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Measurement of the conformation of DNA in protein-DNA complexes is important to decipher the role of DNA conformation and dynamics in protein recognition and function. In this work we report a rigid nucleotide-independent spin label that places paramagnetic Cu(II) within the DNA helix. The labeling strategy exploits the chelation of Cu(II) to two 8-aminoquinoline moieties, one in each strand. Because the rigidity of the probe avoids potentially confounding motions of the label itself, EPR signals can resolve 2-3 Å changes in interspin distance; the breadth of the distance distribution reports dy-namic fluctuations from the most probable conformation. Continuous wave and pulsed electron paramagnetic resonance spectroscopy (EPR) show that the Cu(II) coordinates properly to the labeling sites. Measurements of the interspin dipolar interaction on DNA oligonucleotides with two labels placed at various distances demonstrate that the label provides accurate and narrow distance distributions sensitive to DNA conformation and flexibility. Molecular dynamics simulations support these interpretations. We utilize this label to measure the conformations of DNA when type II restriction endonuclease EcoRV binds to its specific recognition sequence. The results provide in-solution evidence that EcoRV endonuclease induces axial DNA bending in the absence of metal ions, contrary to long-standing belief. Furthermore, the distance distribution nar-rows upon protein binding and even further on subsequent metal binding, implying that bound protein constrains the bend-ing dynamics of DNA. This method provides a novel and accurate approach to assess DNA conformation and dynamics in solution. 

In‐cell measurements of the relationship between structure and dynamics to protein function is at the forefront of biophysics. Recently, developments in EPR methodology have demonstrated the sensitivity and power of this method to measure structural constraints in‐cell. However, the need to spin label proteins ex‐situ or use noncanonical amino acids to achieve endogenous labeling remains a bottleneck. In this work we expand the methodology to endogenously spin label proteins with Cu(II) spin labels and describe how to assess in‐cell spin labeling. We quantify the amount of Cu(II)‐NTA in cells, assess spin labeling, and account for orientational effects during distance measurements. We compare the efficacy of using heat‐shock and hypotonic swelling to deliver spin label, showing that hypotonic swelling is a facile and reproducible method to efficiently deliver Cu(II)‐NTA into E. coli. Notably, over six repeats we accomplish a bulk average of 57 μM spin labeled sites, surpassing existing endogenous labeling methods. The results of this work open the door for endogenous spin labeling that is easily accessible to the broader biophysical community. 

Herein, we develop an efficient rotamer library-based approach to predict distance distributions from Cu(ii) protein labels. 

Recently, there have been remarkable improvements in pulsed ESR sensitivity, paving the way for broader applicability of ESR in the measurement of biological distance constraints, for instance, at physiological concentrations and in more complex systems. Nevertheless, submicromolar distance measurements with the commonly used nitroxide spin label take multiple days. Therefore, there remains a need for rapid and reliable methods of measuring distances between spins at nanomolar concentrations. In this work, we demonstrate the power of double quantum coherence (DQC) experiments at Q-band frequencies. With the help of short and intense pulses, we showcase DQC signals on nitroxide-labeled proteins with modulation depths close to 100%. We show that the deep dipolar modulations aid in the resolution of bimodal distance distributions. Finally, we establish that distance measurements with protein concentrations as low as 25 nM are feasible. This limit is approximately 4-fold lower than previously possible. We anticipate that nanomolar concentration measurements will lead to further advancements in the use of ESR, especially in cellular contexts. 

Combining rigid Cu( ii ) labels and pulsed-EPR techniques enables distance constraint measurements that are incisive probes of protein structure and dynamics. However, the labels can lead to a dipolar signal that is biased by the relative orientation of the two spins, which is typically unknown a priori in a bilabeled protein. This effect, dubbed orientational selectivity, becomes a bottleneck in measuring distances. This phenomenon also applies to other pulsed-EPR techniques that probe electron–nucleus interactions. In this work, we dissect orientational selectivity by generating an in silico sample of Cu( ii )-labeled proteins to evaluate pulse excitation in the context of double electron–electron resonance (DEER) at Q-band frequencies. This approach enables the observation of the contribution of each protein orientation to the dipolar signal, which provides direct insights into optimizing acquisition schemes to mitigate orientational effects. Furthermore, we incorporate the excitation profile of realistic pulses to identify the excited spins. With this method, we show that rectangular pulses, despite their imperfect inversion capability, can sample similar spin orientations as other sophisticated pulses with the same bandwidth. Additionally, we reveal that the efficiency of exciting spin-pairs in DEER depends on the frequency offset of two pulses used in the experiment and the relative orientation of the two spins. Therefore, we systematically examine the frequency offset of the two pulses used in this double resonance experiment to determine the optimal frequency offset for optimal distance measurements. This procedure leads to a protocol where two measurements are sufficient to acquire orientational-independent DEER at Q-band. Notably, this procedure is feasible with any commercial pulsed-EPR spectrometer. Furthermore, we experimentally validate the computational results using DEER experiments on two different proteins. Finally, we show that increasing the amplitude of the rectangular pulse can increase the efficiency of DEER experiments by almost threefold. Overall, this work provides an attractive new approach for analyzing pulsed-EPR spectroscopy to obtain microscopic nuances that cannot be easily discerned from analytical or numerical calculations. 

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. 

Cooperative gas adsorption in metal–organic frameworks (MOFs) is a rare phenomenon that generally involves long-range communication between multiple binding sites. We demonstrate a MOF containing cobalt(II)–methyl sites that selectively and reversibly capture two carbon monoxide (CO) molecules per site, leading to record-high adsorption capacities at ambient temperatures and pressures. Gas adsorption and structural, spectroscopic, and computational analyses support a mechanism in which binding of one CO molecule triggers a spin transition, followed by binding of a second CO molecule and migratory insertion of the first CO molecule into the cobalt–methyl bond to form an acetyl. The greater binding affinity associated with the second CO results in sigmoidal adsorption isotherms, a hallmark of cooperativity and phase-change materials, despite the absence of long-range interactions within the framework.