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Pulsed electron–electron double resonance (PELDOR) spectroscopy is a powerful method for determining nucleic acid (NA) structure and conformational dynamics. PELDOR with molecular dynamics (MD) simulations opens up unique possibilities for defining the conformational ensembles of flexible, three-dimensional, self-assembled complexes of NA. Understanding the diversity and structure of these complexes is vital for uncovering matrix and regulative biological processes in the human body and artificially influencing them for therapeutic purposes. To explore the reliability of PELDOR and MD simulations, we site-specifically attached nitroxide spin labels to oligonucleotides, which form self-assembled complexes between NA chains and exhibit significant conformational flexibility. The DNA complexes assembled from a pair of oligonucleotides with different linker sizes showed excellent agreement between the distance distributions obtained from PELDOR and calculated from MD simulations, both for the mean inter-spin distance and the distance distribution width. These results prove that PELDOR with MD simulations has significant potential for studying the structure and dynamics of conformational flexible complexes of NA. [ABSTRACT FROM AUTHOR]

Pulsed electron–electron double resonance (PELDOR) spectroscopy is a powerful method for determining nucleic acid (NA) structure and conformational dynamics. PELDOR with molecular dynamics (MD) simulations opens up unique possibilities for defining the conformational ensembles of flexible, three-dimensional, self-assembled complexes of NA. Understanding the diversity and structure of these complexes is vital for uncovering matrix and regulative biological processes in the human body and artificially influencing them for therapeutic purposes. To explore the reliability of PELDOR and MD simulations, we site-specifically attached nitroxide spin labels to oligonucleotides, which form self-assembled complexes between NA chains and exhibit significant conformational flexibility. The DNA complexes assembled from a pair of oligonucleotides with different linker sizes showed excellent agreement between the distance distributions obtained from PELDOR and calculated from MD simulations, both for the mean inter-spin distance and the distance distribution width. These results prove that PELDOR with MD simulations has significant potential for studying the structure and dynamics of conformational flexible complexes of NA.

This study presents a new algorithm for group decision-making solutions using Pythagorean Fuzzy Soft Matrices (PFSMs) and confident weight is given by experts. Pythagorean Fuzzy Set (PFS) is a generalization of the intuitionistic fuzzy set (IFS). Therefore, in real-life problems for uncertainty, the decision-making mechanism in PFSs outcomes better than IFS decision-making. Pythagorean Fuzzy Soft Set (PFSS) is deriving from the combination of PFS and Soft Set. PFSM is also the matrix representation of PFSSs. Based on the cardinalities of the PFSS, experts have been given a new method that assigns confident weight. Confident weight is given according to the experience and knowledge of each expert. For this process, the choice matrix and the combined choice matrix are created first. PFSMs and choice matrices given for each expert are multiplied and the matrices obtained are summed. Pythagorean distance measurements were used to check the accuracy of the results obtained by applying the algorithm. A medical case was studied to see if the proposed method for group decision-making is feasible. In the section of medical case, infectious diseases that were common before COVID-19 were selected. The newly given algorithm was applied to the opinions of physicians about these diseases. According to the Hamming Distance values, the results of three out of four physicians are the same; In the values obtained with Euclidean distance, it was seen that the opinions of all physicians were the same. It has been revealed that the newly proposed algorithm has increased the reliability of the results from the group decision analysis.

We present a new photoswitchable spin label for light-induced pulsed electron paramagnetic resonance dipolar spectroscopy (LiPDS), the photoexcited triplet state of erythrosin B (EB), which is ideal for biological applications. With this label, we perform an in-depth study of the orientational effects in dipolar traces acquired using the refocused laser-induced magnetic dipole technique to obtain information on the distance and relative orientation between the EB and nitroxide labels in a rigid model peptide, in good agreement with density functional theory predictions. Additionally, we show that these orientational effects can be averaged to enable an orientation-independent analysis to determine the distance distribution. Furthermore, we demonstrate the feasibility of these experiments above liquid nitrogen temperatures, removing the need for expensive liquid helium or cryogen-free cryostats. The variety of choices in photoswitchable spin labels and the affordability of the experiments are critical for LiPDS to become a widespread methodology in structural biology. [ABSTRACT FROM AUTHOR]

