title: High-resolution proton and laser photochemically induced dynamic nuclear polarization NMR studies of cation binding to bovine alpha-lactalbumin.
abstract: alpha-Lactalbumin (alpha-LA) is a calcium binding protein that also binds Mn(II), lanthanide ions, A1(III), Zn(II), Co(II). The structural implications of cation binding were studied by high-resolution proton (200 MHz) NMR and photochemically induced dynamic nuclear polarization (CIDNP) spectroscopy. Marked changes were observed in the NMR spectra of the apoprotein upon addition of a stoichiometric amount of calcium to yield Ca(II)-alpha-LA, manifested particularly in ring current shifted aliphatic peaks and in several shifts in the aromatic region, all of which were under slow exchange conditions. The CIDNP results showed that two surface-accessible tyrosine residues, assigned as Tyr-18 and -36, became inaccessible to the solvent upon addition of 1:1 Ca(II) to apo-alpha-lactalbumin, while Tyr-103 and Trp-104 remained completely accessible in both conformers. The proton NMR spectra of apo-alpha-LA and A1(III)-alpha-LA were extremely similar, which was also consistent with intrinsic fluorescence results [Murakami, K., & Berliner, L. J. (1983) Biochemistry 22, 3370-3374]. The paramagnetic cation Mn(II) bound to the strong calcium binding site on apo-alpha-LA but also to the weak secondary Ca(II) binding site(s) on Ca(II)-alpha-LA. It was also found that Co(II) bound to some secondary sites on Ca(II)-alpha-LA that overlapped the weak calcium site. All of the lanthanide shift reagents [Pr(III), Eu(III), Tb(III), Dy(III), Tm(III), Yb(III)] bound under slow exchange conditions; their relative affinities for apo-alpha-lactalbumin from competitive binding experiments were Dy(III), Tb(III), and Pr(III) greater than Ca(II) greater than Yb(III).
authors: Department of Chemistry, Ohio State University, Columbus 43210.
authors: L J
authors: LJ
authors: Berliner
authors: K
authors: K
authors: Koga
authors: H
authors: H
authors: Nishikawa
authors: J E
authors: JE
authors: Scheffler
mesh_terms: D000818:Animals; D002412:Cations; D002417:Cattle; D003035:Cobalt / Q000378:metabolism; D007768:Lactalbumin / Q000378:metabolism*; D007834:Lasers; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008345:Manganese / Q000378:metabolism; D010777:Photochemistry; D011485:Protein Binding
chemical_list: D002412:Cations; D003035:Cobalt; D008345:Manganese; D007768:Lactalbumin