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Thaumatin, a potent sweet tasting protein extracted from the Katemfe Plant, is emerging as a natural alternative to synthetic non-nutritive sweeteners and flavor enhancer. As a food additive, its stability within the food matrix during thermal processing is of great interest to the food industry. When heated under neutral or basic conditions, thaumatin was found to lose its sweetness due to protein aggregation caused by sulfhydryl catalyzed disulfide bond interchange. At lower pH, while thaumatin was also found to lose sweetness after heating, it does so at a slower rate and shows more resistance to sweetness loss. SDS-PAGE indicated that thaumatin fragmented into multiple smaller pieces under heating in acidic pH. Using BEMPO-3, a lipophilic spin trap, we were able to detect the presence of a free-radical within the hydrophobic region of the protein during heating. Protein carbonyl content, a byproduct of protein oxidation, also increased upon heating, providing additional evidence for protein cleavage by a radical pathway. Hexyl gallate successfully inhibited the radical generation as well as protein carbonyl formation of thaumatin during heating., Competing Interests: Declaration of competing interest The authors declare no financial interests/personal relationships which may be considered as potential competing interests. Conflict of Interest The authors have no conflicts of interest to declare.

Complexins play a critical role in regulating SNARE-mediated exocytosis of synaptic vesicles. Evolutionary divergences in complexin function have complicated our understanding of the role these proteins play in inhibiting the spontaneous fusion of vesicles. Previous structural and functional characterizations of worm and mouse complexins have indicated the membrane curvature-sensing C-terminal domain of these proteins is responsible for differences in inhibitory function. We have characterized the structure and dynamics of the mCpx1 CTD in the absence and presence of membranes and membrane mimetics using NMR, ESR, and optical spectroscopies. In the absence of lipids, the mCpx1 CTD features a short helix near its N-terminus and is otherwise disordered. In the presence of micelles and small unilamellar vesicles, the mCpx1 CTD forms a discontinuous helical structure in its C-terminal 20 amino acids, with no preference for specific lipid compositions. In contrast, the mCpx1 CTD shows distinct compositional preferences in its interactions with large unilamellar vesicles. These studies identify structural divergences in the mCpx1 CTD relative to the wCpx1 CTD in regions that are known to be critical to the wCpx1 CTD's role in inhibiting spontaneous fusion of synaptic vesicles, suggesting a potential structural basis for evolutionary divergences in complexin function. 1 ., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Coronaviruses are a major infectious disease threat, and include the zoonotic-origin human pathogens SARS-CoV-2, SARS-CoV, and MERS-CoV (SARS-2, SARS-1, and MERS). Entry of coronaviruses into host cells is mediated by the spike (S) protein. In our previous ESR studies, the local membrane ordering effect of the fusion peptide (FP) of various viral glycoproteins including the S of SARS-1 and MERS has been consistently observed. We previously determined that the sequence immediately downstream from the S2' cleavage site is the bona fide SARS-1 FP. In this study, we used sequence alignment to identify the SARS-2 FP, and studied its membrane ordering effect. Although there are only three residue differences, SARS-2 FP induces even greater membrane ordering than SARS-1 FP, possibly due to its greater hydrophobicity. This may be a reason that SARS-2 is better able to infect host cells. In addition, the membrane binding enthalpy for SARS-2 is greater. Both the membrane ordering of SARS-2 and SARS-1 FPs are dependent on Ca 2+ , but that of SARS-2 shows a greater response to the presence of Ca 2+ . Both FPs bind two Ca 2+ ions as does SARS-1 FP, but the two Ca 2+ binding sites of SARS-2 exhibit greater cooperativity. This Ca 2+ dependence by the SARS-2 FP is very ion-specific. These results show that Ca 2+ is an important regulator that interacts with the SARS-2 FP and thus plays a significant role in SARS-2 viral entry. This could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca 2+ channel., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

