Interconversion of the ligand arrays in the CD and EF sites of oncomodulin. Influence on Ca2+-binding affinity

The parvalbumin metal ion-binding sites differ at the +z and -x residues: Whereas the CD site employs serine and glutamate (or aspartate), respectively, the EF site employs aspartate and glycine. Although frequently indistinguishable in Ca2+- and Mg2+-binding assays, the CD and EF sites nonetheless exhibit markedly different preferences for members of the lanthanide series [Williams et al. (1984) J. Am. Chem. Soc. 106, 5698-5702], underscoring an intrinsic nonequivalence. This nonequivalence reaches its pinnacle in the mammalian beta-parvalbumin (oncomodulin). Whereas the oncomodulin EF site exhibits the expected Ca2+/Mg2+ signature, the Ca2+ affinity of the CD site is severely attenuated. To obtain insight into the structural factors responsible for this reduction in binding affinity, oncomodulin variants were examined in which the CD and EF site ligand arrays had been exchanged. Our data suggest that binding affinity may be dictated either by ligand identity or by the binding site environment. For example, the Ca2+ affinity of the quasi-EF site resulting from the combined S55D and D59G mutations is substantially lower than that of the authentic EF site. This finding implies that other local environmental variables (e.g., binding loop flexibility, electrostatic potentials) within the CD binding site supersede the influence of ligand identity. However, the CD site ligand array does not acquire a high-affinity signature when imported into the EF site, as in the D94S/G98D variant. Instead, it retains its Ca2+-specific signature, implying that this constellation of ligands is less sensitive to placement within the protein molecule. The D59G and D94S single mutations substantially lower binding affinity, consistent with removal of a liganding carboxylate. By contrast, the S55D and G98D mutations substantially increase binding affinity, a finding at odds with corresponding data collected on model peptide systems. Significantly, the Ca2+ affinity of the oncomodulin CD site is increased by mutations that weaken binding at the EF site, indicating a negatively cooperative interaction between the two sites.     

Phase structure and temperature dependent luminescence properties of Sr2LiSiO4F: Eu2+ and Sr2MgSi2O7: Eu2+ phosphors

Green-emitting Sr2LiSiO4F:Eu2+ and blue-emitting Sr2MgSi2O7:Eu2+ phosphors were synthesized by the conventional high temperature solid-state route,respectively.Their structures and photoluminescenee properties were comparatively investigated.It was found that the mixture phases of Sr2MgSi2O7 and SrF2 were obtained when a part of Sr2+ in Sr2LiSiO4F was replaced by some amount of Mg2+ in order to design the possible SrMgLiSiO4F:Eu2+ phosphor.Based on the photoluminescence analysis,Sr2LiSiO4F:Eu2+ phosphor exhibited a green broad emission band of main peak at 513 nm under the excitation of 365 nm,while the Sr2MgSi2O7:Eu2+ and SrMgLiSiO4F:Eu2+ phosphor showed blue emission centered at 467 nm.The temperature dependent photoluminescence properties and room temperature decay time for the three kinds of phosphors were also discussed in this paper.     

Werner syndrome protein. II. Characterization of the integral 3' --> 5' DNA exonuclease

In addition to its DNA helicase activity, Werner syndrome protein (WRN) also possesses an exonuclease activity (Shen, J.-C., Gray, M. D., Kamath-Loeb, A. S., Fry, M., Oshima, J., and Loeb, L. A. (1998) J. Biol. Chem. 273, 34139-34144). Here we describe the properties of nearly homogeneous WRN exonuclease. WRN exonuclease hydrolyzes a recessed strand in a partial DNA duplex but does not significantly digest single-stranded DNA, blunt-ended duplex, or a protruding strand of a partial duplex. Although DNA is hydrolyzed in the absence of nucleoside triphosphates, nuclease activity is markedly stimulated by ATP, dATP, or CTP. WRN exonuclease digests DNA with a 3' --> 5' directionality to generate 5'-dNMP products, and DNA strands terminating with either a 3'-OH or 3'-PO4 group are hydrolyzed to similar extents. A recessed DNA strand with a single 3'-terminal mismatch is hydrolyzed more efficiently by WRN than one with a complementary nucleotide, but the enzyme fails to hydrolyze a DNA strand terminating with two mismatched bases. WRN exonuclease is distinguished from known mammalian DNA nucleases by its covalent association with a DNA helicase, preference for a recessed DNA strand, stimulation by ATP, ability to equally digest DNA with 3'-OH or 3'-PO4 termini, and its preferential digestion of DNA with a single 3'-terminal mismatch.