Compact, hydrophilic, lanthanide-binding tags for paramagnetic NMR spectroscopy

Abstract

The design, synthesis and evaluation of four novel lanthanide-binding tags for paramagnetic NMR spectroscopy are reported. Each tag is based on the ((2<i>S</i>,2'<i>S</i>,2''<i>S</i>,2'''<i>S</i>)-1,1',1'',1'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetrakis(propan-2-ol)) scaffold, featuring small chiral alcohol coordinating pendants to minimise the size and hydrophobic character of each tag. The tags feature different linkers of variable length for conjugation to protein <i>via</i> a single cysteine residue. Each tag's ability to induce pseudocontact shifts (PCS) was assessed on a ubiquitin A28C mutant. Two enantiomeric tags of particular note, <b>C7</b> and <b>C8</b>, produced significantly larger Δ<i>χ</i>-tensors compared to a previously developed tag, <b>C1</b>, attributed to the extremely short linker utilised, limiting the mobility of the bound lanthanide ion. The <b>C7</b> and <b>C8</b> tags' capacity to induce PCSs was further demonstrated on GB1 Q32C and 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) S112C/C80A mutants. Whilst factors such as the choice of lanthanide ion, pH and site of conjugation influence the size of the PCSs obtained, the tags represent a significant advance in the field.