[Re(CO)(3)](+) labelling of a novel cysteine/hexahistidine tag: insights into binding mode by liquid chromatography-mass spectrometry.
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)](+).
Division of Imaging Sciences and Biomedical Engineering, King's College London, St. Thomas' Hospital, London SE1 7EH, UK.
Richard
R
Tavaré
Jennifer
J
Williams
Kevin
K
Howland
Philip J
PJ
Blower
Gregory E D
GE
Mullen
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D007202:Indicators and Reagents / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry*; D009942:Organometallic Compounds / Q000737:chemistry*; D010718:Phosphatidylserines / Q000737:chemistry; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization; D050863:Synaptotagmin I / Q000737:chemistry; D017556:Technetium Compounds / Q000737:chemistry*; D014357:Trypsin
C471213:His-His-His-His-His-His; D007202:Indicators and Reagents; D009842:Oligopeptides; D009942:Organometallic Compounds; D010718:Phosphatidylserines; D050863:Synaptotagmin I; D017556:Technetium Compounds; C476042:triaquatricarbonylrhenium(I); C543202:triaquatricarbonyltechnetium(I); D006639:Histidine; D014357:Trypsin; D003545:Cysteine