Amino group environments and metal binding properties of carbon-13 reductively methylated bovine alpha-lactalbumin.
13C NMR spectroscopy has been used to study the amino group environments and metal binding properties of 13C reductively methylated bovine alpha-lactalbumin. Bovine alpha-lactalbumin is a Ca2+ metalloprotein containing 12 lysyl amino groups and a free amino terminus. All 13 amino groups can be 13C-dimethylated without altering Ca2+ binding or biological activity. pH titrations (chemical shift vs. pH) of this dimethylated protein reveal unique behavior for each of the 13 amino groups. The pKa values for the lysyl amino groups range from 9.1 to 10.8 while the pKa for the N-terminal amino group is 8.3. This relatively high pKa (by 1 pH unit) for the N-terminal supports its interaction in an ion pair as proposed by Warme et al. [Warme, P. K., Momany, F. A., Rumball, S. V., Tuttle, R. W., & Scheraga, H. A. (1974) Biochemistry 13, 768-782]. Carbon-13 NMR studies further show that the removal of Ca2+ from the high-affinity binding site results in a conformational change, with the disruption of the N-terminal ion pair interaction (pKa decreased to 7.4). The study of Zn2+ binding to Ca2+-saturated protein suggests that Zn2+ binds initially at a low-affinity Ca2+ site while maintaining the N-terminal ion pair interaction. The further addition of Zn2+ leads to the disruption of this ion pair forming a presumed apoprotein-like conformation. Finally on the basis of the specific effects of added Mn2+ on the 13C NMR spectra of the methylated protein, a low-affinity divalent metal binding site is proposed about 7.5 A from the amino terminus.
D000818:Animals; D002118:Calcium / Q000032:analysis; D002247:Carbon Isotopes; D002250:Carbon Radioisotopes; D002417:Cattle; D002712:Chlorides; D005557:Formaldehyde / Q000494:pharmacology*; D005700:Galactosyltransferases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007768:Lactalbumin / Q000302:isolation & purification / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000494:pharmacology; D017895:Manganese Compounds; D008745:Methylation; D008958:Models, Molecular; D011487:Protein Conformation; D014531:Uridine Diphosphate Galactose / Q000378:metabolism; D015032:Zinc / Q000032:analysis
D002247:Carbon Isotopes; D002250:Carbon Radioisotopes; D002712:Chlorides; D017895:Manganese Compounds; D005557:Formaldehyde; D014531:Uridine Diphosphate Galactose; D008345:Manganese; D007768:Lactalbumin; D005700:Galactosyltransferases; D015032:Zinc; C025340:manganese chloride; D002118:Calcium