Due to impaired mobility caused by aging, it is very important to employ early detection and monitoring of gait parameters to prevent the inevitable huge amount of medical cost at a later age. For gait training and potential tele-monitoring application outside clinical settings, low-cost yet highly reliable gait analysis systems are needed. This research proposes using a single LiDAR system to perform automatic gait analysis with polynomial fitting. The experimental setup for this study consists of two different walking speeds, fast walk and normal walk, along a 5-m straight line. There were ten test subjects (mean age 28, SD 5.2) who voluntarily participated in the study. We performed polynomial fitting to estimate the step length from the heel projection cloud point laser data as the subject walks forwards and compared the values with the visual inspection method. The results showed that the visual inspection method is accurate up to 6 cm while the polynomial method achieves 8 cm in the worst case (fast walking). With the accuracy difference estimated to be at most 2 cm, the polynomial method provides reliability of heel location estimation as compared with the observational gait analysis. The proposed method in this study presents an improvement accuracy of 4% as opposed to the proposed dual-laser range sensor method that reported 57.87 cm ± 10.48, an error of 10%. Meanwhile, our proposed method reported ±0.0633 m, a 6% error for normal walking.

This paper investigates the problem of localization and circumnavigation of an unknown stationary target in 3D space using distance measurements. An estimator is first designed in 2D space to locate the target, with which a control protocol is proposed to force the agent to travel along a circular trajectory around the target. Then the algorithm is extended to 3D space to circumnavigate the target under the condition of obtaining gravity direction additionally. In particular, the agent moves alternately in the X-Y horizontal planes and the X-Z vertical planes in different patterns under different switching conditions, which is beneficial to monitor the target from a broader perspective. Meanwhile, the rigorous stability analyses of two cases are given in terms of the Lyapunov method. Finally, the effectiveness of the proposed algorithms is verified through numerical simulations and a real experiment. [ABSTRACT FROM AUTHOR]

This study presents a new algorithm for group decision-making solutions using Pythagorean Fuzzy Soft Matrices (PFSMs) and confident weight is given by experts. Pythagorean Fuzzy Set (PFS) is a generalization of the intuitionistic fuzzy set (IFS). Therefore, in real-life problems for uncertainty, the decision-making mechanism in PFSs outcomes better than IFS decision-making. Pythagorean Fuzzy Soft Set (PFSS) is deriving from the combination of PFS and Soft Set. PFSM is also the matrix representation of PFSSs. Based on the cardinalities of the PFSS, experts have been given a new method that assigns confident weight. Confident weight is given according to the experience and knowledge of each expert. For this process, the choice matrix and the combined choice matrix are created first. PFSMs and choice matrices given for each expert are multiplied and the matrices obtained are summed. Pythagorean distance measurements were used to check the accuracy of the results obtained by applying the algorithm. A medical case was studied to see if the proposed method for group decision-making is feasible. In the section of medical case, infectious diseases that were common before COVID-19 were selected. The newly given algorithm was applied to the opinions of physicians about these diseases. According to the Hamming Distance values, the results of three out of four physicians are the same; In the values obtained with Euclidean distance, it was seen that the opinions of all physicians were the same. It has been revealed that the newly proposed algorithm has increased the reliability of the results from the group decision analysis. [ABSTRACT FROM AUTHOR]

Pulse dipolar electron paramagnetic resonance spectroscopy (PDS) is continuously furthering the understanding of chemical and biological assemblies through distance measurements in the nanometer range. New paramagnets and pulse sequences can provide structural insights not accessible through other techniques. In the pursuit of alternative spin centers for PDS, we synthesized a low-spin CoII complex bearing a nitroxide (NO) moiety, where both the CoII and NO have an electron spin S of 1/2. We measured CoII-NO distances with the well-established double electron–electron resonance (DEER aka PELDOR) experiment, as well as with the five- and six-pulse relaxation-induced dipolar modulation enhancement (RIDME) spectroscopies at Q-band frequencies (34 GHz). We first identified challenges related to the stability of the complex in solution via DEER and X-ray crystallography and showed that even in cases where complex disproportionation is unavoidable, CoII-NO PDS measurements are feasible and give good signal-to-noise (SNR) ratios. Specifically, DEER and five-pulse RIDME exhibited an SNR of ~100, and while the six-pulse RIDME exhibited compromised SNR, it helped us minimize unwanted signals from the RIDME traces. Last, we demonstrated RIDME at a 10 μM sample concentration. Our results demonstrate paramagnetic CoII to be a feasible spin center in medium magnetic fields with opportunities for PDS studies involving CoII ions. [ABSTRACT FROM AUTHOR]

