The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by ¹H Nuclear Magnetic Resonance

Abstract

1. The aromatic proton resonances in the 360-MHz 'H 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-1 19) and two tyrosines are accessible 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-1 15 and the C-4 proton of His-12 were identified by comparison with subtilisin-modified and nitrated ribonucleases. Other resonances were assigned tentatively to 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" about their CP-Cc" 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 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.

15N‐ and 1H‐NMR investigations of the active‐site amino acids in semisynthetic RNase S′ and RNase A

“…As has been discussed in former publications a conformational change, in which Asp-14, Tyr-25 and His-48 are involved, takes place at neutral pH [2,14,69,70]. However, the fact that the inflection points are dependent on ionic strength in the same manner as the pK values of the activesite histidines makes it more likely that the inflection is correlated with the titration behaviour of the histidines.…”

Sequential 1H‐NMR assignment and solution structure of bovine pancreatic ribonuclease A

“…A large single resonance in the aromatic region at 6 = 6.89 ppm was assigned by Lenstra et al [19] to the ortho and meta protons of Y92, accidentally coincident. A strong NOE effect is actually observed at this position with the CcrH resonance (6 = 3.64 ppm) already assigned to R39.…”

“…Interestingly, Lenstra et al [19] had assigned the corresponding ortho and meta proton resonances to Tyrl15 on an independent basis (titration behaviour, chemically induced dynamic nuclear polarisation, solvent exposure, chemical modification, etc. ).…”

“…overlapping such as the aromatic or methyl regions (19,21,261. Two-dimensional (2D) 'H-NMR sequence-specific procedures [27] were applied to RNase A by Hahn and Ruterjans [24], who assigned proton resonances to 21 amino acid moieties.…”

Two-dimensional 1H NMR investigation of ribonuclease A and ribonuclease-A-pyrimidine-nucleotide complexes

“…Based on the 2D procedures, developed by Wuthrich et al [22,23], and more recently developed methods [24] we were able to identify many amino acids spin systems as well as to assign a number of side-chain and backbone 'H NMR resonances to specific amino acid residues even in this large protein. This information provides a powerful tool to monitor structural changes in the protein.…”