We report on a new approach to the analysis of dynamic NMR lineshapes from polycrystalline (i.e., macroscopically disordered) samples in the presence of Magic Angle Spinning (MAS). This is an application of the Stochastic Liouville Equation developed by Freed and co-workers for treating restricted (i.e., microscopically ordered) motions. The 2 H nucleus in an internally-mobile C-CD 3 moiety serves as a prototype probe. The acronym is 2 H/MOMD/MAS, where MOMD stands for "microscopic-order-macroscopic-disorder." The key elements describing internal motions - their type, the local spatial restrictions, and related features of local geometry - are treated in MOMD generally, within their rigorous three-dimensional tensorial requirements. Based on this representation a single physically well-defined model of local motion has the capability of reproducing experimental spectra. There exist other methods for analyzing dynamic 2 H/MAS spectra which advocate simple motional modes. Yet, to reproduce satisfactorily the experimental lineshapes, one has either to use unusual parameter values, or combine several simple motional modes. The multi-simple-mode reasoning assumes independence of the constituent modes, features ambiguity as different simple modes may be used, renders inter-system comparison difficult as the overall models differ, and makes possible model-improvement only by adding yet another simple mode, i.e., changing the overall model. 2 H/MOMD/MAS is free of such limitations and inherently provides a clear physical interpretation. These features are illustrated. The advantage of 2 H/MOMD/MAS in dealing with sensitive but hardly investigated slow-motional lineshapes is demonstrated by applying it to actual experimental data. The results differ from those obtained previously with a two-site exchange scheme that yielded unusual parameters., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Coronaviruses (CoVs) are a major infectious disease threat and include the pathogenic human pathogens of zoonotic origin: severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV). Entry of CoVs into host cells is mediated by the viral spike (S) protein, which is structurally categorized as a class I viral fusion protein, within the same group as influenza virus and HIV. However, S proteins have two distinct cleavage sites that can be activated by a much wider range of proteases. The exact location of the CoV fusion peptide (FP) has been disputed. However, most evidence suggests that the domain immediately downstream of the S2' cleavage site is the FP (amino acids 798-818 SFIEDLLFNKVTLADAGFMKQY for SARS-CoV, FP1). In our previous electron spin resonance spectroscopic studies, the membrane-ordering effect of influenza virus, HIV, and Dengue virus FPs has been consistently observed. In this study, we used this effect as a criterion to identify and characterize the bona fide SARS-CoV FP. Our results indicate that both FP1 and the region immediately downstream (amino acids 816-835 KQYGECLGDINARDLICAQKF, FP2) induce significant membrane ordering. Furthermore, their effects are calcium dependent, which is consistent with in vivo data showing that calcium is required for SARS-CoV S-mediated fusion. Isothermal titration calorimetry showed a direct interaction between calcium cations and both FPs. This Ca 2+ -dependency membrane ordering was not observed with influenza FP, indicating that the CoV FP exhibits a mechanistically different behavior. Membrane-ordering effects are greater and penetrate deeper into membranes when FP1 and FP2 act in a concerted manner, suggesting that they form an extended fusion "platform.", (Copyright © 2017 Elsevier Ltd. All rights reserved.)

