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摘要
The previously published IDA-SH and NTA-SH tags are small synthetic lanthanide-binding tags derived from cysteine, which afford site-specific lanthanide labelling by disulfide-bond formation with a cysteine residue of the target protein. Following attachment to a single cysteine in an alpha-helix, sizeable pseudocontact shifts (PCS) can be observed, if the lanthanide is immobilized by additional coordination to a negatively charged amino-acid side chain that is located in a neighboring turn of the helix. To identify the best labelling strategy for PCS measurements, we performed a systematic study, where IDA-SH or NTA-SH tags were ligated to a cysteine residue in position i of an alpha-helix, and aspartate or glutamate residues were placed in the positions i - 4 or i + 4. The largest anisotropy components of the magnetic susceptibility tensor were observed for an NTA-SH tag in position i with a glutamate residue in position i - 4. While the NTA-SH tag produced sizeable PCSs regardless of the presence of nearby carboxyl groups of the protein, the IDA-SH tag generated a good lanthanide binding site only if an aspartate was placed in position i + 4. The findings provide a firm basis for the design of site-directed mutants that are suitable for the reliable generation of PCSs in proteins with paramagnetic lanthanides.
- 出版日期2013-2
