The Dynamic Plasticity of P. aeruginosa CueR Copper Transcription Factor upon Cofactor and DNA Binding.

Bacteria use specialized proteins, like transcription factors, to rapidly control metal ion balance. CueR is a Gram-negative bacterial copper regulator. The structure of E. coli CueR complexed with Cu(I) and DNA was published, since then many studies have shed light on its function. However, P. aeruginosa CueR, which shows high sequence similarity to E. coli CueR, has been less studied. Here, we applied room-temperature electron paramagnetic resonance (EPR) measurements to explore changes in dynamics of P. aeruginosa CueR in dependency of copper concentrations and interaction with two different DNA promoter regions. We showed that P. aeruginosa CueR is less dynamic than the E. coli CueR protein and exhibits much higher sensitivity to DNA binding as compared to its E. coli CueR homolog. Moreover, a difference in dynamical behavior was observed when P. aeruginosa CueR binds to the copZ2 DNA promoter sequence compared to the mexPQ-opmE promoter sequence. Such dynamical differences may affect the expression levels of CopZ2 and MexPQ-OpmE proteins in P. aeruginosa. Overall, such comparative measurements of protein-DNA complexes derived from different bacterial systems reveal insights about how structural and dynamical differences between two highly homologous proteins lead to quite different DNA sequence-recognition and mechanistic properties.

Modeling of Cu(II)-based protein spin labels using rotamer libraries.

The bifunctional spin label double-histidine copper-(II) capped with nitrilotriacetate [dHis-Cu(II)-NTA], used in conjunction with electron paramagnetic resonance (EPR) methods can provide high-resolution distance data for investigating protein structure and backbone conformational diversity. Quantitative utilization of this data is limited due to a lack of rapid and accurate dHis-Cu(II)-NTA modeling methods that can be used to translate experimental data into modeling restraints. Here, we develop two dHis-Cu(II)-NTA rotamer libraries using a set of recently published molecular dynamics simulations and a semi-empirical meta-dynamics-based conformational ensemble sampling tool for use with the recently developed chiLife bifunctional spin label modeling method. The accuracy of both the libraries and the modeling method are tested by comparing model predictions to experimentally determined distance distributions. We show that this method is accurate with absolute deviation between the predicted and experimental modes between 0.0-1.2 Å with an average of 0.6 Å over the test data used. In doing so, we also validate the generality of the chiLife bifunctional label modeling method. Taken together, the increased structural resolution and modeling accuracy of dHis-Cu(II)-NTA over other spin labels promise improvements in the accuracy and resolution of protein models by EPR.

Differentiating between Label and Protein Conformers in Pulsed Dipolar EPR Spectroscopy with the dHis-Cu2+ (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 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, that is, 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; this result suggests 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. Our results therefore suggest 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.

Efficient sampling of molecular orientations for Cu(II)-based DEER on protein labels.

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.

Veillonella atypica causing retropharyngeal abscess: A rare case presentation.

Veillonella species are obligate anaerobes which are part of the human oral, gut and vaginal microbiota. The genus Veillonella consists of 16 characterized species. Very few infections due to Veillonella atypica have been reported till date. Here we present a case of retropharyngeal abscess due to this organism in a 55-year-old lady.

Going the dHis-tance: Site-Directed Cu2+ Labeling of Proteins and Nucleic Acids.

