Characterization of pH-dependent conformational heterogeneity in Rhodospirillum rubrum cytochrome c2 using 15N and 1H NMR

The 15N-enriched ferricytochrome c2 from Rhodospirillum rubrum has been studied by 15N and 1H NMR spectroscopy as a function of pH. The 15N resonances of the heme and ligand tau nitrogen are broadened beyond detection because of paramagnetic relaxation. The 15N resonance of the ligand histidine phi nitrogen was unambiguously identified at 184 ppm (pH 5.6). The 15N resonances of the single nonligand histidine are observed only at low pH, as in the ferrocytochrome because of the severe broadening caused by tautomerization. The dependence of the 15N and 1H spectra of the ferricytochrome on pH indicated that the ligand histidine tau NH does not dissociate in the neutral pH range and is involved in a hydrogen bond, similar to that in the reduced state. Because neither deprotonated nor non-hydrogen-bonded forms of the ligand histidine are observed in the spectra of either oxidation state, the participation of such forms in producing heterogeneous populations having different electronic g tensors is ruled out. Transitions having pKa's of 6.2, 8.6, and 9.2 are observed in the ferricytochrome. The localized conformational change around the omega loops is observed in the neutral pH range, as in the ferrocytochrome. Structural heterogeneity leads to multiple resonances of the heme ring methyl at position 8. The exchange rate between the conformations is temperature dependent. The transition with a pKa of 6.2 is assigned to the His-42 imidazole group. The displacement of the ligand methionine, which occurs with a pKa of 9.2, causes gross conformational change near the heme center.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assignments of 15N and 1H NMR resonances and a neutral pH ionization in Rhodospirillum rubrum cytochrome c2

The phi NH proton and 15N resonances of the ligand histidine of Rhodospirillum rubrum fericytochrome c2 are found at 14.7 and 184 ppm, respectively, contradicting the proposal that this proton is absent in the R. rubrum ferricytochrome. Substitution of the deuterium atom for this proton causes small upfield shifts of the phi nitrogen in both oxidation states, indicating that the phi NH-peptide carboxyl hydrogen bond is not substantially weakened by the substitution. The proton and 15N resonances of the indolic NH group of the invariant tryptophan-62 and numerous proton resonances of the heme and extraheme ligands in the spectrum of the ferricytochrome are also assigned. An ionization in the ferrocytochrome occurring at neutral pH is assigned to the single nonligand histidine. This attribution is supported by the direct measurement of the ionization by NOE difference spectroscopy and by comparative structural arguments involving closely related cytochromes and chemically modified cytochromes.     

Proton nuclear magnetic resonance studies of Rhodospirillum rubrum cytochrome.

Rhodospirillum rubrum cytochrome c2 was studied by proton nuclear magnetic resonance at 220 MHz. Assignments were made to the resonances of heme c by double-resonance techniques and by temperature-dependence studies. The aromatic resonances of Trp-62 and Tyr-70 of ferrocytochrome c2 were identified by spin-decoupling experiments. The resonances of the Met-91 methyl group of the ferri- and ferrocytochromes were assigned by saturation-transfer experiments. The assignments are compared to those made for cytochromes c. A pH titration showed that the methionine methyl resonance of ferricytochrome c2 shifted with a pK of 6.25 and disappeared above pH 9. No histidine CH resonances that titrated normally over the neutral pH range were observed in the spectrum of either oxidation state of the protein. The possible origins of the ionizations at pH 6.25 and 9 are discussed.     

Fluorescence spectrum of barnase: contributions of three tryptophan residues and a histidine-related pH dependence

Fluorescence spectra of wild-type barnase and mutants in which tryptophan and histidine residues have been substituted have been analyzed to give the individual contributions of the three tryptophan residues. The spectrum is dominated by the contribution of Trp-35. The fluorescence intensity varies with pH according to an ionization of a pKa of 7.75. This pKa is close to that previously determined by NMR titration of the C2-H resonances of His-18 as a function of pH (Sali et al., 1989). This histidine residue is close to Trp-94. The pH dependence of the spectrum is abolished when either His-18 or Trp-94 is mutated, and so appears to be caused by the His-18/Trp-94 interaction. The spectral response of this interaction can serve as a probe of the folding pathway and of electrostatic effects within the protein. Changes in the fluorescence spectra on substitution of Trp-94 and His-18 suggest that there is net energy transfer from Trp-71 to Trp-94.     