We present a new photoswitchable spin label for light-induced pulsed electron paramagnetic resonance dipolar spectroscopy (LiPDS), the photoexcited triplet state of erythrosin B (EB), which is ideal for biological applications. With this label, we perform an in-depth study of the orientational effects in dipolar traces acquired using the refocused laser-induced magnetic dipole technique to obtain information on the distance and relative orientation between the EB and nitroxide labels in a rigid model peptide, in good agreement with density functional theory predictions. Additionally, we show that these orientational effects can be averaged to enable an orientation-independent analysis to determine the distance distribution. Furthermore, we demonstrate the feasibility of these experiments above liquid nitrogen temperatures, removing the need for expensive liquid helium or cryogen-free cryostats. The variety of choices in photoswitchable spin labels and the affordability of the experiments are critical for LiPDS to become a widespread methodology in structural biology.

The first comprehensive solid‐state nuclear magnetic resonance (NMR) characterization of geminal alane‐phosphane frustrated Lewis pairs (Al/P FLPs) is reported. Their relevant NMR parameters (isotropic chemical shifts, direct and indirect 27Al‐31P spin‐spin coupling constants, and 27Al nuclear electric quadrupole coupling tensor components) have been determined by numerical analysis of the experimental NMR line shapes and compared with values computed from the known crystal structures by using density functional theory (DFT) methods. Our work demonstrates that the 31P NMR chemical shifts for the studied Al/P FLPs are very sensitive to slight structural inequivalences. The 27Al NMR central transition signals are spread out over a broad frequency range (>200 kHz), owing to the presence of strong nuclear electric quadrupolar interactions that can be well‐reproduced by the static 27Al wideband uniform rate smooth truncation (WURST) Carr‐Purcell‐Meiboom‐Gill (WCPMG) NMR experiment. 27Al chemical shifts and quadrupole tensor components offer a facile and clear distinction between three‐ and four‐coordinate aluminum environments. For measuring internuclear Al⋅⋅⋅P distances a new resonance‐echo saturation‐pulse double‐resonance (RESPDOR) experiment was developed by using efficient saturation via frequency‐swept WURST pulses. The successful implementation of this widely applicable technique indicates that internuclear Al⋅⋅⋅P distances in these compounds can be measured within a precision of ±0.1 Å. [ABSTRACT FROM AUTHOR]

Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain "K1," and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron-electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite reagents, should make K1 labeling an increasingly viable alternative to cysteine-based methods for SDSL in proteins.

Pulse dipolar electron paramagnetic resonance spectroscopy (PDS) is continuously furthering the understanding of chemical and biological assemblies through distance measurements in the nanometer range. New paramagnets and pulse sequences can provide structural insights not accessible through other techniques. In the pursuit of alternative spin centers for PDS, we synthesized a low-spin CoII complex bearing a nitroxide (NO) moiety, where both the CoII and NO have an electron spin S of 1/2. We measured CoII-NO distances with the well-established double electron–electron resonance (DEER aka PELDOR) experiment, as well as with the five- and six-pulse relaxation-induced dipolar modulation enhancement (RIDME) spectroscopies at Q-band frequencies (34 GHz). We first identified challenges related to the stability of the complex in solution via DEER and X-ray crystallography and showed that even in cases where complex disproportionation is unavoidable, CoII-NO PDS measurements are feasible and give good signal-to-noise (SNR) ratios. Specifically, DEER and five-pulse RIDME exhibited an SNR of ~100, and while the six-pulse RIDME exhibited compromised SNR, it helped us minimize unwanted signals from the RIDME traces. Last, we demonstrated RIDME at a 10 μM sample concentration. Our results demonstrate paramagnetic CoII to be a feasible spin center in medium magnetic fields with opportunities for PDS studies involving CoII ions.

