Calmodulin tagging provides a general method of using lanthanide induced magnetic field orientation to observe residual dipolar couplings in proteins in solution
Calmodulin tagging provides a general method of using lanthanide induced magnetic field orientation to observe residual dipolar couplings in proteins in solution
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DOI：
10.1023/a:1011924017938
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发表时间：
2001-09-01
影响因子：
2.7
通讯作者：
Bayley, PM
中科院分区：
文献类型：
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作者：
Feeney, J;Birdsall, B;Bayley, PM
提出了一种蛋白质溶液磁场取向的一般方法。通过用顺磁性镧系离子饱和的钙调蛋白标记靶蛋白，可以测量大量的残余偶极偶联（RDC），同时最小化伪接触位移对靶蛋白的影响。制备由钙调蛋白结合肽（来自sk-MLCK的M13）连接到靶蛋白（在这种情况下为二氢叶酸还原酶）组成的构建体。这种工程蛋白质与被铽（一种顺磁性镧系离子）饱和的钙调蛋白紧密结合。通过在M13和靶蛋白之间仅使用短的接头区域，在CaM（Tb 3+）（4）中诱导的一些磁场排列被有效地传递到靶蛋白（DHFR）。对含有N-15标记的DHFR-M13蛋白和未标记的CaM（Tb 3+）（4）的标记复合物进行的H-1-N-15 HSQC IPAP实验允许测量比对复合物中的RDC贡献。在600 MHz下测量+4.0至-7.4 Hz范围内的RDC值。单独的N-15-DHFR-M13及其与CaM（Ca 2+）（4）和CaM（Tb 3+）（4）的复合物的H-1-N-15 HSQC光谱的比较表明：（i）靶蛋白的结构不受复合物形成的影响，（ii）靶蛋白的光谱不受假接触位移的严重干扰。使用相对较大的标记基团（CaM）允许我们使用具有非常高的磁化率各向异性的镧系元素离子（例如Tb 3+）来给出大的比对，同时保持与靶蛋白核的相对较长的距离（并且因此仅给出小的伪接触位移贡献）。
A general method is presented for magnetic field alignment of proteins in solution. By tagging a target protein with calmodulin saturated with paramagnetic lanthanide ions it is possible to measure substantial residual dipolar couplings (RDC) whilst minimising the effects of pseudocontact shifts on the target protein. A construct was made consisting of a calmodulin-binding peptide (M13 from sk-MLCK) attached to a target protein, dihydrofolate reductase in this case. The engineered protein binds tightly to calmodulin saturated with terbium, a paramagnetic lanthanide ion. By using only a short linker region between the M13 and the target protein, some of the magnetic field alignment induced in the CaM(Tb3+)(4) is effectively transmitted to the target protein (DHFR). H-1-N-15 HSQC IPAP experiments on the tagged complex containing N-15-labelled DHFR-M13 protein and unlabelled CaM(Tb3+)(4) allow one to measure RDC contributions in the aligned complex. RDC values in the range +4.0 to -7.4 Hz were measured at 600 MHz. Comparisons of H-1-N-15 HSQC spectra of N-15-DHFR-M13 alone and its complexes with CaM(Ca2+)(4) and CaM(Tb3+)(4) indicated that (i) the structure of the target protein is not affected by the complex formation and (ii) the spectra of the target protein are not seriously perturbed by pseudocontact shifts. The use of a relatively large tagging group (CaM) allows us to use a lanthanide ion with a very high magnetic susceptibility anisotropy (such as Tb3+) to give large alignments while maintaining relatively long distances from the target protein nuclei (and hence giving only small pseudocontact shift contributions).