At the base of the bacterial flagella, a cytoplasmic rotor (the C-ring) generates torque and reverses rotation sense in response to stimuli. The bulk of the C-ring forms from many copies of the proteins FliG, FliM, and FliN, which together constitute the switch complex. To help resolve outstanding issues regarding C-ring architecture, we have investigated interactions between FliM and FliG from Thermotoga maritima with X-ray crystallography and pulsed dipolar ESR spectroscopy (PDS). A new crystal structure of an 11-unit FliG:FliM complex produces a large arc with a curvature consistent with the dimensions of the C-ring. Previously determined structures along with this new structure provided a basis to test switch complex assembly models. PDS combined with mutational studies and targeted cross-linking reveal that FliM and FliG interact through their middle domains to form both parallel and antiparallel arrangements in solution. Residue substitutions at predicted interfaces disrupt higher-order complexes that are primarily mediated by contacts between the C-terminal domain of FliG and the middle domain of a neighboring FliG molecule. Spin separations among multi-labeled components fit a self-consistent model that agree well with electron microscopy images of the C-ring. An activated form of the response regulator CheY destabilizes the parallel arrangement of FliM molecules to perturb FliG alignment in a process that may reflect the onset of rotation switching. These data suggest a model of C-ring assembly in which intermolecular contacts among FliG domains provide a template for FliM assembly and cooperative transitions., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Bacterial receptors typically contain modular architectures with distinct functional domains that combine to send signals in response to stimuli. Although the properties of individual components have been investigated in many contexts, there is little information about how diverse sets of modules work together in full-length receptors. Here, we investigate the architecture of Aer2, a soluble gas-sensing receptor that has emerged as a model for PAS (Per-Arnt-Sim) and poly-HAMP (histidine kinase-adenylyl cyclase-methyl-accepting chemotaxis protein-phosphatase) domain signaling. The crystal structure of the heme-binding PAS domain in the ferric, ligand-free form, in comparison to the previously determined cyanide-bound state, identifies conformational changes induced by ligand binding that are likely essential for the signaling mechanism. Heme-pocket alternations share some similarities with the heme-based PAS sensors FixL and EcDOS but propagate to the Iβ strand in a manner predicted to alter PAS-PAS associations and the downstream HAMP junction within full-length Aer2. Small-angle X-ray scattering of PAS and poly-HAMP domain fragments of increasing complexity allow unambiguous domain assignments and reveal a linear quaternary structure. The Aer2 PAS dimeric crystal structure fits well within ab initio small-angle X-ray scattering molecular envelopes, and pulsed dipolar ESR measurements of inter-PAS distances confirm the crystallographic PAS arrangement within Aer2. Spectroscopic and pull-down assays fail to detect direct interactions between the PAS and HAMP domains. Overall, the Aer2 signaling mechanism differs from the Escherichia coli Aer paradigm, where side-on PAS-HAMP contacts are key. We propose an in-line model for Aer2 signaling, where ligand binding induces alterations in PAS domain structure and subunit association that is relayed through the poly-HAMP junction to downstream domains., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Pulsed dipolar ESR spectroscopy, DEER and DQC, require frozen samples. An important issue in the biological application of this technique is how the freezing rate and concentration of cryoprotectant could possibly affect the conformation of biomacromolecule and/or spin-label. We studied in detail the effect of these experimental variables on the distance distributions obtained by DEER from a series of doubly spin-labeled T4 lysozyme mutants. We found that the rate of sample freezing affects mainly the ensemble of spin-label rotamers, but the distance maxima remain essentially unchanged. This suggests that proteins frozen in a regular manner in liquid nitrogen faithfully maintain the distance-dependent structural properties in solution. We compared the results from rapidly freeze-quenched (≤100 μs) samples to those from commonly shock-frozen (slow freeze, 1 s or longer) samples. For all the mutants studied we obtained inter-spin distance distributions, which were broader for rapidly frozen samples than for slowly frozen ones. We infer that rapid freezing trapped a larger ensemble of spin label rotamers; whereas, on the time-scale of slower freezing the protein and spin-label achieve a population showing fewer low-energy conformers. We used glycerol as a cryoprotectant in concentrations of 10% and 30% by weight. With 10% glycerol and slow freezing, we observed an increased slope of background signals, which in DEER is related to increased local spin concentration, in this case due to insufficient solvent vitrification, and therefore protein aggregation. This effect was considerably suppressed in slowly frozen samples containing 30% glycerol and rapidly frozen samples containing 10% glycerol. The assignment of bimodal distributions to tether rotamers as opposed to protein conformations is aided by comparing results using MTSL and 4-Bromo MTSL spin-labels. The latter usually produce narrower distance distributions., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A liquid-helium temperature study of Fe2+ and Mn2+ ions has been carried out on a single crystal of Fe2+-doped ZnSiF(6).6H2O at 5-35K at 170, 222.4 and 333.2G Hz. The spectra are found to be an overlap of two magnetically inequivalent Fe2+ ions, as well as that of an Mn2+ ion. From the simulation of the EPR line positions for the Fe2+ (d6, S=2) ion the spin-Hamiltonian parameters were estimated for the two inequivalent Fe2+ ions at the various temperatures. From the relative intensities of lines the absolute sign of the fine-structure parameters have been estimated. In addition, the fine-structure and hyperfine-structure spin-Hamiltonian parameters for the Mn2+ ion, present as impurity at interstitial sites, were estimated from the hyperfine allowed and forbidden line positions. The particular virtues of such a single-crystal study vs. that on powders are noted., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Recent progress in developing 2D-ELDOR (2D electron-electron double resonance) techniques to better capture molecular dynamics in complex fluids, particularly in model and biological membranes, is reported. The new "full S(c-) method", which corrects the spectral analysis for the phase distortion effects present in the experiments, is demonstrated to enhance the sensitivity of 2D-ELDOR in reporting on molecular dynamics in complex membrane environments. That is, instead of performing spectral fitting in the magnitude mode, our new method enables simultaneous fitting of both the real and imaginary components of the S(c-) signal. The full S(c-) fitting not only corrects the phase distortions in the experimental data but also more accurately determines instrumental dead times. The phase corrections applied to the S(c-) spectrum enable the extraction of the pure absorption-mode spectrum, which is characterized by much better resolution than the magnitude-mode spectrum. In the absorption mode, the variation of homogeneous broadening, which reports on the dynamics of the spin probe, can even be observed by visual inspection. This new method is illustrated with results from model membranes of dipalmitoyl-sn-glycero-phosphatidylcholine (DPPC)-cholesterol binary mixtures, as well as with results from plasma membrane vesicles of mast cells. In addition to the dynamic parameters, which provide quantitative descriptions for membranes at the molecular level, the high-resolution absorption spectra themselves may be used as a "fingerprint" to characterize membrane phases and distinguish coexisting components in biomembranes. Thus we find that 2D-ELDOR is greatly improved with the new "full S(c-) method" especially for exploring the complexity of model and biological membranes.

