a relatively slow diffusion coefficient and that of the water solvent with a faster diffusion coefficient. Although in slow exchange on the NMR chemical shift timescale, the magnetic field gradient dependence of the NH peak intensities in an experiment used to measure diffusion coefficients reflects the relative time periods spent in the two environments and this allows the determination of the relative solvent accessibility of exchangeable protons in peptides or proteins. To test this approach, the magnetic field gradient dependent intensities of the chemically shifted amide and amine NH protons of the peptide antibiotic viomycin have been measured using the high resolution longitudinal-eddy-current-delay (LED) NMR method incorporating solvent water peak elimination by non-excitation. The NH resonances of viomycin have been assigned previously and their relative exchange rates determined. Here, the gradient dependence of each NH proton intensity is reported, and these, after a bi- exponential least squares fitting, yield the fractional lifetimes of the protons spent in the peptide and water environments during the diffusion period of the experiment.     

Infrared investigation of poly-L-histidine structure dependent on protonation

Poly-L -histidine (PLH) films at different degrees of protonation were produced mid subjected to infrared spectroscopic investigation (range 4000-650 cm^?1). In addition, the N-deuterated film spectra were plotted. The amide II and III bands show that the peptide group is present in the trans form. The amide I and II bands show that at 0% and 50% protonation the PLH occurs as an α-helix and at 100% protonation as a random coil with some ranges in β structure. At 0% and 50% protonation, no hydration water is bound to the backbone. At 0% protonation all NH groups are linked to each other or to water molecules via hydrogen bonds. At 50% protonation NH⁺?N bonds form between the imidazole rings. These protons are present in continuous energy level distribution. Such bonds with tunneling protons are extremely polarizable and between these bonds may act proton dispersion forces. The Cl^? ions are bonded to the NH groups of the imidazole groups. The hydration water is bonded to the Cl^?? ions and to the NH groups. At 100% protonation, hydration water is bonded also to the CO groups of the backbone. The NH groups of the backbone, like those of the rings, endeavor especially in the dry state to bond to the Cl^? ions. This leads to a strong steric constraint of the random coil.