On the Use of Q-Band Double Electron–Electron Resonance To Resolve the Relative Orientations of Two Double Histidine-Bound Cu²⁺ Ions in a Protein

Abstract

In this work, we explore the potential of a rigid Cu 2+ spin-labeling technique, the double histidine (dHis) motif, along with Q-band electron paramagnetic resonance to report on the relative orientations of the spin labels. We show that the precision of the dHis motif, coupled with the sensitivity and resolution of Q-band frequencies, may allow for the straightforward determination of the relative orientation of the dHis-Cu 2+ labels using double electron− electron resonance (DEER). We performed Q-band DEER measurements at different magnetic fields on a protein containing two dHis Cu 2+ sites. These measurements exhibited orientational selectivity such that each discrete magnetic field yielded a unique DEER signal. We determined the relative orientation of the two metal centers by simulating the orientationally selective DEER data. These relative orientations were validated by visual analysis of the protein crystal structure modified with dHis sites. The simple visual analysis was shown to agree well with the angular values determined via simulation of the experimental data. The combination of the dHis-Cu 2+ motif along with the advantages of the Q-band can aid in the accurate measurement of protein structural and conformational dynamics.

Light‐Induced Pulsed EPR Dipolar Spectroscopy on a Paradigmatic Hemeprotein

“…[12][13][14] The most commonly used spin label is (1-oxyl-2,2,5,5-tetra-methylpyrroline-3-methyl)-methanethiosulfonate (MTSSL), which specifically reacts with the thiol group of cysteine residues. [20][21][22][23] The search for alternative spin labels is an active area of research. [19] Recently, Cu(II) and high spin Mn(II) tags have also been successfully employed.…”

“…[19] Recently, Cu(II) and high spin Mn(II) tags have also been successfully employed. [20][21][22][23] The search for alternative spin labels is an active area of research. [24] One important new development is the demonstration that the triplet state of porphyrin chromophores can be exploited to determine inter-spin distances.…”

EPR Spectroscopy Detects Various Active State Conformations of the Transcriptional Regulator CueR

“…The experimental data presented here was performed at a fixed observer and pump frequencies for a precise comparison (at the g┴ region). Although the use of Qband dHis based DEER is nascent, a range of 500G around g┴ should show the presence of orientational effects (see Figure S3 in SI) [11] . Such effects are likely inhibited in the CueR system because of orientational flexibility, possibly due to the loop regions of the protein.…”

Accessing distributions of exchange and dipolar couplings in stiff molecular rulers with Cu(ii) centres

“…[24][25][26][27] Significant progress in performing and analysing Cu(II)-based PDS experiments has been achieved over the past few years. [28][29][30][31][32][33] Exchange interactions are often observed for Cu(II) ions that are bound to conjugated p-systems, [34][35][36][37][38][39] or in strongly coupled systems, such as molecular magnets. 7,40,41 The exchange coupling may give additional information on the system of interest, yet, the dipolar and exchange couplings can become difficult to disentangle.…”