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A systematic study of labelling an [alpha]-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
Ort / Verlag
Dordrecht: Springer Nature B.V
Erscheinungsjahr
2013
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 [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.[PUBLICATION ABSTRACT]
Sprache
Englisch
Identifikatoren
ISSN: 0925-2738
eISSN: 1573-5001
DOI: 10.1007/s10858-012-9697-3
Titel-ID: cdi_proquest_journals_1285235381
Format
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