PHOSPHOPHORYN, AN ACIDIC BIOMINERALIZATION REGULATORY PROTEIN - CONFORMATIONAL FOLDING IN THE PRESENCE OF CD(II)

被引:20
作者
EVANS, JS [1 ]
CHIU, T [1 ]
CHAN, SI [1 ]
机构
[1] CALTECH,ARTHUR AMOS NOYES LAB CHEM PHYS,DIV CHEM & CHEM ENGN,PASADENA,CA 91125
关键词
D O I
10.1002/bip.360341008
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The divalent cation-induced protein folding properties of the template macromolecule, bovine dentine phosphophoryn (BDPP), have been examined by H-1/P-31/C-113/Cd-113-nmr spectroscopy. Cd(II) was employed, exploiting the sensitivity of Cd-113-nmr to ligand-binding interactions and kinetics. Cation binding was studied over the stoichiometric range of 0-50 : 1 Cd(II) :protein (mole ratio), well below the range of Cd(II) concentration required to induce protein precipitation. The stepwise titration of divalent cation-depleted phosphophoryn at pH 7.2 in H2O/D2O with (CdCl2)-Cd-113 revealed that (PSer)(n), (PSerAsp)(n), and (Asp)(n), polyelectrolyte cation-binding domains undergo two major transitions in their secondary and tertiary structures: the first transition, occurring between 1 : 10 and 1 : 1 Cd(II) : protein stoichiometry, and the second, between 10 : 1 and 50 : 1. By monitoring the amide NH intensities, P-31-nmr chemical shift, and C-13 Asp-C-gamma resonances, it was concluded that Cd(II) ions exhibit a binding-site preference for polyelectrolyte cation-binding domains, in the order (PSer)(n) > (PSerAsp)(n) > (Asp)(n) This preference correlates with the degree of negative charge density for each sequence motif. Accompanying the backbone conformational transitions at the polyelectrolyte regions were conformational transitions in the flanking hinge domains, indicating that the hinge domains participate in the folding of the phosphophoryn molecule as divalent cation binding occurs at the polyelectrolyte domains. We were unsuccessful in detecting phosphophoryn-bound Cd(II) species by Cd-113-nmr because of chemical exchange modulation. However, using a smaller 21-residue peptide mimetic of phosphophoryn, we have observed three stoichiometric-dependent Cd-113 resonances that differ in terms of the oxoanion coordination number. Our observation of multiple Cd(II) species in the presence of the peptide supports our contention that Cd(II) has many chemically distinct coordination sites on phosphophoryn, each in multiple equilibria with H2O, Cl-, and side-chain oxoatoms. (C) 1994 John Wiley and Sons, Inc.
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页码:1359 / 1375
页数:17
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