Two-dimensional (2D) Fourier transform ESR techniques, such as 2D-ELDOR, have considerably improved the resolution of ESR in studies of molecular dynamics in complex fluids such as liquid crystals and membrane vesicles and in spin labeled polymers and peptides. A well-developed theory based on the stochastic Liouville equation (SLE) has been successfully employed to analyze these experiments. However, one fundamental assumption has been utilized to simplify the complex analysis, viz. the pulses have been treated as ideal non-selective ones, which therefore provide uniform irradiation of the whole spectrum. In actual experiments, the pulses are of finite width causing deviations from the theoretical predictions, a problem that is exacerbated by experiments performed at higher frequencies. In the present paper we provide a method to deal with the full SLE including the explicit role of the molecular dynamics, the spin Hamiltonian and the radiation field during the pulse. The computations are rendered more manageable by utilizing the Trotter formula, which is adapted to handle this SLE in what we call a "Split Super-Operator" method. Examples are given for different motional regimes, which show how 2D-ELDOR spectra are affected by the finite pulse widths. The theory shows good agreement with 2D-ELDOR experiments performed as a function of pulse width.

Tikhonov regularization (TIKR) has been demonstrated as a powerful and valuable method for the determination of distance distributions of spin-pairs in bi-labeled biomolecules directly from pulsed ESR signals. TIKR is a direct method, which requires no iteration, and, therefore, provides a rapid and unique solution. However, the distribution obtained tends to exhibit oscillatory excursions with negative portions in the presence of finite noise, especially in the peripheral regions of the distribution. The Shannon-Jaynes entropy of a probability distribution provides an intrinsic non-negativity constraint on the probability distribution and an unbiased way of obtaining information from incomplete data. We describe how the maximum entropy regularization method (MEM) may be applied to solve the ill-posed nature of the dipolar signal in pulsed ESR. We make use of it to suppress the negative excursions of the distance distribution and to increase the tolerance to noise in the dipolar signal. Model studies and experimental data are investigated, and they show that, with the initial or "seed" probability distribution that is required for MEM taken as the TIKR result, then MEM is able to provide a regularized solution, subject to the non-negativity constraint, and it is effective in dealing with noise that is problematic for TIKR. In addition we have incorporated into our MEM method the ability to extract the intermolecular dipolar component, which is embedded in the raw experimental data. We find that MEM minimization, which is implemented iteratively, is greatly accelerated using the TIKR result as the seed, and it converges more successfully. Thus we regard the MEM method as a complement to TIKR by securing a positive pair distance distribution and enhancing the accuracy of TIKR.