Proton nuclear magnetic resonance study of cobrotoxin.

Cobrotoxin (Mr 6949), which binds tightly to the acetylcholine receptors, contains no phenylalanines and only two histidines, two tyrosines, and one tryptophan that result in well-resolved aromatic proton resonances in D2O at 360 MHz. His-32, Tyr-25, and the Trp are essential for toxicity and may interact with the acetylcholine receptor. We assign two titratable resonances (pKa = 5.1) at delta = 9.0 and 7.5 ppm at pH 2.5 and at 7.7 and 7.1 ppm at pH 9.5 to the C-2 and C-4 ring protons, respectively, of His-4. Two other titratable resonances (pKa = 5.7) at delta = 8.8 and 6.9 ppm at pH 2.5 and at 7.8 and 6.7 ppm at pH 9.5 are assigned to the C-2 and C-4 ring protons of His-32, respectively. The differences in delta values of the two histidines reflect chemically different microenvironments while their low pKa values could arise from nearby positive charges. A methyl resonance gradually shifts upfield to delta approximately 0.4 ppm as His-4 is deprotonated and is tentatively assigned to the methyl group of Thr-14 or Thr-15 which, from published X-ray studies of neurotoxins, are located in the vicinity of His-4. Further, we have identified the aromatic resonances of the invariant tryptophan and individual tyrosines and the methyl resonance of one of the two isoleucines in the molecule. Several broad nontitrating resonances of labile protons which disappear at pH greater than 9 may arise from amide groups of the beta sheet in cobrotoxin.     

High-resolution 1H- and 15N-NMR studies of Rhodospirillum rubrum cytochrome c2.

Rhodospirillum rubrum cytochrome c2 was uniformly enriched in 15N and studied by 1H- and 15N-NMR spectroscopy. Relaxation and NOE data allowed determination of the rotational correlation time and indicated more rapid side-chain motion in the native protein and increased segmental motion in the base-denatured protein. The pi nitrogen of the ligand histidine and the indolic nitrogen of the invariant tryptophan both remain protonated and act as proton-donors in hydrogen bonds over a wide pH range and therefore do not contribute to pH-related changes in the midpoint potential. pK values identified by numerous methods in the ferrocytochrome at pH 6.9 and in the ferricytochrome at pH 6.2 arise from His-42. At pH values below the pK, the imidazolium group participates in a salt bridge or in a hydrogen bond with the carboxylate group of the inner propionate of the heme. Loss of the proton causes a local conformational change which alters the midpoint potential. The pK values of the amino terminus and lysines were also determined from pH titrations monitored by 15N-NMR. Similar titrations of the ferricytochrome monitored by 1H-NMR showed structural heterogeneity in that the resonance of heme ring methyl 8 split into a doublet as the pH was raised.     

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.     

Heme pocket interactions in cytochrome c peroxidase studied by site-directed mutagenesis and resonance Raman spectroscopy

Resonance Raman spectra are reported for FeII and FeIII forms of cytochrome c peroxidase (CCP) mutants prepared by site-directed mutagenesis and cloning in Escherichia coli. These include the bacterial "wild type", CCP(MI), and mutations involving groups on the proximal (Asp-235----Asn, Trp-191----Phe) and distal (Trp-51----Phe, Arg-48----Leu and Lys) side of the heme. These spectra are used to assess the spin and ligation states of the heme, via the porphyrin marker band frequencies, especially v3, near 1500 cm-1, and, for the FeII forms, the status of the Fe-proximal histidine bond via its stretching frequency. The FeII-His frequency is elevated to approximately 240 cm-1 in CCP(MI) and in all of the distal mutants, due to hydrogen-bonding interactions between the proximal His-175 N delta and the carboxylate acceptor group on Asp-235. The FeII-His RR band has two components, at 233 and 246 cm-1, which are suggested to arise from populations having H-bonded and deprotonated imidazole; these can be viewed in terms of a double-well potential involving proton transfer coupled to protein conformation. The populations shift with changing pH, possibly reflecting structure changes associated with protonation of key histidine residues, and are influenced by the Leu-48 and Phe-191 mutations. A low-spin FeII form is seen at high pH for the Lys-48, Leu-48, Phe-191, and Phe-51 mutants; for the last three species, coordination of the distal His-52 is suggested by a approximately 200-cm-1 RR band assignable to Fe(imidazole)2 stretching.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton-nuclear-magnetic-resonance study of the conformation of neurotoxin II from Middle-Asian cobra (Naja naja oxiana) venom.