In this Account, we showcase site-directed Cu2+ labeling in proteins and DNA, which has opened new avenues for the measurement of the structure and dynamics of biomolecules using electron paramagnetic resonance (EPR) spectroscopy. In proteins, the spin label is assembled in situ from natural amino acid residues and a metal complex and requires no post-expression synthetic modification or purification procedures. The labeling scheme exploits a double histidine (dHis) motif, which utilizes endogenous or site-specifically mutated histidine residues to coordinate a Cu2+ complex. Pulsed EPR measurements on such Cu2+-labeled proteins potentially yield distance distributions that are up to 5 times narrower than the common protein spin label-the approach, thus, overcomes the inherent limitation of the current technology, which relies on a spin label with a highly flexible side chain. This labeling scheme provides a straightforward method that elucidates biophysical information that is costly, complicated, or simply inaccessible by traditional EPR labels. Examples include the direct measurement of protein backbone dynamics at ß-sheet sites, which are largely inaccessible through traditional spin labels, and rigid Cu2+-Cu2+ distance measurements that enable higher precision in the analysis of protein conformations, conformational changes, interactions with other biomolecules, and the relative orientations of two labeled protein subunits. Likewise, a Cu2+ label has been developed for use in DNA, which is small, is nucleotide independent, and is positioned within the DNA helix. The placement of the Cu2+ label directly reports on the biologically relevant backbone distance. Additionally, for both of these labeling techniques, we have developed models for interpretation of the EPR distance information, primarily utilizing molecular dynamics (MD) simulations. Initial results using force fields developed for both protein and DNA labels have agreed with experimental results, which has been a major bottleneck for traditional spin labels. Looking ahead, we anticipate new combinations of MD and EPR to further our understanding of protein and DNA conformational changes, as well as working synergistically to investigate protein-DNA interactions.

An optimal acquisition scheme for Q-band EPR distance measurements using Cu2+-based protein labels.

Recent advances in site-directed Cu2+ 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 Cu2+ 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 Cu2+ 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 Cu2+ 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 Cu2+ systems. Furthermore, the concepts presented here may be applied to other metals and pulsed EPR experiments.

Cu2+-based distance measurements by pulsed EPR provide distance constraints for DNA backbone conformations in solution.

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.

Internal drainage of retropharyngeal abscess secondary to esophageal foreign bodies: a case series.

INTRODUCTION AND OBJECTIVES: Foreign body oesophagus is a commonly seen emergency in ENT. It is seen both in children and adults. When sharp bony foreign bodies such as chicken, fish, and mutton bone gets impacted in the oesophagus, it predisposes the patient to various complications. The foreign body can migrate extraluminally with time and cause retropharyngeal abscess. MATERIALS AND METHODS: Retrospective study over a period of 6 months from November 2019 to April 2020 of patients with foreign body oesophagus. We came across 20 patients with oesophageal foreign bodies and five of them had associated retropharyngeal abscess. Rigid esophagoscopy with foreign body removal and internal drainage of pus through the oesophageal rent followed by conservative management with intravenous antibiotics based on culture and sensitivity was done. RESULTS: Patients improved drastically as the pus drained into the oesophagus via the rent in the posterior oesophageal wall and did not require an external incision and drainage. They were discharged in a week. CONCLUSION: Removal of partial extraluminally migrated foreign body oesophagus and internal drainage of the abscess followed by nasogastric feeds till the rent resolves and intravenous pus culture-sensitive antibiotics fastens patient recovery and reduces the morbidity associated with external incision and drainage and oesophageal rent repair.

'Shifting from anxiety to the new normal': A qualitative exploration on personal protective equipment use by otorhinolaryngology health-care professionals during COVID-19 pandemic.

Background: The novel coronavirus pandemic has influenced the working practice of health-care professionals who come across symptomatic and asymptomatic COVID patients in their day-to-day practice. Especially, among HCWs in otorhinolaryngology, with the risk of exposure being high, hence were mandated to use personal protective equipment (PPE). Materials and Methods: The change in perceptions and patterns of PPE use throughout the COVID-19 pandemic was studied in detail through interviews conducted among 15 key informants, and the data were analyzed using health belief model in our study. Results: A health belief model explains the trajectory of PPE use by otorhinolaryngology health care providers during the COVID-19 pandemic. The course of usage of PPE by otorhinolaryngology health-care professionals during the COVID-19 pandemic was explained through the health belief model. During the initial days of the COVID-19 pandemic, intense perceived severity and susceptibility to COVID infection led to PPE use, and otorhinolaryngology HCWs resorted to higher grade PPEs which gave optimal protection; but in course of time with a better understanding of the natural course of illness, minimal PPEs without compromising HCW safety were used with minimal discomfort. Perceived severity of COVID infection on self and family, health knowledge, influence of peers, and support from the institution encouraged them in using PPEs. Conclusion: We found that various aspects of health belief model such as the perceived susceptibility, perceived severity of the disease, perceived barriers and benefits in PPE use, self-efficacy, health-related knowledge, and the cues to action influence PPE use among otorhinolaryngology HCWs. The key findings can be applied in behavior change models to promote the use of PPE in the hospitals, especially during the time of pandemic.