EPR measurements have been carried out on a single crystal of Mn(2+)-doped NH(4)Cl(0.9)I(0.1) at 170-GHz in the temperature range of 312-4.2K. The spectra have been analyzed (i) to estimate the spin-Hamiltonian parameters; (ii) to study the temperature variation of the zero-field splitting (ZFS) parameter; (iii) to confirm the negative absolute sign of the ZFS parameter unequivocally from the temperature-dependent relative intensities of hyperfine sextets at temperatures below 10K; and (iv) to detect the occurrence of a structural phase transition at 4.35K from the change in the structure of the EPR lines with temperature below 10K.

Pulsed ESR techniques with the aid of site-directed spin labeling have proven useful in providing unique structural information about proteins. The determination of distance distributions in electron spin pairs directly from the dipolar time evolution of the pulsed ESR signals by means of the Tikhonov regularization method is reported. The difficulties connected with numerically inverting this ill-posed mathematical problem are clearly illustrated. The Tikhonov regularization with the regularization parameter determined by the L-curve criterion is then described and tested to confirm its accuracy and reliability. The method is applied to recent experimental results on doubly labeled proteins that have been studied using two pulsed ESR techniques, double quantum coherence (DQC) ESR and double electron-electron resonance (DEER). The extracted distance distributions are able to provide valuable information about the conformational constraints in various partially folded states of proteins. This study supplies a mathematically reliable method for extracting pair distributions from pulsed ESR experimental data and has extended the use of pulsed ESR to provide results of greater value for structural biology.

NMR microscopy is routinely employed in fields of science such as biology, botany, and materials science to observe magnetic parameters and transport phenomena in small scale structures. Despite extensive efforts, the resolution of this method is limited (>10 microm for short acquisition times), and thus cannot answer many key questions in these fields. We show, through theoretical prediction and initial experiments, that ESR microscopy, although much less developed, can improve upon the resolution limits of NMR, and successfully undertake the 1 mum resolution challenge. Our theoretical predictions demonstrate that existing ESR technology, along with advanced imaging probe design (resonator and gradient coils), using solutions of narrow linewidth radicals (the trityl family), should yield 64 x 64 pixels 2D images (with z slice selection) with a resolution of 1 x 1 x 10 microm at approximately 60 GHz in less than 1h of acquisition. Our initial imaging results, conducted by CW ESR at X-band, support these theoretical predictions and already improve upon the previously reported state-of-the-art for 2D ESR image resolution achieving approximately 10 x 10 mum, in just several minutes of acquisition time. We analyze how future progress, which includes improved resonators, increased frequency of measurement, and advanced pulsed techniques, should achieve the goal of micron resolution.

The 19F nuclear spin-lattice relaxation rate constants were measured as a function of magnetic field strength for 1,12-diaminododecane labeled at one end with a nitroxide radical and at the other with a trifluoromethyl group. The magnetic relaxation dispersion profile (MRD) reports the spectral density function appropriate to the end-to-end correlation function for the doubly labeled molecule. After extrapolation to zero concentration to eliminate the intermolecular relaxation contribution to relaxation, the resulting intramolecular MRD profile was compared with several model approaches. The rotational model for the spectral density functions as included in the Solomon-Bloembergen-Morgan equations does not describe the data well. The earlier model of Freed for nuclear spin relaxation induced by a freely diffusing paramagnetic co-solute is not rigorous for this case because the paramagnet is tethered to the observed nuclear spin and only a restricted space in the immediate vicinity of the nuclear spin is accessible for pseudo-translational diffusion of one end of the molecule with respect to the other. A generalization of the Torrey model for magnetic relaxation by translational diffusion developed by Nevzorov and Freed, which includes the effect of restrictions imposed by the finite length of the chain, describes the experiment within experimental errors. A simple modification of the Hwang-Freed model that does not specifically include the dynamical effects of the finite tether also provides a good approximation to the data when the tether chain is sufficiently long.