A proton nuclear magnetic resonance (NMR) study at 100 and 300 MHz of neurotoxin II from the venom of Middle-Asian cobra Naja naja oxiana has been performed in 2H2O and H2O solutions. By means of chemical modification and double resonance all the aromatic residue resonances have been assigned. From the NMR titration curves, pK values of histidine 4 and histidine 31 residues have been determined. For one of the two neighbouring tryptophan residues pH dependence (in the 2-8-pH range) of the chemical shifts of indole protons has been revealed. According to the different sensitivity of the linewidth of indole NH resonances to pH in H2O solution, the accessibility of each of the tryptophan residues has been estimated. Temperature dependence has been observed for the linewidth of the aromatic resonances of the tyrosine 24 residue. Deuterium exchange rates have been measured for amide protons as well as for C(2)H histidine resonances. The NMR data obtained have allowed the conclusions to be made that the two histidine residues and one of the tryptophan residues should be localized on the surface of the protein globule, that arginine residues should be present in the environment of histidine 4, that histidine 31 and the buried tryptophan are possibly localized in close spatial proximity and that the side chain of tyrosine 24 is buried within the protein globule.     

Proton nuclear magnetic resonance characterization of phospholipase A2 from Laticauda semifasciata

The molecular properties of phospholipases (PLases) A2 I and A2 III from a sea snake, Laticauda semifasciata, have been characterized by gel-filtration, as well as proton NMR, CD, UV absorption, and fluorescence spectroscopic methods. PLase A2 I exists as a monomer in aqueous solution in the presence or in the absence of Ca2+. The dissociation constants of the Ca2+-enzyme complexes have been determined for the two enzymes. The 270-mHz proton NMR spectra of PLases A2 I and A2 III have been measured, and the aromatic proton resonances of His-21 and His-48 in the active site have been assigned. By analyzing the pH dependence of the chemical shifts of the histidine proton resonances, pKa values have been determined for His-21 and His-48 with and without Ca2+. The conformational transitions have been found to take place at low pH or at high temperature (at approximately 65 degrees C). Fluorescence change of PLase A2 I upon addition of substrate analogs suggests that Trp-70 in PLase A2 I is involved in the binding to micellar substrates. The lack of Trp-70 in PLase A2 III is probably related to the low enzymatic activity as compared with that of PLase A2 I.     

15N NMR spectroscopy of Pseudomonas cytochrome c-551

15N-1H correlation spectroscopy with detection at the 1H frequency has been used at natural abundance to detect nitrogen nuclei bonded to protons in the ferrocytochrome c-551 from Pseudomonas aeruginosa (ATCC 19429). Side-chain aromatic nitrogens, main-chain amides, and side-chain amides have been assigned to specific residues by comparison to previous proton assignments. Assignment ambiguities arising from overlap in the proton dimension have been resolved by examining spectra as a function of temperature and pH. Nitrogen chemical shifts are reported at pH 4.6 and 9.4 and three temperatures, 32, 50, and 60 degrees C. Significant differences arise from the observed protein shifts and expected shifts in the random coil polypeptide.     

Proton NMR assignments of heme contacts and catalytically implicated amino acids in cyanide-ligated cytochrome c peroxidase determined from one- and two-dimensional nuclear Overhauser effects.

