Ionization properties of titratable groups in ribonuclease T1 -: I.: pKa values in the native state determined by two-dimensional heteronuclear NMR spectroscopy

pK(a) values of amino acid side chains of ribonuclease TI have been determined from the pH dependence of C-13 and N-15 resonances. It was possible to derive pK(a) values of single protonation or deprotonation sites of carboxylate and imidazole groups. Deviations from pK(a) values of free amino acids could be interpreted with electrostatic interactions of corresponding side chains with the protein environment. In particular, the interaction between H27 and E82 led to an increase of the H27 pK(a) and a decrease of the E82 pK(a). The pK(a) of E28 at the C-terminal end of the cc-helix was increased because of the dipolar character of the alpha -helix. D76 did not titrate in the investigated pH range of about 2-9. From the chemical shift value this buried side chain seems to be protonated. The pK(a) values of side chains in the active site deviate from a normal behaviour. The lower pK(a) value of E58 may be interpreted with the close proximity of this side chain with positively charged H40 and R77. A novel two-dimensional H-1(C-13(delta))C-13(gamma) correlation experiment was developed to observe the pH dependence of the chemical shifts of the C-gamma resonances of histidine residues. From the inspection of the C-gamma chemical shift-pH profiles it was possible to determine the predominant tautomeric form for the histidine residues at higher pH values.