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[Re(CO)\(_3\)]\(^+\) labelling of a novel cysteine/hexahistidine tag: insights into binding mode by liquid chromatography-mass spectrometry.

Tavaré, Richard, Williams, Jennifer, Howland, Kevin, Blower, Philip J, Mullen, Gregory E D (2012) [Re(CO)\(_3\)]\(^+\) labelling of a novel cysteine/hexahistidine tag: insights into binding mode by liquid chromatography-mass spectrometry. Journal of Inorganic Biochemistry, 114 . pp. 24-27. ISSN 0162-0134. (doi:10.1016/j.jinorgbio.2012.04.006) (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided) (KAR id:34168)

The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided.
Official URL
http://dx.doi.org/10.1016/j.jinorgbio.2012.04.006

Abstract

We recently described a novel amino acid sequence, KCKLAAALEHHHHHH, for site-specific radiolabelling of proteins with [\(^{99m}\)Tc(CO)\(_3\)(OH\(_2\))\(_3\)]\(^+\) or [Re(CO)\(_3\)(OH\(_2\))\(_3\)]\(^+\) with improved efficiency compared to conventional hexahistidine tags (His-tag). C2AH, a modification of the protein C2A (the phosphatidylserine (PS)-binding domain of rat synaptotagmin I) engineered to contain this novel C-terminal tag, was produced. Rhenium tricarbonyl conjugates of C2AH were analysed post tryptic digest by liquid chromatography-electrospray mass spectrometry (LC-MS), giving rise to a peak with the molecular weight corresponding to M\(^+\)=[Re(CO)\(_3\)+CK+LAAALEHHHHHH]\(^+\). This species arises as a result of trypsin cleavage on the C-terminus of both the lysine (Lys) residues on either side of the Cys while both fragments still remain bound to the rhenium. This confirmed that cysteine (Cys) was directly involved in the coordination of the rhenium tricarbonyl. To demonstrate the superiority of the cysteine containing His-tag sequences for binding [Re(CO)\(_3\)]\(^+\), two peptides CKLAAALEHHHHHH and LAAALEHHHHHH were synthesised. In a competition experiment the mixed peptides were incubated with one molar equivalent of [Re(CO)\(_3\)(H\(_2\)O)\(_3\)]\(^+\), and LC-ESMS demonstrated that 92% and 9% of CKLAAALEHHHHHH and LAAALEHHHHHH respectively were co-ordinated by one [Re(CO)\(_3\)]\(^+\).

Item Type: Article
DOI/Identification number: 10.1016/j.jinorgbio.2012.04.006
Uncontrolled keywords: Rhenium and technetium tricarbonyl; Histag; ESI
Subjects: Q Science
Divisions: Divisions > Division of Natural Sciences > Biosciences
Depositing User: Kevin Howland
Date Deposited: 15 May 2020 14:51 UTC
Last Modified: 16 Nov 2021 10:11 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/34168 (The current URI for this page, for reference purposes)
Howland, Kevin: https://orcid.org/0000-0003-4370-0974
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