Electron paramagnetic resonance (EPR) based distance measurements have been exploited to measure protein–protein docking, protein–DNA interactions, substrate binding and metal coordination sites. Here, we use EPR to locate a native paramagnetic metal binding site in a protein with less than 2 Å resolution. We employ a rigid Cu 2+ binding motif, the double histidine (dHis) motif, in conjunction with double electron electron resonance (DEER) spectroscopy. Specifically, we utilize a multilateration approach to elucidate the native Cu 2+ binding site in the immunoglobulin binding domain of protein G. Notably, multilateration performed with the dHis motif required only the minimum number of four distance constraints, whereas comparable studies using flexible nitroxide-based spin labels require many more for similar precision. This methodology demonstrates a significant increase in the efficiency of structural determinations via EPR distance measurements using the dHis motif.
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Differentiating between Label and Protein Conformers in Pulsed Dipolar EPR Spectroscopy with the dHis‐Cu 2+ (NTA) Motif
Pulsed dipolar EPR spectroscopy (PDS) in combination with site‐directed spin labeling is a powerful tool in structural biology. However, the commonly used spin labels are conjugated to biomolecules via rather long and flexible linkers, which hampers the translation of distance distributions into biomolecular conformations. In contrast, the spin label copper(II)‐nitrilotriacetic acid [Cu2+(NTA)] bound to two histidines (dHis) is rigid and yields narrow distance distributions, which can be more easily translated into biomolecular conformations. Here, we use this label on the 71 kDa
- Award ID(s):
- 2006154
- NSF-PAR ID:
- 10472942
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Chemistry – A European Journal
- Volume:
- 29
- Issue:
- 72
- ISSN:
- 0947-6539
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract 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 in
g 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. -
Spin labels attached to two residues of a protein chain have less conformational flexibility than those attached to a single residue and thus lead to a narrower spatialdistribution of the unpaired electron. The case of Cu(II) labels based on the double-histidine (dHis) motif is of particular interest, as it combines the advantage of precise localization of the unpaired electron with a labelling scheme orthogonal to the more common cysteine-based labelling. Here, we introduce an approach for in silico spin labelling of a protein by dHis motifs and Cu(II) complexes of iminodiacetic acid or nitrilotriacetic acid. We discuss a computerized scan for native histidine pairs that might be prone to bind such Cu(II) complexes and spin-labelling site pair scans that can identify suitable double mutants for labelling. Predicted distance distributions between two Cu(II) labels are compared to experimental distance distributions. We also test the hypothesis that elastic network modelling of conformational transitions with Cu2(II)- dHis labels can provide more accurate structural models than with nitroxide labels.more » « less
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An optimal acquisition scheme for Q-band EPR distance measurements using Cu 2+ -based protein labelsRecent advances in site-directed Cu 2+ labeling of proteins and nucleic acids have added an attractive new methodology to measure the structure-function relationship in biomolecules. Despite the promise, accessing the higher sensitivity of Q-band Double Electron Electron Resonance (DEER) has been challenging for Cu 2+ labels designed for proteins. Q-band DEER experiments on this label typically require many measurements at different magnetic fields, since the pulses can excite only a few orientations at a given magnetic field. Herein, we analyze such orientational effects through simulations and show that three DEER measurements, at strategically selected magnetic fields, are generally sufficient to acquire an orientational-averaged DEER time trace for this spin label at Q-band. The modeling results are experimentally verified on Cu 2+ labeled human glutathione S-transferase (hGSTA1-1). The DEER distance distribution measured at the Q-band shows good agreement with the distance distribution sampled by molecular dynamics (MD) simulations and X-band experiments. The concordance of MD sampled distances and experimentally measured distances adds growing evidence that MD simulations can accurately predict distances for the Cu 2+ labels, which remains a key bottleneck for the commonly used nitroxide label. In all, this minimal collection scheme reduces data collection time by as much as six-fold and is generally applicable to many octahedrally coordinated Cu 2+ systems. Furthermore, the concepts presented here may be applied to other metals and pulsed EPR experiments.more » « less
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Abstract 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.