This study applied size and shape analyses through traditional and geometric morphometrics to quantify the differences between three taxa: Lathyrus sativus (grass pea), Lens culinaris (lentil) and Vicia faba (broad bean). This analysis represents a pilot study in view of a wider future application of this technique to identify the role of legumes throughout the Western Mediterranean in the Bronze and Iron Ages. Here, we quantify the morphological differences of seed species from the site of La Font de la Canya (Catalonia, Spain), an Iron Age (625–150 bc) centre of storage and trade which delivered a vast assemblage of cereal, fruit and legume remains. Our results reveal that seed outline analyses have a greater potential of prediction than length measurements. The combination of the two, however, leads to even more precise predictions. Moreover, the quantitative differences between the lentils of the different samples confirm the potential of these analyses to further identify intra-species morphotypes. [ABSTRACT FROM AUTHOR]

The extensive use of Internet of things (IoT) devices demonstrates an increasing demand for secure frameworks. Position altering attacks are amongst the most severe vulnerabilities in two‐way time of arrival distance measurement technique, which is the most common in distance measurements by IoT devices. The overshadowing attack as a distance enlargement attack replays the authentic signal with a certain amount of delay and sensible power amplification. Therefore, the attack detection is difficult and requires a precise model to estimate its behaviour. The contribution of this study is a location estimation method based on the overshadowing attack probabilistic model that estimates the attack outcome delay and corrects the erroneous measurements. The overshadow delay, which is the delay between the authentic signal and the attacked one, plays a key role in this process. The method has been evaluated by three attack scenarios and compared to the conventional maximum likelihood (CML) and mixture secure location of things (M‐SLOT) methods. The results of the proposed method demonstrate root mean squared error (RMSE) of 0.48 in position estimation. With the same dataset, RMSEs of M‐SLOT and CML methods are 4.69 and 9.84, respectively, which confirm the better performance of the proposed method. [ABSTRACT FROM AUTHOR]

Face recognition has become an attractive field in computer-based application development in the last few decades. This is because of the wide range of areas in which it is used. In addition, because of the wide variations of faces, face recognition from database images, real data, capture images, and sensor images are a challenging problem and limitation. Image processing, pattern recognition, and computer vision are relevant subjects to the face recognition field. The innovation of new approaches of face authentication technologies is a continuous subject to building much stronger face recognition algorithms. In this work, to identify a face, three major strategies for feature extractions are discussed. Appearance-based, Model-based methods and hybrid methods as feature extractions techniques are discussed too. There is also a review of major person recognition research. The characteristics of good face authentication applications, Classification, Distance measurements, and face databases are discussed while the final suggested methods are presented. This research has six sections ordered as follows: Section one is the introduction. Section two is dedicated to applications related to face recognition. In section three, face recognition techniques are presented by details. Then, classification types are illustrated in section four. In section five, standard face databases are presented. Finally, in section six, the conclusion is presented followed by the list of references.

An existing approach to the deployment of a lunar navigation system for lunar missions is further developed. Attention focuses on radio navigation systems operating with a query model of measurements. The stations in the network are assumed to be located in the vicinity of potential lunar landing sites and possible bases. In a priori analysis of the positioning accuracy, assignment errors of beacon initial coordinates in the local navigation system are taken into account, in the case where coordinate assignment is based on mutual measurements of the distances between the beacons. [ABSTRACT FROM AUTHOR]

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Fluorescence resonance energy transfer in single enzyme molecules (smFRET, single-molecule measurement) allows the measurement of multicomponent distance distributions in complex biomolecules similar to pulsed electron–electron double resonance (PELDOR, ensemble measurement). Both methods use reporter groups: FRET exploits the distance dependence of the electric interaction between electronic transition dipole moments of the attached fluorophores, whereas PELDOR spectroscopy uses the distance dependence of the interaction between the magnetic dipole moments of attached spin labels. Such labels can be incorporated easily to cysteine residues in the protein. Comparison of distance distributions obtained with both methods was carried out with the H+-ATPase from Escherichia coli (EF0F1). The crystal structure of this enzyme is known. It contains endogenous cysteines, and as an internal reference two additional cysteines were introduced (EF0F1–γT106C–εH56C). These positions were chosen to allow application of both methods under optimal conditions. Both methods yield very similar multicomponent distance distributions. The dominating distance distribution (> 50%) is due to the two cysteines introduced by site-directed mutagenesis and the distance is in agreement with the crystal structure. Two additional distance distributions are detected with smFRET and with PELDOR. These can be assigned by comparison with the structure to labels at endogenous cysteines. One additional distribution is detected only with PELDOR. The comparison indicates that under optimal conditions smFRET and PELDOR result in the same distance distributions. PELDOR has the advantage that different distributions can be obtained with ensemble measurements, whereas FRET requires single-molecule techniques. [ABSTRACT FROM AUTHOR]

