A ~(15)N NMR Mobility Study on the Dicalcium P43M Calbindin D_(9k) and Its Mono-La~(3+)-Substituted Form

Ivano Bertini; Carl J. Carrano; Claudio Luchinat; Mario Piccioli; Luisa Poggi

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A ~(15)N NMR Mobility Study on the Dicalcium P43M Calbindin D_(9k) and Its Mono-La~(3+)-Substituted Form

机译：A〜（15）NMR流动性研究P43M Calbindin D_（9K）及其单 - La〜（3 +）取代的形式

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Calbindin D_(9k) is a dicalcium binding protein consisting of two helix-loop-helix EF-hand motifs joined together by a flexible linker region where one metal ion can bind to each of the two loops. A proline residue at position 43 in the linker region displays cis-trans isomerism in the wild-type (WT) protein. Such isomerism appeared to be removed by substituting the proline with a glycine or a methionine in the P43G or P43M mutant. We have extended the available mobility studies on the P43M mutant through amide ~(15)N R_1, R_2, and R_(1#sigma#) measurements. This has revealed unexpected conformational equilibria on the millisecond time scale involving residues 38, 42-44, and 46 in the linker region and residues 18 and 19 in calcium binding site I with similar energy barriers. These data are discussed in comparison with those available for the WT, as well as the apo-, mono-, and disubstituted P43G mutant. Quantification of water-amide proton exchange rates using saturation transfer and qualitative application of ~(15)N-(CLEANEX-PM)-FHSQC shows the values are in agreement with high mobility for the above-mentioned residues. Cross correlation between N-H dipole-dipole relaxation and ~(15)N CSA relaxation indicates that some of these mobility differences may extend to the sub-nanosecond time scale. Similar data were also obtained for the derivative where the calcium ion in the C-terminal lop was replaced with lanthanum. The results presented here show that, contrary to expectations, there are significant differences in dynamics between the dicalcium state of P43G and P43M and that these differences are not confined to the flexible linker region containing the point mutation. They also demonstrate that substitution of a lanthanide ion for calcium, which is a common procedure, does not significantly alter the mobility of the native protein.

机译：Calbindin D_（9K）是由两个螺旋环 - 螺旋EF手图案组成的二钙粘合蛋白，其通过柔性接头区域连接在一起，其中一个金属离子可以与两个环中的每一个结合。在接头区域中的位置43处的脯氨酸残基显示在野生型（WT）蛋白中的顺式反式异构体。通过在P43G或P43M突变体中用甘氨酸或甲硫氨酸代替脯氨酸来除去这种异构态。我们通过酰胺〜（15）n r_1，r_2和r_2（1＃sigma＃）测量来扩展了P43M突变体的可用移动性研究。这揭示了在具有相似能量屏障的钙结合位点I中的链接区和残基18和19中的残留物38,42-44和46的毫秒时间标度上揭示了意外的构象平衡。与可用于WT的那些，以及APO-，单 - 和二取代的P43G突变体进行讨论这些数据。使用饱和转移的水酰胺质子交换率的定量和〜（15）n-（Cleanex-PM）-fhsqc的定性施加显示该值与上述残留物的高迁移率一致。 N-H偶极子 - 偶极松弛和〜（15）N CSA弛豫之间的互相关表明，这些迁移率差异可以延伸到亚纳秒时尺度。还可以获得类似的数据，其中C末端循环中的钙离子被镧替代的衍生物。此处呈现的结果表明，与期望相反，P43G和P43M的DICICLCIUM状态之间的动态存在显着差异，并且这些差异不限于包含点突变的柔性接头区域。他们还证明，钙砜离子的钙离子是一种常见程序，不会显着改变天然蛋白的迁移率。

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来源
《Biochemistry》 |2002年第16期|共8页
作者
Ivano Bertini; Carl J. Carrano; Claudio Luchinat; Mario Piccioli; Luisa Poggi;
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作者单位

Magnetic Resonance Center (CERM) and Department of Chemistry University of Florence Sesto fiorentino Italy Department of Chemistry and Biochemistry Southwest Texas State University San Marcos Texas 78666 and Magnetic Resonance Center (CERM) a;

Magnetic Resonance Center (CERM) and Department of Chemistry University of Florence Sesto fiorentino Italy Department of Chemistry and Biochemistry Southwest Texas State University San Marcos Texas 78666 and Magnetic Resonance Center (CERM) a;

Magnetic Resonance Center (CERM) and Department of Chemistry University of Florence Sesto fiorentino Italy Department of Chemistry and Biochemistry Southwest Texas State University San Marcos Texas 78666 and Magnetic Resonance Center (CERM) a;

Magnetic Resonance Center (CERM) and Department of Chemistry University of Florence Sesto fiorentino Italy Department of Chemistry and Biochemistry Southwest Texas State University San Marcos Texas 78666 and Magnetic Resonance Center (CERM) a;

Magnetic Resonance Center (CERM) and Department of Chemistry University of Florence Sesto fiorentino Italy Department of Chemistry and Biochemistry Southwest Texas State University San Marcos Texas 78666 and Magnetic Resonance Center (CERM) a;

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中图分类生物化学;
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A ~(15)N NMR Mobility Study on the Dicalcium P43M Calbindin D_(9k) and Its Mono-La~(3+)-Substituted Form

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