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Site‐specific dynamics in proteins are at the heart of protein function. While electron paramagnetic resonance (EPR) has potential to measure dynamics in large protein complexes, the reliance on flexible nitroxide labels is limitating especially for the accurate measurement of site‐specific β‐sheet dynamics. Here, we employed EPR spectroscopy to measure site‐specific dynamics across the surface of a protein, GB1. Through the use of the double Histidine (dHis) motif, which enables labeling with a Cu(II) – nitrilotriacetic acid (NTA) complex, dynamics information was obtained for both α‐helical and β‐sheet sites. Spectral simulations of the resulting CW‐EPR report unique site‐specific fluctuations across the surface of GB1. Additionally, we performed molecular dynamics (MD) simulations to complement the EPR data. The dynamics observed from MD agree with the EPR results. Furthermore, we observe small changes ing_ǁvalues for different sites, which may be due to small differences in coordination geometry and/or local electrostatics of the site. Taken together, this work expands the utility of Cu(II)NTA‐based EPR measurements to probe information beyond distance constraints.

 

Sensitive in-cell distance measurements in proteins using pulsed-Electron Spin Resonance (ESR) require reduction-resistant and cleavage-resistant spin-labels. Among the reduction-resistant moieties, the hydrophilic trityl core known as OX063 is promising due to its long phase-memory relaxation time (T_m). This property leads to a sufficiently intense ESR signal for reliable distance measurements. Furthermore, the T_m of OX063 remains sufficiently long at higher temperatures, opening the possibility for measurements at temperatures above 50 K. In this work, we synthesized deuterated OX063 with a maleimide linker (mOX063-d24). We show that the combination of the hydrophilicity of the label and the maleimide linker enables high protein labeling that is cleavage-resistant in-cells. Distance measurements performed at 150 K using this label are more sensitive than the measurements at 80 K. The sensitivity gain is due to the significantly short longitudinal relaxation time (T_1) at higher temperatures, which enables more data collection per unit of time. In addition to in vitro experiments, we perform distance measurements in Xenopus laevis oocytes. Interestingly, the T_m of mOX063-d24 is sufficiently long even in the crowded environment of the cell, leading to signals of appreciable intensity. Overall, mOX063-d24 provides highly sensitive distance measurements both in vitro and in-cells. 

This review describes the use of Electron Paramagnetic Resonance (EPR) to measure residue specific dynamics in proteins with a specific focus on Cu(II)‐based spin labels. First, we outline approaches used to measure protein motion by nitroxide‐based spin labels. Here, we describe conceptual details and outline challenges that limit the use of nitroxide spin labels to solvent‐exposed α‐helical sites. The bulk of this review showcases the use of newly developed Cu(II)‐based protein labels. In this approach, the strategic mutation of native residues on a protein to generate two neighboring Histidine residues (i.e., the dHis motif) is exploited to enable a rigid site‐selective binding of a Cu(II) complex. The chelation of the Cu(II) complex to dHis directly anchors the Cu(II) spin label to the protein backbone. The improvement in rigidity expands both the spin‐labeling toolkit as well as the resolution of many EPR measurements. We describe how EPR measurements of the Cu(II) label directly reflect backbone motion and fluctuations. The EPR are complemented by Molecular Dynamics simulations. Finally, the dHis motif provides access to the measurement of site‐specific dynamics at both α‐helices and β‐sheets. The review outlines the limitations of the dHis method and provides an outlook for future developments.

 

Abstract Electron paramagnetic resonance (EPR) has become an important tool to probe conformational changes in nucleic acids. An array of EPR labels for nucleic acids are available, but they often come at the cost of long tethers, are dependent on the presence of a particular nucleotide or can be placed only at the termini. Site directed incorporation of Cu2+-chelated to a ligand, 2,2′dipicolylamine (DPA) is potentially an attractive strategy for site-specific, nucleotide independent Cu2+-labelling in DNA. To fully understand the potential of this label, we undertook a systematic and detailed analysis of the Cu2+-DPA motif using EPR and molecular dynamics (MD) simulations. We used continuous wave EPR experiments to characterize Cu2+ binding to DPA as well as optimize Cu2+ loading conditions. We performed double electron-electron resonance (DEER) experiments at two frequencies to elucidate orientational selectivity effects. Furthermore, comparison of DEER and MD simulated distance distributions reveal a remarkable agreement in the most probable distances. The results illustrate the efficacy of the Cu2+-DPA in reporting on DNA backbone conformations for sufficiently long base pair separations. This labelling strategy can serve as an important tool for probing conformational changes in DNA upon interaction with other macromolecules. 

Protein dynamics is at the heart of all cellular processes. Here, we utilize the dHis‐Cu^IINTA label to obtain site‐specific information on dynamics for both an α‐helix and β‐sheet site of GB1, the immunoglobulin binding domain of protein G. Spectral features found in our CW‐EPR measurements were consistent with the overall rigid nature of GB1 and with predictions from molecular dynamics simulations. Using this information, we show the potential of this approach to elucidate the role of dynamics in substrate binding of a functionally necessary α‐helix in human glutathione transferase A1‐1 (hGSTA1‐1). We observe two dynamical modes for the helix. The addition of the inhibitor GS‐Met and GS‐Hex resulted in hGSTA1‐1 to favor the more rigid active state conformation, while the faster mode potentially aids the search for substrates. Together the results illustrate the remarkable potential of the dHis‐based labelling approach to measure site‐specific dynamics using room temperature lineshape analysis.