Spin‐label electron spin resonance (ESR) has emerged as a powerful tool to characterize protein dynamics. One recent advance is the development of ESR for resolving dynamical components that occur or coexist during a biological process. It has been applied to study the complex structural and dynamical aspects of membranes and proteins, such as conformational changes in protein during translocation from cytosol to membrane, conformational exchange between equilibria in response to protein‐protein and protein‐ligand interactions in either soluble or membrane environments, protein oligomerization, and temperature‐ or hydration‐dependent protein dynamics. As these topics are challenging but urgent for understanding the function of a protein on the molecular level, the newly developed ESR methods to capture individual dynamical components, even in low‐populated states, have become a great complement to other existing biophysical tools. [ABSTRACT FROM AUTHOR]

Precise nanoscale distance measurements by pulsed electron paramagnetic resonance (EPR) spectroscopy play a crucial role in structural studies of biomolecules. The properties of the spin labels used in this approach determine the sensitivity limits, attainable distances, and proximity to biological conditions. Herein, we propose and validate the use of photoexcited fullerenes as spin labels for pulsed dipolar (PD) EPR distance measurements. Hyperpolarization and the narrower spectrum of fullerenes compared to other triplets (e.g., porphyrins) boost the sensitivity, and superior relaxation properties allow PD EPR measurements up to a near‐room temperature. This approach is demonstrated using fullerene–nitroxide and fullerene–triarylmethyl pairs, as well as a supramolecular complex of fullerene with nitroxide‐labeled protein. Photoexcited triplet fullerenes can be considered as new spin labels with outstanding spectroscopic properties for future structural studies of biomolecules. [ABSTRACT FROM AUTHOR]

• Will pulse EPR cross the diffusion between the experts and the users? • Hyperfine spectroscopy applications are likely to remain within the expert court. • Pulse-dipolar EPR can become a widely used technique beyond the experts circle. • A dedicated, low cost, bench top DEER spectrometer is envisioned. Application of EPR to biological systems includes many techniques and applications. In this short perspective, which dares to look into the future, I focus on pulse EPR, which is my field of expertise. Generally, pulse EPR techniques can be divided into two main groups: (1) hyperfine spectroscopy, which explores electron-nuclear interactions, and (2) pulse-dipolar (PD) EPR spectroscopy, which is based on electron-electron spin interactions. Here I focus on PD-EPR because it has a better chance of becoming a widely applied, easy-to-use table-top method to study the structural and dynamic aspects of bio-molecules. I will briefly introduce this technique, its current state of the art, the challenges it is facing, and finally I will describe futuristic scenarios of low-cost PD-EPR approaches that can cross the diffusion barrier from the core of experts to the bulk of the scientific community. [ABSTRACT FROM AUTHOR]

This study develops an approach that incorporates power aggregation operators with the evaluation based on distance from average solution (EDAS) method under linguistic neutrosophic situations to solve fuzzy multi-criteria group decision-making problems. Firstly, the existing operational laws and comparison methods of linguistic neutrosophic numbers (LNNs) are analysed. Secondly, the distance measurement between two LNNs is defined. Thirdly, the power-weighted averaging operator and the power-weighted geometric operator with LNNs are developed to support the decision makers' evaluation information. The models to derive the criteria weights are also constructed based on the proposed distance measurements. Finally, the EDAS method is extended to resolve group decision-making problems in the linguistic neutrosophic environment. An illustrative example of the property management company selection is given to verify the effectiveness and practicality of the proposed approach. [ABSTRACT FROM AUTHOR]

