The Cu²⁺-nitrilotriacetic acid complex improves loading of α-helical double histidine site for precise distance...

Site-directed spin labeling using two strategically placed natural histidine residues allows for the rigid attachment of paramagnetic Cu²⁺. This double histidine (dHis) motif enables extremely precise, narrow distance distributions resolved by Cu²⁺-based pulsed ESR. Furthermore, the distance measurements are easily relatable to the protein backbone-structure. The Cu²⁺ ion has, till now, been introduced as a complex with the chelating agent iminodiacetic acid (IDA) to prevent unspecific binding. Recently, this method was found to have two limiting concerns that include poor selectivity towards α-helices and incomplete Cu²⁺-IDA complexation. Herein, we introduce an alternative method of dHis-Cu²⁺ loading using the nitrilotriacetic acid (NTA)-Cu²⁺ complex. We find that the Cu²⁺-NTA complex shows a four-fold increase in selectivity toward α-helical dHis sites. Furthermore, we show that 100% Cu²⁺-NTA complexation is achievable, enabling precise dHis loading and resulting in no free Cu²⁺ in solution. We analyze the optimum dHis loading conditions using both continuous wave and pulsed ESR. We implement these findings to show increased sensitivity of the Double Electron-Electron Resonance (DEER) experiment in two different protein systems. The DEER signal is increased within the immunoglobulin binding domain of protein G (called GB1). We measure distances between a dHis site on an α-helix and dHis site either on a mid-strand or a non-hydrogen bonded edge-strand β-sheet. Finally, the DEER signal is increased twofold within two α-helix dHis sites in the enzymatic dimer glutathione S-transferase exemplifying the enhanced α-helical selectivity of Cu²⁺-NTA.