A 15N NMR mobility study on the dicalcium P43M calbindin D9k and its mono-La3+-substituted form.
A 15N NMR mobility study on the dicalcium P43M calbindin D9k and its mono-La3+-substituted form.
复制标题
二钙 P43M 钙结合蛋白 D9k 及其单 La3 取代形式的 15N NMR 迁移率研究。
DOI：
10.1021/bi015945n
复制
发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Poggi,Luisa
中科院分区：
文献类型：
--
作者：
Bertini,Ivano;Carrano,CarlJ;Luchinat,Claudio;Piccioli,Mario;Poggi,Luisa
Calbindin D9kis 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 amide15NR1,R2, andR1ρmeasurements. 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 of15N-(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 and15N 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 loop 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.