The DEER (double electron-electron resonance, also called PELDOR) experiment, which probes the dipolar interaction between two spins and thus reveals distance information, is an important tool for structural studies. In recent years, shaped pump pulses have become a valuable addition to the DEER experiment. Shaped pulses offer an increased excitation bandwidth and the possibility to precisely adjust pulse parameters, which is beneficial especially for demanding biological samples. We have noticed that on our home built W-band spectrometer, the dead-time free 4-pulse DEER sequence with chirped pump pulses suffers from distortions at the end of the DEER trace. Although minor, these are crucial for Gd(III)-Gd(III) DEER where the modulation depth is on the order of a few percent. Here we present a modified DEER sequence--referred to as reversed DEER (rDEER)--that circumvents the coherence pathway which gives rise to the distortion. We compare the rDEER (with two chirped pump pulses) performance values to regular 4-pulse DEER with one monochromatic as well as two chirped pulses and investigate the source of the distortion. We demonstrate the applicability and effectivity of rDEER on three systems, ubiquitin labeled with Gd(III)-DOTA-maleimide (DOTA, 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid) or with Gd(III)-DO3A (DO3A, 1,4,7,10-Tetraazacyclododecane-1,4,7-triyl) triacetic acid) and the multidrug transporterMdfA, labeled with a Gd(III)-C2 tag, and report an increase in the signal-to-noise ratio in the range of 3 to 7 when comparing the rDEER with two chirped pump pulses to standard 4-pulse DEER. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • REL axation O ptimized A cquisition (Length) D istribution is a new DEER detection scheme. • DEER-RELOAD is acquired in segments which lengths are relaxation-optimized. • DEER-RELOAD detection scheme is demonstrated for standard 4-pulse DEER sequence. • Signal-to-noise improves by two- to threefold for two membrane protein complexes. Abstract Over the past decades pulsed electron-electron double resonance (PELDOR), often called double electron-electron resonance (DEER), became one of the major spectroscopic tools for measurements of nanometer-scale distances and distance distributions in non-crystalline biological and chemical systems. The method is based on detecting the amplitude of the primary (3-pulse DEER) or refocused (4-pulse DEER) spin echo for the so-called "observer" spins when the other spins coupled to the former by a dipolar interaction are flipped by a "pump" pulse at another EPR frequency. While the timing of the pump pulse is varied in steps, the positions of the observer pulses are typically fixed. For such a detection scheme the total length of the observer pulse train and the electron spin memory time determine the amplitude of the detected echo signal. Usually, the distance range considerations in DEER experiments dictate the total length of the observer pulse train to exceed the phase memory time by a factor of few and this leads to a dramatic loss of the signal-to-noise ratio (SNR). While the acquisition of the DEER signal seems to be irrational under such conditions, it is currently the preferred way to conduct DEER because of an effective filtering out of all other unwanted interactions. Here we propose a novel albeit simple approach to improve DEER sensitivity and decrease data acquisition time by introducing the signal acquisition scheme based on REL axation O ptimized A cquisition (Length) D istribution (DEER-RELOAD). In DEER-RELOAD the dipolar phase evolution signal is acquired in multiple segments in which the observer pulses are fixed at the positions to optimize SNR just for that specific segment. The length of the segment is chosen to maximize the signal acquisition efficiency according the phase relaxation properties of the spin system. The total DEER trace is then obtained by "stitching" the multiple segments into a one continuous trace. The utility of the DEER-RELOAD acquisition scheme has been demonstrated on an example of the standard 4-pulse DEER sequence applied to two membrane protein complexes labeled with nitroxides. While theoretical gains from the DEER-RELOAD scheme increase with the number of stitched segments, in practice, even dividing the acquisition of the DEER trace into two segments may improve SNR by a factor of >3, as it has been demonstrated for one of these two membrane proteins. [ABSTRACT FROM AUTHOR]

