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1. An optimal acquisition scheme for Q-band EPR distance measurements using Cu ²⁺ -based protein labels

   https://doi.org/10.1039/d2cp01032a  

   Bogetti, Xiaowei; Hasanbasri, Zikri; Hunter, Hannah R.; Saxena, Sunil (January 2022, Physical Chemistry Chemical Physics)

   Recent 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. 

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2. Cleavage-Resistant Protein Labeling With Hydrophilic Trityl Enables Distance Measurements In-Cell

   https://doi.org/10.1021/acs.jpcb.1c02371  

   Hasanbasri, Zikri; Singewald, Kevin; Gluth, Teresa D.; Driesschaert, Benoit; Saxena, Sunil (April 2021, The Journal of Physical Chemistry B)

   null (Ed.)

   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. 

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3. The effect of spin polarization on double electron–electron resonance (DEER) spectroscopy

   https://doi.org/10.5194/mr-3-101-2022  

   Sweger, Sarah R.; Denysenkov, Vasyl P.; Maibaum, Lutz; Prisner, Thomas F.; Stoll, Stefan (January 2022, Magnetic Resonance)

   Abstract. Double electron–electron resonance (DEER) spectroscopy measures the distribution of distances between two electron spins in the nanometer range, often on doubly spin-labeled proteins, via the modulation of a refocused spin echo by the dipolar interaction between the spins. DEER is commonly conducted under conditions where the polarization of the spins is small. Here, we examine the DEER signal under conditions of high spin polarization, thermally obtainable at low temperatures and high magnetic fields, and show that the signal acquires a polarization-dependent out-of-phase component both for the intramolecular and intermolecular contributions. For the latter, this corresponds to a phase shift of the spin echo that is linear in the pump pulse position. We derive a compact analytical form of this phase shift and show experimental measurements using monoradical and biradical nitroxides at several fields and temperatures. The effect highlights a novel aspect of the fundamental spin physics underlying DEER spectroscopy. 

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4. Gd ³⁺ –Gd ³⁺ distances exceeding 3 nm determined by very high frequency continuous wave electron paramagnetic resonance

   https://doi.org/10.1039/c6cp07119h  

   Clayton, Jessica A.; Qi, Mian; Godt, Adelheid; Goldfarb, Daniella; Han, Songi; Sherwin, Mark S. (January 2017, Physical Chemistry Chemical Physics)

   Electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling is a very powerful tool for elucidating the structure and organization of biomolecules. Gd 3+ complexes have recently emerged as a new class of spin labels for distance determination by pulsed EPR spectroscopy at Q- and W-band. We present CW EPR measurements at 240 GHz (8.6 Tesla) on a series of Gd-rulers of the type Gd-PyMTA–spacer–Gd-PyMTA, with Gd–Gd distances ranging from 1.2 nm to 4.3 nm. CW EPR measurements of these Gd-rulers show that significant dipolar broadening of the central |−1/2〉 → |1/2〉 transition occurs at 30 K for Gd–Gd distances up to ∼3.4 nm with Gd-PyMTA as the spin label. This represents a significant extension for distances accessible by CW EPR, as nitroxide-based spin labels at X-band frequencies can typically only access distances up to ∼2 nm. We show that this broadening persists at biologically relevant temperatures above 200 K, and that this method is further extendable up to room temperature by immobilizing the sample in glassy trehalose. We show that the peak-to-peak broadening of the central transition follows the expected 1/ r 3 dependence for the electron–electron dipolar interaction, from cryogenic temperatures up to room temperature. A simple procedure for simulating the dependence of the lineshape on interspin distance is presented, in which the broadening of the central transition is modeled as an S = 1/2 spin whose CW EPR lineshape is broadened through electron–electron dipolar interactions with a neighboring S = 7/2 spin. 

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5. Bayesian Probabilistic Inference of Nonparametric Distance Distributions in DEER Spectroscopy

   https://doi.org/10.1021/acs.jpca.4c05056  

   Sweger, Sarah R; Cheung, Julian C; Zha, Lukas; Pribitzer, Stephan; Stoll, Stefan (October 2024, The Journal of Physical Chemistry A)

   Double electron−electron resonance (DEER) spectroscopy measures distance distributions between spin labels in proteins, yielding important structural and energetic information about conformational landscapes. Analysis of an experimental DEER signal in terms of a distance distribution is a nontrivial task due to the ill-posed nature of the underlying mathematical inversion problem. This work introduces a Bayesian probabilistic inference approach to analyze DEER data, assuming a nonparametric distance distribution with a Tikhonov smoothness prior. The method uses Markov Chain Monte Carlo sampling with a compositional Gibbs sampler to determine a posterior probability distribution over the entire parameter space, including the distance distribution, given an experimental data set. This posterior contains all of the information available from the data, including a full quantification of the uncertainty about the model parameters. The corresponding uncertainty about the distance distribution is visually captured via an ensemble of posterior predictive distributions. Several examples are presented to illustrate the method. Compared with bootstrapping, it performs faster and provides slightly larger uncertainty intervals. 

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