Multifrequency electron paramagnetic resonance studies on the Mn(2+) impurity ion in a mixed single crystal NH(4)Cl(0.9)I(0.1) were carried out at 9.62 (X-band) in the range 120-295 K, at 35.87 (Q-band) at 77 and 295 K, and at 249.9 GHz (far-infrared band) at 253 K. The high-field EPR spectra at 249.9 GHz are well into the high-field limit leading to a considerable simplification of the spectra and their interpretation. Three magnetically inequivalent, but physically equivalent, Mn(2+) ions with their respective magnetic Z-axes oriented along the crystallographic [100], [010], [001] axes were observed. Simultaneous fitting of EPR line positions observed at X-, Q-, and far infra-red bands was performed using a least-squares procedure and matrix diagonalization to estimate accurately the Mn(2+) spin-Hamiltonian parameters. The temperature variation of the linewidth and peak-to-peak intensities of the EPR lines indicate the presence of lambda-transitions in the mixed NH(4)Cl(0.9)I(0.1) crystal at 242 and 228 K consistent with those observed in the pure NH(4)Cl and NH(4)I crystals, respectively. A superposition-model analysis of the spin-Hamiltonian parameters reveals that the local environment of the Mn(2+) ion is considerably reorganized to produce axially symmetric crystal fields about the respective Z-axes of the three magnetically inequivalent ions as a consequence of the vacancy created due to charge-compensation when the divalent Mn(2+) ion substitutes for a monovalent NH(4)(+) ion in the NH(4)Cl(0.9)I(0.1) crystal. This reorganization is almost the same as that observed in NH(4)Cl and NH(4)I single crystals, although the latter two are characterized by different, simple cubic and face-centered cubic, structures.

Adenylate kinase from Escherichia coli (AKeco), consisting of a single 23.6 kDa polypeptide chain folded into domains CORE, AMPbd and LID, catalyzes the reaction AMP+ATP-->2ADP. In the ligand-free enzyme the domains AMPbd and LID execute large-amplitude movements controlling substrate binding and product release during catalysis. Domain flexibility is investigated herein with the slowly relaxing local structure (SRLS) model for (15)N relaxation. SRLS accounts rigorously for coupling between the global and local N-H motions through a local ordering potential exerted by the protein structure at the N-H bond. The latter reorients with respect to its protein surroundings, which reorient on the slower time scale associated with the global protein tumbling. AKeco diffuses globally with correlation time tau(m)=15.1 ns, while locally two different dynamic cases prevail. The domain CORE features ordering about the equilibrium N-H bond orientation with order parameters, S(2), of 0.8-0.9 and local motional correlation times, tau, mainly between 5-130 ps. This represents a conventional rigid protein structure with rapid small-amplitude N-H fluctuations. The domains AMPbd and LID feature small parallel (Z(M)) ordering of S(2)=0.2-0.5 which can be reinterpreted as high perpendicular (Y(M)) ordering. M denotes the local ordering/local diffusion frame. Local motion about Z(M) is given by tau( parallel) approximately 5 ps and local motion of the effective Z(M) axis about Y(M) by tau( perpendicular)=6-11 ns. Z(M) is tilted at approximately 20 degrees from the N-H bond. The orientation of the Y(M) axis may be considered parallel to the C(alpha)(i-1)-C(alpha)(i) axis. The tau( perpendicular) mode reflects collective nanosecond peptide-plane motions, interpretable as domain motion. A powerful new model of protein flexibility/domain motion has been established. Conformational exchange (R(ex)) processes accompany the tau( perpendicular) mode. The SRLS analysis is compared with the conventional model-free analysis., (Copyright 2002 Academic Press.)