Proton NMR assignments of the heme pocket and catalytically relevant amino acid protons have been accomplished for cyanide-ligated yeast cytochrome c peroxidase. This form of the protein, while not enzymatically active itself, is the best model available (that displays a resolvable proton NMR spectrum) for the six-coordinate low-spin active intermediates, compounds I and II. The assignments were made with a combination of one- and two-dimensional nuclear Overhauser effect methods and demonstrate the utility of NOESY experiments for paramagnetic proteins of relatively large size (Mr 34,000). Assignments of both isotope exchangeable and nonexchangeable proton resonances were obtained by using enzyme preparations in both 90% H2O/10% D2O and, separately, in 99.9% D2O solvent systems. Complete resonance assignments have been achieved for the proximal histidine, His-175, and His-52, which is a member of the catalytic triad on the distal side of the heme. In addition, partial assignments are reported for Trp-51 and Arg-48, catalytically important residues, both on the distal side. Aside from His-175, partial assignments for amino acids on the proximal side of the heme are proposed for the alanines at primary sequence positions 174 and 176 and for Thr-180 and Leu-232.     

pH dependence of tritium exchange with the C-2 protons of the histidines in bovine trypsin.

At pH 8.9 and 37 degrees C the half-times for tritium exchange with the C-2 protons of the histidines of trypsin are 73 days for His-57, and greater than 1000 days for His-40 and His-91. These half-times are much longer than the half-life of exchange for the C-2 proton of free histidine (2.8 days at pD 8.2), and longer than any previously reported half-time of exchange at pH greater than 8. These very low rates of exchange are discussed with reference to the refined structure of trypsin. The tritium exchange of His-57 depends on an apparent pKa of 6.6. This pKa may represent the pKa of the imidazole of His-57 in an inactive conformation of the enzyme.     

Sequential NMR resonance assignment and secondary structure of ferrocytochrome c553 from Desulfovibrio vulgaris Hildenborough.

Two-dimensional nuclear magnetic resonance spectroscopy was used to assign the proton resonances of ferrocytochrome c553 from Desulfovibrio vulgaris Hildenbourough at 37 degrees C and pH = 5.9. Only a few side-chain protons were not identified because of degeneracy or overlap. The spin systems of the 79 amino acids were identified by DQF-COSY and HOHAHA spectra in H2O and D2O. Sequential assignments were obtained from NOESY connectivities between adjacent amide, C alpha H, and C beta H protons. From sequential NH(i)----NH(i + 1) and long-range C alpha H(i)----NH(i + 3) connectivities, four stretches of helices were identified (2----8, 34----46, 53----59, 67----77). Long-range NOE between residues in three different helices provide qualitative information on the tertiary structure, in agreement with the general folding pattern of cytochrome c. The heme protons, including the propionate groups, were assigned, and the identification of Met 57 as sixth heme ligand was established. The dynamical behavior of the ring protons of the six tyrosines was analyzed in detail in terms of steric hindrance. The NMR data for ferrocytochrome c553 are consistent with the X-ray structure for the homologous cytochrome from D. vulgaris Miyazaki. On the basis of the secondary structure element and of observed chemical shift due to the heme ring current, a structural alignment of eukaryotic and prokaryotic cytochromes c is proposed.     

Conformation of cobrotoxin in aqueous solution as studied by nuclear magnetic resonance.

