Ionization properties of titratable groups in ribonuclease T-1 - I. pK(a) values in the native state determined by two-dimensional heteronuclear NMR spectroscopy
N. Spitzner et al., Ionization properties of titratable groups in ribonuclease T-1 - I. pK(a) values in the native state determined by two-dimensional heteronuclear NMR spectroscopy, EUR BIOPHYS, 30(3), 2001, pp. 186-197
Citations number
63
Categorie Soggetti
Biochemistry & Biophysics
Journal title
EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS
pK(a) values of amino acid side chains of ribonuclease TI have been determi
ned 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 carboxy
late 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 betwee
n 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 increase
d 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 t
his buried side chain seems to be protonated. The pK(a) values of side chai
ns in the active site deviate from a normal behaviour. The lower pK(a) valu
e of E58 may be interpreted with the close proximity of this side chain wit
h 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 dependenc
e of the chemical shifts of the C-gamma resonances of histidine residues. F
rom the inspection of the C-gamma chemical shift-pH profiles it was possibl
e to determine the predominant tautomeric form for the histidine residues a
t higher pH values.