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A systematic study of labelling an α-helix in a protein with a lanthanide using IDA-SH or NTA-SH tags
Ist Teil von
Journal of biomolecular NMR, 2013-02, Vol.55 (2), p.157-166
Ort / Verlag
Dordrecht: Springer Netherlands
Erscheinungsjahr
2013
Link zum Volltext
Quelle
SpringerLink
Beschreibungen/Notizen
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 α-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 α-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.