The 270-MHz proton NMR spectra of cobrotoxin from Naja naja atra were observed in 2H2O solution. The pKa value (5.93) of His-32 is slightly lower than the pKa value (6.65) of the reference model of N-acetylhistidine methylamide, because of the electrostatic interaction with Arg-33 and Asp-31. The pKa value (5.3--5.4) of His-4 is appreciably low, because of the interaction with the positively charged guanidino group possibly of Arg-59. The hydrogen-deuterium exchange rates in 2H2O solution were measured of cobrotoxin and imidazole-bearing models. The second-order rate constants of N-acetylhistidine methylamide, N-acetylhistidine and imidazole acetic acid satisfy the Br?nsted relation. With reference to this Br?nsted relation, the imidazole ring of His-32 is confirmed to be exposed. The imidazole ring of His-4 is also exposed and the exchange rate is excessively promoted by the presence possibly of Arg-59 in the proximity. All the methyl proton resonances are assigned to amino-acid types, by conventional double-resonance method and more effectively by the spin-echo double-resonance method. Eight methyl proton resonances are identified as due to the gamma and/or delta-methyl groups of Val-46, Leu-1, Ile-50 and Ile-52 residues. The proximity of aromatic ring protons and methyl protons is elucidated by the analyses of nulcear Overhauser effect enhancements. The aromatic proton resonances of Trp-29 are affected by the ionizable groups of Asp-31, His-32 and Tyr-35. The methyl groups of Ile-50 are in the proximity to the aromatic ring of Trp-29 and the methyl groups of Ile-52 are in the proximity to Tyr-25. The highest-field methyl proton resonance is due to a threonine residue in the proximity to His-4. The appreciable temperature-dependent chemical shift of this methyl proton resonance suggests a temperature-dependent local conformational equilibrium around the His-4 residue of the first loop of the cobrotoxin molecule.     

Identification by proton nuclear magnetic resonance of the histidines in cytochrome b5 modified by diethyl pyrocarbonate

Diethyl pyrocarbonate (DEP) is an electrophilic reagent that is used to modify reversibly the histidine residues of proteins. Unfortunately, the lability of the acylated histidine adduct usually does not permit the isolation and identification of the modified histidine. By use of 500-MHz proton NMR spectroscopy, it has been possible to identify the C-H resonances of the nonaxial histidines of trypsin-solubilized bovine, rabbit, and porcine cytochrome b5 and therefore observe the interaction of DEP with specific histidine residues of cytochrome b5. In addition, the pKa of the peripheral histidines of bovine and rabbit cytochrome b5 have been measured in D2O. In the bovine protein it was found that the histidines are modified sequentially with increasing DEP concentration in the order His-26 greater than His-15 greater than His-80. This order is maintained in the rabbit protein with the following additions: His-26 approximately His-27 greater than His-15 greater than or equal to His-17 greater than His-80. The relative reactivity of the peripheral histidines with DEP was rationalized by considering three of their characteristics: (1) the pKa of the histidine, (2) the fraction of the side chain exposed to the solvent, and (3) the hydrogen-bond interactions of the imidazole ring.     

1H, 13C, and 15N resonance assignments for a ferrocytochrome c553 heme by multinuclear NMR spectroscopy

A novel strategy has been used to assign the 1H, 13C, and 15N resonances of the heme in Anabaena 7120 ferrocytochrome c553. 13C[13C] double-quantum coherence spectroscopy was used to delineate the heme carbons, 1H[13C] single-bond correlation spectroscopy was used to define the attached protons, and 1H[15N] multiple-bond correlation spectroscopy was used to assign the nitrogens. 1H[13C] multiple-bond correlation spectroscopy confirmed many of the assignments. Proteins were labeled uniformly with 13C or 15N to obtain the required spectral sensitivity.     

Assignment of 1H NMR resonances of histidine and other aromatic residues in met-, cyano-, oxy-, and (carbon monoxy)myoglobins