[Display omitted] • 5- and 6-pulse variable time RIDME experiments are introduced. • Variable-time RIDME has about double the SNR of the constant time experiment. • Artefact suppression can be combined with removal of nuclear modulation and relaxation contributions. • Variable-time acquisition can measure dipolar evolution window to arbitrary length before truncation. Pulse dipolar EPR spectroscopy (PDS) measurements are an important complementary tool in structural biology and are increasingly applied to macromolecular assemblies implicated in human health and disease at physiological concentrations. This requires ever higher sensitivity, and recent advances have driven PDS measurements into the mid-nanomolar concentration regime, though optimization and acquisition of such measurements remains experimentally demanding and time expensive. One important consideration is that constant-time acquisition represents a hard limit for measurement sensitivity, depending on the maximum measured distance. Determining this distance a priori has been facilitated by machine-learning structure prediction (AlphaFold2 and RoseTTAFold) but is often confounded by non-representative behaviour in frozen solution that may mandate multiple rounds of optimization and acquisition. Herein, we endeavour to simultaneously enhance sensitivity and streamline PDS measurement optimization to one-step by benchmarking a variable-time acquisition RIDME experiment applied to CuII-nitroxide and CuII-CuII model systems. Results demonstrate marked sensitivity improvements of both 5- and 6-pulse variable-time RIDME of between 2- and 5-fold over the constant-time analogues. [ABSTRACT FROM AUTHOR]

Recently, the photoexcited triplet state of porphyrin was proposed as a promising spin-label for pulsed dipolar electron paramagnetic resonance (EPR). Herein, we report the factors that determine the electron spin echo dephasing of the photoexcited porphyrin in a water–glycerol matrix. The electron spin relaxation of a water-soluble porphyrin was measured by Q-band EPR, and the temperature dependence and the effect of solvent deuteration on the relaxation times were studied. The phase memory relaxation rate (1/Tm) is noticeably affected by solvent nuclei and is substantially faster in protonated solvents than in deuterated solvents. The Tm is as large as 13–17 μs in deuterated solvent, potentially expanding the range of distances available for measurement by dipole spectroscopy with photoexcited porphyrin. The 1/Tm depends linearly on the degree of solvent deuteration and can be used to probe the environment of a porphyrin in or near a biopolymer, including the solvent accessibility of porphyrins used in photodynamic therapy. We characterized the noncovalent binding of porphyrin to human serum albumin (HSA) from 1/Tm and electron spin echo envelope modulation (ESEEM) and found that porphyrin is quite exposed to solvent on the surface of HSA. The 1/Tm and ESEEM are equally effective and provide complementary methods to determine the solvent accessibility of a porphyrin bound to protein or to determine the location of the porphyrin.

The substitution of Mg2+ by Mn2+ in the bacterial DnaB helicase from Helicobacter pylori, an ATP:Mg2+-fuelled protein engine, allows electron paramagnetic resonance (EPR) spectroscopy to be performed on this system. EPR experiments make it possible to monitor nucleotide binding and to estimate the fraction of bound Mn2+ through relaxation measurements. Furthermore, by measuring spin–spin distances we probe the geometry within such multimeric assemblies using ultra-wideband double electron-electron resonance (DEER) and relaxation induced dipolar modulation enhancement (RIDME). The extraction of distance distributions from RIDME experiments on high-spin paramagnetic centres is influenced by the presence of dipolar frequency overtones. We show herein that we can correct for these overtones by using a modified kernel function in Tikhonov regularization analysis routines, and that the overtone coefficients for Mn2+ in the DnaB helicase are practically the same as in the previously studied Mn2+–Mn2+ model compounds.

Electronic canopy characterization to determine structural properties is an important issue in tree crop management. Ultrasonic and optical sensors are the most used sensors for this purpose. The objective of this work was to assess the performance of an ultrasonic sensor under laboratory and field conditions in order to provide reliable estimations of distance measurements to apple tree canopies. To achieve this purpose, a methodology has been designed to analyze sensor performance in relation to foliage distance and to the effects of interference with adjacent sensors when working simultaneously. Results showed that the average error in distance measurement using the ultrasonic sensor in laboratory conditions was 0.64 cm. However, the increase of variability in field conditions reduced the accuracy of this kind of sensors when estimating distances to canopies. The average error in such situations was 3.19 cm. When analyzing interferences of adjacent sensors 30 cm apart, the average error was ±14.65 cm. When adjacent sensors were placed apart by 60 cm, the average error became 6.73 cm. The ultrasonic sensor tested has been proven to be suitable to estimate distances to the canopy in pistachio garden conditions when sensors are 60 cm apart or more and can, therefore, be used in a system to estimate structural canopy parameters in precision horticulture.

