Small neutral Gd(iii) tags for distance measurements in proteins by double electron–electron resonance experiments†
Spin labels containing a Gd(III) ion have become important for measuring nanometer distances in proteins by double electron–electron resonance (DEER) experiments at high EPR frequencies. The distance resolution and sensitivity of these measurements strongly depend on the Gd(III) tag used. Here we report the performance of two Gd(III) tags, propargyl-DO3A and C11 in DEER experiments carried out at W-band (95 GHz). Both tags are small, uncharged and devoid of bulky hydrophobic pendants. The propargyl-DO3A tag is designed for conjugation to the azide-group of an unnatural amino acid. The C11 tag is a new tag designed for attachment to a single cysteine residue. The tags delivered narrower distance distributions in the E. coli aspartate/glutamate binding protein and the Zika virus NS2B–NS3 protease than previously established Gd(III) tags. The improved performance is consistent with the absence of specific hydrophobic or charge–charge interactions with the protein. In the case of the Zika virus NS2B–NS3 protease, unexpectedly broad Gd(III)–Gd(III) distance distributions observed with the previously published charged C9 tag, but not the C11 tag, illustrate the potential of tags to perturb a labile protein structure and the importance of different tags. The results obtained with the C11 tag demonstrate the closed conformation in the commonly used linked construct of the Zika virus NS2B–NS3 protease, both in the presence and absence of an inhibitor.