The resolved 1H NMR resonances of the aromatic region in the 270-MHz NMR spectrum of sperm whale, horse, and pig metmyoglobin (metMb) have been assigned, including the observable H-2 and H-4 histidine resonances, the tryptophan H-2 resonances, and upfield-shifted resonances from one tyrosine residue. The use of different Mb species, carboxymethylation, and matching of pK values allows the assignment of the H-4 resonances, which agree in only three cases out of seven with scalar-correlated two-dimensional NMR spectroscopy assignments by others. The conversion to hydroxymyoglobin at high pH involves rearrangements throughout the molecule and is observed by many assigned residues. In sperm whale ferric cyanomyoglobin, nine H-2 and eight H-4 histidine resonances have been assigned, including the His-97 H-2 resonance and tyrosine resonances from residues 103 and 146. The hyperfine-shifted resonances from heme and near-heme protons observe a shift with a pK = 5.3 +/- 0.3 (probably due to deprotonation of His-97, pK = 5.6) and another shift at pK = 10.8 +/- 0.3. The spectrum of high-spin ferrous sperm whale deoxymyoglobin is very similar to that of metMb, which allows the assignment of seven surface histidine H-2 and H-4 resonances and also resonances from the two tryptophan residues and one tyrosine. In diamagnetic sperm whale (carbon monoxy)myoglobin (COMb), 10 His H-2 and 11 His H-4 resonances are observed, and 8 H-2 and 9 H-4 resonances are assigned, including His-64 H-4, the distal histidine. This important resonance is not observed in sperm whale oxymyoglobin, which in general shows very similar titration curves to COMb. Histidine-36 shows unusual titration behavior in the paramagnetic derivatives but normal behavior in the diamagnetic derivatives, which is discussed in the accompanying paper [Bradbury, J. H., & Carver, J. A. (1984) Biochemistry (following paper in this issue)].     

An oxidized tryptophan facilitates copper binding in Methylococcus capsulatus-secreted protein MopE

Proteins can coordinate metal ions with endogenous nitrogen and oxygen ligands through backbone amino and carbonyl groups, but the amino acid side chains coordinating metals do not include tryptophan. Here we show for the first time the involvement of the tryptophan metabolite kynurenine in a protein metal-binding site. The crystal structure to 1.35 angstroms of MopE* from the methane-oxidizing Methylococcus capsulatus (Bath) provided detailed information about its structure and mononuclear copper-binding site. MopE* contains a novel protein fold of which only one-third of the structure displays similarities to other known folds. The geometry around the copper ion is distorted tetrahedral with one oxygen ligand from a water molecule, two histidine imidazoles (His-132 and His-203), and at the fourth distorted tetrahedral position, the N1 atom of the kynurenine, an oxidation product of Trp-130. Trp-130 was not oxidized to kynurenine in MopE* heterologously expressed in Escherichia coli, nor did this protein bind copper. Our findings indicate that the modification of tryptophan to kynurenine and its involvement in copper binding is an innate property of M. capsulatus MopE*.     

The aromatic residues of bovine pancreatic ribonuclease studied by 1H nuclear magnetic resonance.

1. The aromatic proton resonances in the 360-MHz 1H nuclear magnetic resonance (NMR) spectrum of bovine pancreatic ribonuclease were divided into histidine, tyrosine and phenylalanine resonances by means of pH titrations and double resonance experiments. 2. Photochemically induced dynamic nuclear polarization spectra showed that one histidine (His-119) and two tyrosines are accessibly to photo-excited flavin. This permitted the identification of the C-4 proton resonance of His-119. 3. The resonances of the ring protons of Tyr-25, Tyr-76 and Tyr-115 and the C-4 proton of His-12 were identified by comparison with subtilisin-modified and nitrated ribonucleases. Other resonances were assigned tentatively to Tyr-73, Tyr-92 and Phe-46. 4. On addition of active-site inhibitors, all phenylalanine resonances broadened or disappeared. The resonance that was most affected was assigned tentatively to Phe-120. 5. Four of the six tyrosines of bovine RNase, identified as Tyr-76, Tyr-115 and, tentatively, Tyr-73 and Tyr-92, are titratable above pH 9. The rings of Tyr-73 and Tyr-115 are rapidly rotating or flipping by 180 degrees about their C beta--C gamma bond and are accessible to flavin in photochemically induced dynamic nuclear polarization experiments. Tyr-25 is involved in a pH-dependent conformational transition, together with Asp-14 and His-48. A scheme for this transition is proposed. 6. Binding of active-site inhibitors to bovine RNase only influences the active site and its immediate surroundings. These conformational changes are probably not connected with the pH-dependent transition in the region of Asp-14, Tyr-25 and His-48. 7. In NMR spectra of RNase A at elevated temperatures, no local unfolding below the temperature of the thermal denaturation was observed. NMR spectra of thermally unfolded RNase A indicated that the deviations from a random coil are small and might be caused by interactions between neighbouring residues.