Differentiating between Label and Protein Conformers in Pulsed Dipolar EPR Spectroscopy with the dHis‐Cu²⁺(NTA) Motif

Abstract

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 flexible linkers, which hampers the translation of distance distributions into biomolecular conformations. In contrast, the spin label (nitrilotriacetic acid)copper(II) [Cu2+(NTA)] bound to two histidines (dHis) is rigid and can be more easily translated into biomolecular conformations. Here, we use this label on the 71 kDa Yersinia outer protein O (YopO) to decipher whether a previously experimentally observed bimodal distance distribution is due to two conformations of the biomolecule or of the flexible spin labels. Two different PDS experiments, i.e., Pulsed Electron‐Electron Double Resonance (PELDOR aka DEER) and Relaxation Induced Dipolar Modulation Enhancement (RIDME), yield unimodal distance distribution with the dHis‐Cu2+(NTA) motif, suggesting that the α‐helical backbone of YopO adopts a single conformation in frozen solution. In addition, we show that the Cu2+(NTA)‐label preferentially binds to the target double Histidine (dHis) sites even in the presence of 22 competing native histidine residues. This suggests that the generation of a His‐null background is not required for this spin labeling methodology. Together these results highlight the value of the dHis‐Cu2+(NTA) motif in PDS experiments.

Integrating Electron Paramagnetic Resonance Spectroscopy and Computational Modeling to Measure Protein Structure and Dynamics

“…[136,137] Finally, a His-null mutant is not required for labelling. [249] In addition to protein labels, SDSL has been utilized to introduce a multitude of spin labels in DNAs and RNAs to probe conformational dynamics, and there are several comprehensive reviews. [107,[138][139][140][141] Only a broad summary of nucleic acid labels is provided here.…”

PDSFit: PDS data analysis in the presence of orientation selectivity, g‐anisotropy, and exchange coupling

“…In the last test, PDSFit was applied to the orientationselective Q-band RIDME and PELDOR time traces of the Yersinia outer protein O mutant, YopO(C219A/A595H/ V599H/S620H/N624H), labeled with Cu 2+ NTA at two introduced double histidine sites 77 (Figure 6a). The RIDME time traces were acquired at 14 equidistant positions in the Cu 2+ spectrum (Figure 6b).…”

Pulse Dipolar Electron Paramagnetic Resonance Spectroscopy Distance Measurements at Low Nanomolar Concentrations: The CuII-Trityl Case

“…In most cases, paramagnetic centers are introduced via site-directed spin-labeling (SDSL) of specific residues, usually employing stable organic radicals such as nitroxides ,− or trityls ,, or paramagnetic metal ions such as Gd III , ,,− Mn II , or Cu II . − In most cases, SDSL involves stable radicals introduced via cysteine-specific chemistry, where the label-specific length of the linker contributes degrees of freedom to the dynamics of the protein, which artificially broadens distance distributions or allows only distinct label conformations that could make interpretation of distance distributions ambiguous. , Additionally, different spin-labels may lead to different degrees of perturbation of the native structure due to their size and interactions with protein residues, e.g., trityl labels. Another approach uses Cu II complexed with nitrilotriacetic acid (CuNTA) coordinated to a site-specifically introduced double-histidine (dHis) motif (dHis-CuNTA), posing more stringent requirements on the labeling site but yielding very narrow distance distributions due to the rigidity of this labeled side chain. ,,− CuNTA increases specificity to dHis sites compared to free Cu II in solution, which has a stronger propensity for unspecific binding. ,, Depending on the secondary structural elements (α-helix or β-sheet), dissociation constants ( K d ) were determined to be on the order of 10 –5 to 10 –7 under EPR conditions. , An advantage of using spectroscopically orthogonal labels is that binding sites can be saturated by an excess of CuNTA without overlap with the detected signal .…”

“…Furthermore, the dHis-CuNTA labeling has been shown to be robust against competing ligands and to retain its high affinity binding over a wide pH range, thus demonstrating biologically relevant compatibility . While a majority of the benchmarking studies on dHis-CuNTA have been performed on different constructs of a model protein (GB1, vide infra ), this labeling approach has also been applied to a variety of more complex biological systems. ,, Despite its vulnerability to reduction, CuNTA has been used for in-cell PDS …”

“…GB1 I6C/K28H/Q32H was expressed and purified as described previously and labeled with SLIM and CuNTA according to established protocols (for more details, see the Supporting Information). ,, Quantitative labeling with SLIM could be achieved as demonstrated by sample characterization using continuous wave (CW) EPR spectroscopy (Figure S1), electrospray ionization (ESI) mass spectrometry (Figure S2), and ultraviolet–visible (UV–vis) spectroscopy (Figure S3) for concentration determination and estimation of labeling efficiency.…”