Expression and characterisation of functional lanthanide-binding tags fused to a Galpha-protein and muscarinic (M2) receptor

Abstract

Lanthanide-binding tags (LBTs) are small peptides showing great promise for use in novel imaging technologies due to their high affinity for terbium, europium and gadolinium ions which have unique luminescent and/or paramagnetic properties. When fused to functional proteins the LBT can act as a site directed location for the binding of lanthanide ions which could be utilised in assays (usually luminescence-based) to derive data about protein interactions and functionality. This study shows the lanthanide-binding tag, LBT2, to have an apparent Kd for terbium of 9.3 nM. Other common ions showed much lower affinities for LBT2; however, gadolinium provided competitive binding with an IC50 of 13.7 nM. LBTs were then fused to both the M2-muscarinic receptor and a chimeric Gα-subunit allowing them to be labelled with terbium. The presence of this LBT did not disrupt either the ligand binding or signalling of the receptor. The construction of a LBT motif in association with specific proteins presents exciting possibilities to employ bimodal (luminescent and magnetic) imaging properties in relation to such areas as biosensor development and design.