Synthesis of Square Planar Cu4 Clusters.

Template-assisted synthesis of well-defined polynuclear clusters remains a challenge for [M4 ] square planar topologies. Herein, we present a tetraamine scaffold R L(NH2 )4 , where L is a rigidified resorcin[4]arene, to direct the formation of C4 -symmetric R L(NH)4 Cu4 clusters with Cu-Cu distances around 2.7 Å, suggesting metal-metal direct interactions are operative since the sum of copper's van der Waals radii is 2.8 Å. DFT calculations display HOMO to HOMO-3 residing all within a 0.1 eV gap. These four orbitals display significant electron density contribution from the Cu centers suggesting a delocalized electronic structure. The one-electron oxidized [Cu4 ]+ species was probed by variable temperature X-band continuous wave-electron paramagnetic resonance (CW-EPR), which displays a multiline spectrum at room temperature. This work presents a novel synthetic strategy for [M4 ] clusters and a new platform to investigate activation of small molecules.

Role of endotracheal tube size on nasal and laryngeal morbidity during awake fiberoptic nasotracheal intubation: A Randomized controlled trial.

Background and Aims: Awake fibreoptic nasotracheal intubation is associated with adverse airway and hemodynamic complications. The aim of this study was to evaluate the role of endotracheal tube size on nasal and laryngeal morbidity during awake fibreoptic-guided nasotracheal intubation. Material and Methods: Eighty patients recruited to undergo awake fibreoptic intubation were randomly allocated to Group C (standard size endotracheal tube) and Group S (small size endotracheal tube followed by exchange to standard size using Airway Exchange Catheter under general anesthesia). Nasal morbidity was assessed by incidence of epistaxis, olfactory acuity, and mucociliary clearance. Patient discomfort during intubation was assessed using grimace score and hemodynamic parameters were recorded. Postoperatively, the incidence of nasal and laryngeal injury was recorded using nasendoscopy and telelaryngoscopy, respectively. Results: Demographic profile between the two groups was comparable. Epistaxis was noted in 47.5% of patients in group C as compared to 12.5% in group S. Postoperative olfactory acuity was decreased [2 (1-4) vs 4 (2-5)] and saccharin clearance time was prolonged (314 s vs 134 s) in Group C as compared to Group S. (P-value <0.001) Higher grimace score [2 (1-3) vs 1 (0-2)] and increased hemodynamic response was demonstrated in Group C. (P-value <0.001) Incidence of nasal injury [2 (1-4) vs 1 (0-2)] and laryngeal injury [1 (0-2) vs 0 (0-2)] was more in Group C as compared to Group S. Conclusion: Awake fibreoptic nasotracheal intubation with small size endotracheal tube followed by exchange to standard size under general anesthesia reduces nasal, laryngeal, and hemodynamic complications.

Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules.

Distance distribution information obtained by pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology. Increasingly, such information is used in integrative structural modeling, where it delivers unique restraints on the width of conformational ensembles. In order to ensure reliability of the structural models and of biological conclusions, we herein define quality standards for sample preparation and characterization, for measurements of distributed dipole-dipole couplings between paramagnetic labels, for conversion of the primary time-domain data into distance distributions, for interpreting these distributions, and for reporting results. These guidelines are substantiated by a multi-laboratory benchmark study and by analysis of data sets with known distance distribution ground truth. The study and the guidelines focus on proteins labeled with nitroxides and on double electron-electron resonance (DEER aka PELDOR) measurements and provide suggestions on how to proceed analogously in other cases.

Change in dysphagia and laryngeal function after radical radiotherapy in laryngo pharyngeal malignancies - a prospective observational study.