A novel double-emitter ultrasonic system for distance measurements based on the correlation method is presented. The proposed distance measurement method may be particularly useful in difficult conditions, e.g. for media parameters undergoing fast changes or in cases when obstacles and mechanical interference produce false reflections. The system is a development of a previously studied single-head idea. The present article covers a comparison of the two systems in terms of efficiency and precision. Experimental research described in this paper indicated that adding the second head improved the measurement exactness – standard deviation decreased by 40%. The correlation method is also described in detail, also giving the criterion for the quality of the measurement signal.

EPR pulsed dipolar spectroscopy (PDS) is indispensable for measurements of nm-scale distances between electronic spins in biological and other systems. While several useful modifications and pulse sequences for PDS have been developed in recent years, DEER experiments utilizing pump and observer pulses at two different frequencies remain the most popular for practical applications. One of the major drawbacks of all the available DEER approaches is the presence of a significant unmodulated fraction in the detected signal that arises from an incomplete inversion of the coupled spins by the pump pulse. The latter fraction is perceived as one of the major sources of error for the reconstructed distance distributions. We describe an alternative detection scheme – a R efocused O ut- O f- Ph ase DEER (ROOPh-DEER) – to acquire only the modulated fraction of the dipolar DEER signal. When Zeeman splitting is small compared to the temperature, the out-of-phase magnetization components cancel each other and are not observed in 4-pulse DEER experiment. In ROOPh-DEER these components are refocused by an additional pump pulse while the in-phase component containing an unmodulated background is filtered out by a pulse at the observed frequency applied right at the position of the refocused echo. Experimental implementation of the ROOPh-DEER detection scheme requires at least three additional pulses as was demonstrated on an example of a 7-pulse sequence. The application of 7-pulse ROOPh-DEER sequence to a model biradical yielded the interspin distance of 1.94 ± 0.07 nm identical to the one obtained with the conventional 4-pulse DEER, however, without the unmodulated background present as a dominant fraction in the latter signal. [ABSTRACT FROM AUTHOR]

Viologen dendrimers (G0* to G2*) bearing (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) spin labels are successfully synthesized via a divergent approach. 4-amino TEMPO is successfully grafted onto the periphery of the dendrimer moieties using Zincke reaction under dark conditions. The final products are characterized by cyclic voltammetry and the spin-label efficiencies are characterized by electron paramagnetic resonance (EPR) measurements. Cyclic voltammetric studies show the coexistence of both viologen as well as TEMPO electroactive subunits, and evidence for the intramolecular electrocatalytic reduction of TEMPO by viologen subunit is presented. EPR measurements show moderate to good spin-label efficiencies (72-85%), but the spin-labeled samples are not stable in the presence of air and light. The measured spin-label efficiency decreases with increasing dendrimer generation. Double electron resonance measurements provide interspin distances for zeroth generation G0* dendrimers. MM+ calculations perform using Hyperchem show an averaged single spin distance (2.34 nm) for G0* dendrimers and a more complex scenario of several possible interspin distances in the case of G1* and G2* dendrimers. [ABSTRACT FROM AUTHOR]

Pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) in combination with site-directed spin labeling (SDSL) of proteins and oligonucleotides is a powerful tool in structural biology. Instead of using the commonly employed gem-dimethyl-nitroxide labels, triarylmethyl (trityl) spin labels enable such studies at room temperature, within the cells and with single-frequency electron paramagnetic resonance (EPR) experiments. However, it has been repeatedly reported that labeling of proteins with trityl radicals led to low labeling efficiencies, unspecific labeling and label aggregation. Therefore, this work introduces the synthesis and characterization of a maleimide-functionalized trityl spin label and its corresponding labeling protocol for cysteine residues in proteins. The label is highly cysteine-selective, provides high labeling efficiencies and outperforms the previously employed methanethiosulfonate-functionalized trityl label. Finally, the new label is successfully tested in PDS measurements on a set of doubly labeled Yersinia outer protein O (YopO) mutants.