Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine
Magnetic Resonance
; 3(2):169-182, 2022.
Article
in English
| ProQuest Central | ID: covidwho-2030255
ABSTRACT
The paramagnetism of a lanthanoid tag site-specifically installed on a protein provides a rich source of structural information accessible by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy. Here we report a lanthanoid tag for selective reaction with cysteine or selenocysteine with formation of a (seleno)thioether bond and a short tether between the lanthanoid ion and the protein backbone. The tag is assembled on the protein in three steps, comprising (i) reaction with 4-fluoro-2,6-dicyanopyridine (FDCP);(ii) reaction of the cyano groups withα-cysteine, penicillamine or β-cysteine to complete the lanthanoid chelating moiety;and (iii) titration with a lanthanoid ion. FDCP reacts much faster with selenocysteine than cysteine, opening a route for selective tagging in the presence of solvent-exposed cysteine residues. Loaded with Tb3+ and Tm3+ ions, pseudocontact shifts were observed in protein NMR spectra, confirming that the tag delivers good immobilisation of the lanthanoid ion relative to the protein, which was also manifested in residual dipolar couplings. Completion of the tag with different 1,2-aminothiol compounds resulted in different magnetic susceptibility tensors. In addition, the tag proved suitable for measuring distance distributions in double electron–electron resonance experiments after titration with Gd3+ ions.
Physics; Cysteine; Gadolinium; Titration; Severe acute respiratory syndrome coronavirus 2; Electron paramagnetic resonance; Binding sites; Amino acids; E coli; Electron spin resonance; Route selection; Nuclear magnetic resonance--NMR; Chelation; Protein expression; Proteins; Cyano groups; Plasmids; Protein synthesis; Magnetic permeability; Immobilization; Selenocysteine; Experiments; Paramagnetism; Cloning; Spectroscopy; Tensors; Couplings; Magnetic susceptibility; United States--US
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Databases of international organizations
Database:
ProQuest Central
Language:
English
Journal:
Magnetic Resonance
Year:
2022
Document Type:
Article
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