BACKGROUND: Intensity modulated radiotherapy (IMRT) has the perceived advantage of function preservation by reduction of toxicities in the treatment of laryngo-pharyngeal malignancies. The aim of the study was to assess changes in dysphagia from baseline (i.e. prior to start of treatment) at three and six months post treatment in patients with laryngo-pharyngeal malignancies treated with radical radiotherapy ± chemotherapy. Functional assessment of other structures involved in swallowing was also studied. MATERIALS AND METHODS: 40 patients were sampled consecutively. 33 were available for final analysis. Dysphagia, laryngeal edema, xerostomia and voice of patients were assessed at baseline and at three and six months after treatment. Radiation was delivered with simultaneous integrated boost (SIB) using volumetric modulated radiation therapy (VMAT). Concurrent chemotherapy was three weekly cisplatin 100 mg/m2. RESULTS: Proportion of patients with dysphagia rose significantly from 45.5% before the start of treatment to 57.6% at three months and 60.6% at six months post treatment (p = 0.019). 67% patients received chemotherapy and addition of chemotherapy had a significant correlation with dysphagia (p = 0.05, r = -0.336). Severity of dysphagia at three and six months correlated significantly with the mean dose received by the superior constrictors (p = 0.003, r = 0.508 and p = 0.024, r = 0.391) and oral cavity (p = 0.001, r = 0.558 and p = 0.003, r = 0.501). There was a significant worsening in laryngeal edema at three and six months post treatment (p < 0.01) when compared to the pre-treatment examination findings with 60.6% of patients having grade two edema at six months. Significant fall in the mean spoken fundamental frequency from baseline was seen at 6 months (p = 0.04), mean fall was 21.3 Hz (95% CI: 1.5-41 Hz) with significant increase in roughness of voice post treatment (p = 0.01). CONCLUSION: There was progressive worsening in dysphagia, laryngeal edema and voice in laryngo-pharyngeal malignancies post radical radiotherapy ± chemotherapy.

Orientation and dynamics of Cu2+ based DNA labels from force field parameterized MD elucidates the relationship between EPR distance constraints and DNA backbone distances.

Pulsed electron paramagnetic resonance (EPR) based distance measurements using the recently developed Cu2+-DPA label present a promising strategy for measuring DNA backbone distance constraints. Herein we develop force field parameters for Cu2+-DPA in order to understand the features of this label at an atomic level. We perform molecular dynamics (MD) simulations using the force field parameters of Cu2+-DPA on four different DNA duplexes. The distance between the Cu2+ centers, extracted from the 2 µs MD trajectories, agrees well with the experimental distance for all the duplexes. Further analyses of the trajectory provide insight into the orientation of the Cu2+-DPA inside the duplex that leads to such agreement with experiments. The MD results also illustrate the ability of the Cu2+-DPA to report on the DNA backbone distance constraints. Furthermore, measurement of fluctuations of individual residues showed that the flexibility of Cu2+-DPA in a DNA depends on the position of the label in the duplex, and a 2 µs MD simulation is not sufficient to fully capture the experimental distribution in some cases. Finally, the MD trajectories were utilized to understand the key aspects of the double electron electron resonance (DEER) results. The lack of orientational selectivity effects of the Cu2+-DPA at Q-band frequency is rationalized in terms of fluctuations in the Cu2+ coordination environment and rotameric fluctuations of the label linker. Overall, a combination of EPR and MD simulations based on the Cu2+-DPA labelling strategy can contribute towards understanding changes in DNA backbone conformations during protein-DNA interactions.

Comparison of efficacy of potassium titanyl phosphate laser & diode laser in the management of inferior turbinate hypertrophy: A randomized controlled trial.

Background & objectives: Inferior turbinate hypertrophy (ITH) is a common condition causing nasal obstruction. This study was undertaken to compare the efficacy of potassium titanyl phosphate (KTP) laser and diode laser in the reduction of the turbinate size. Methods: This randomized controlled trial included 209 patients with ITH. Pre-operative symptoms were assessed based on the Nasal Obstruction Symptom Evaluation (NOSE) score. Diagnostic nasal endoscopy was done to rule out other nasal sinuses. Nasal mucociliary clearance was measured by saccharin transit time (STT). Postoperatively, the NOSE score, STT and complications were assessed at days one and two, at one week, one month and three months. Results: Of the 209 patients analyzed at day one, the median NOSE score was 50 in the diode group and 40 in the KTP group, and at three months, 15 in the diode group and five in the KTP group. KTP laser showed a 93 per cent improvement in the NOSE score as compared to 77 per cent improvement shown by diode laser group. Among the intra-operative complications, of the 104 patients in the diode group, 6.73 per cent had burning sensation and 91.43 per cent had bleeding, and of 105 patients in the KTP group, 54.29 per cent had burning sensation and 36.54 per cent had bleeding. Among the post-operative complications in the KTP group, 32 and 34 per cent had bloody nasal discharge on days one and two, compared to 12 and 14 per cent in diode group. Crusting was present in 61 and 49 per cent on days one and two in KTP group as compared to 9 and 15 per cent in diode group, respectively. In the KTP group 30 per cent had synechiae as compared to 10 per cent in diode group. Interpretation & conclusions: KTP laser was more efficacious than diode laser in improving the NOSE scores but with slightly increased rate of complications in early post-operative period. Both the lasers impaired the mucociliary clearance mechanism of the nose till three months of post-operative follow up.

Comparison of middle ear function and hearing thresholds in children with adenoid hypertrophy after microdebrider and conventional adenoidectomy: a randomised controlled trial.

BACKGROUND: Microdebrider has superior efficacy in clearing the adenoids, compared to curettage. We compared the improvement in middle ear function and hearing thresholds after adenoidectomy, by both methods. MATERIALS AND METHODS: 126 patients (median age-9 years) were randomized into groups A and B, where adenoidectomy was done by microdebrider and curettage, respectively. Middle ear function parameters and hearing thresholds were measured serially. RESULTS: The mean improvement in middle ear pressure, compliance and hearing thresholds were 92.5 ± 67.6 and 84.2 ± 71.4 daPa; (p = 0.40), 0.19 ± 0.34 and 0.27 ± 0.27 mL; (p = 0.07) and 3.20 ± 4.95 and 2.54 ± 3.98 dB; (p = 0.27), in groups A and B, respectively. Reversal of type B tympanograms was noted in both groups. CONCLUSIONS: Middle ear function and hearing thresholds improved in both groups after adenoidectomy. More improvement was noted in the microdebrider group, which, however, was not significant.

Double Histidine Based EPR Measurements at Physiological Temperatures Permit Site-Specific Elucidation of Hidden Dynamics in Enzymes.

Protein dynamics is at the heart of all cellular processes. Here, we utilize the dHis-CuII NTA label to obtain site-specific information on dynamics for both an α-helix and ß-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.

Designing Open Metal Sites in Metal-Organic Frameworks for Paraffin/Olefin Separations.

Incorporating open metal sites (OMS) into metal-organic frameworks allows design of well-defined binding sites for selective molecular adsorption, which has a profound impact on catalysis and separations. We demonstrate that Cu(I) sites incorporated into MFU-4l preferentially adsorb olefins over paraffins. Density functional theory (DFT) calculations show that the OMS are independent, with no dependence of binding energy on olefin loading up to one olefin per Cu(I). Experimentally, increasing Cu(I) loading increased olefin uptake without affecting the binding energy, as predicted by DFT and confirmed by temperature-programmed desorption. The potential of this material for olefin/paraffin separation under ambient conditions was investigated by gas adsorption and column breakthrough experiments for an equimolar ratio of olefin/paraffin. High-grade propylene and ethylene (>99.999%) can be generated using temperature-concentration swing recycling from a Cu(I)-MFU-4l packed column with no measurable paraffin breakthrough.

Efficient localization of a native metal ion within a protein by Cu2+-based EPR distance measurements.

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 Cu2+ 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 Cu2+ 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.