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.

SBA-15 is an hexagonal mesoporous material which is synthesized with nonionic poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymers (Pluronics, EO y PO x EO y ), templates. Pore diameters in the range of 2−30 nm can be obtained with a relatively thick silica wall (up to 6 nm). This material possesses both large, uniform, and ordered channels, along with a complementary net of micropores which provides connectivity between the ordered channels through the silica. This study focuses on the investigation of the formation mechanism of SBA-15 with emphasis on the PEO interactions with the silica and the initiation of the micropores. This was achieved using in situ X-band EPR spectroscopy in combination with electron spin−echo envelope modulation (ESEEM) experiments. The paramagnetic centers were introduced as spin-labeled Pluronic L62 (EO6PO30EO6) where nitroxides replace the OH groups at the end of the polypropylene oxide (PEO) blocks (L62-NO). Initially, the acidic reaction conditions were adjusted to prevent the decomposition of the nitroxide radical, while still producing highly ordered SBA-15. Then, the locations of the nitroxides of L62-NO within the micelles of Pluronic P123 (y = 20, x = 70) and L64 (y = 13, x = 30) were determined through three-pulse ESEEM experiments on solutions prepared in D2O. In these experiments, the 2H modulation induced by D2O was compared with that of a series of small spin-probes with known hydrophilic and hydrophobic characters that were introduced into the micelles. The NO group of L62-NO was found to be close to the core-corona interface in both types of Pluronics. The temporal evolution of the EPR spectrum during the reaction showed that for SBA-15 made with P123 the most significant changes in the L62-NO spectrum occur within the first 100 min. Furthermore, X-ray diffraction measurements of dried materials showed that the hexagonal structure of SBA-15 is also created within the first 2 h. A partitioning of the L62-NO between the precursors of the mesopores and micropores of the SBA-15 structure takes place at the very early stages of the reaction, and a continuous depletion of water within the corona−core interface was observed. In the final product obtained without a thermal stage, the majority of the PEO chains are located in the micropores. The extent of the PEO chains located within the silica micropores depends on the thermal stage temperature and on the Si/P123 molar ratio. In the L64 synthesis, practically all of the NO groups of L62-NO are located within the silica network and experience a single environment.

The evolution of the solution microstructures during the formation of the hexagonal mesoporous material SBA-15 was studied by direct imaging and freeze-fracture replication cryo-TEM. A reaction mixture was sampled at different times after the addition of tetramethoxyorthosilane (TMOS) to an acidic solution of Pluronic P123 held at 50 degrees C. Solution microstructures were detected by direct imaging cryo-TEM in the time window of 6.5-40 min after the addition of the TMOS (t = 0). The micrographs revealed that the initial spheroidal micelles evolve into threadlike micelles, which become longer and straighter with time. Then bundles with the dimensions similar to those found in the final material appeared, although there was no sign of a hexagonal arrangement up to 40 min. Due to the appearance of a precipitate at 40 min the sample became too viscous, preventing clear observation of its content. To observe the structures present after 40 min, freeze-fracture replication was carried out as well. Such samples were collected also at 22 min and showed the presence of threadlike micelles in agreement with the direct imaging cryo-TEM micrographs. The 2 h samples showed some areas of hexagonal ordered structures, which become very clear at 2 h 50 min. The cryo-TEM measurements were carried out under the same reaction conditions used in earlier in situ EPR experiments, thus allowing us to correlate molecular level events with the microstructure shape evolutions. This showed that the elongation of the micelles is a consequence of a reduction of the polarity and the water content within the micelles due to silicate adsorption and polymerization. Similar experiments were carried out also on SBA-15 prepared with HCl and TMOS at 35 degrees C. The appearance of threadlike micelles, followed by clustering of the TLMs, was observed under these conditions as well, but the reaction rate was faster. This suggests that the observed mechanism for the formation of SBA-15 is general.

Gd(III) complexes have emerged as spin labels for distance determination in biomolecules through double-electron-electron resonance (DEER) measurements at high fields. For data analysis, the standard approach developed for a pair of weakly coupled spins with S = 1/2 was applied, ignoring the actual properties of Gd(III) ions, i.e. S = 7/2 and ZFS (zero field splitting) ≠ 0. The present study reports on a careful investigation on the consequences of this approach, together with the range of distances accessible by DEER with Gd(III) complexes as spin labels. The experiments were performed on a series of specifically designed and synthesized Gd-rulers (Gd-PyMTA-spacer-Gd-PyMTA) covering Gd-Gd distances of 2-8 nm. These were dissolved in D2O-glycerol-d8 (0.03-0.10 mM solutions) which is the solvent used for the corresponding experiments on biomolecules. Q- and W-band DEER measurements, followed by data analysis using the standard data analysis approach, used for S = 1/2 pairs gave the distance-distribution curves, of which the absolute maxima agreed very well with the expected distances. However, in the case of the short distances of 2.1 and 2.9 nm, the distance distributions revealed additional peaks. These are a consequence of neglecting the pseudo-secular term in the dipolar Hamiltonian during the data analysis, as is outlined in a theoretical treatment. At distances of 3.4 nm and above, disregarding the pseudo-secular term leads to a broadening of a maximum of 0.4 nm of the distance-distribution curves at half height. Overall, the distances of up to 8.3 nm were determined, and the long evolution time of 16 μs at 10 K indicates that a distance of up to 9.4 nm can be accessed. A large distribution of the ZFS parameter, D, as is found for most Gd(III) complexes in a frozen solution, is crucial for the application of Gd(III) complexes as spin labels for distance determination via Gd(III)-Gd(III) DEER, especially for short distances. The larger ZFS of Gd-PyMTA, in comparison to that of Gd-DOTA, makes Gd-PyMTA a better label for short distances.

The hexagonal, silica-based, mesoporous material SBA-15 is prepared using poly(ethylene oxide)−poly(propylene oxide)−poly(ethylene oxide) block co-polymer (Pluronic P123, PEO20−PPO70−PEO20) as a template and tetramethyl orthosilicate (TMOS) as a silica source. This work focuses on the investigation of its formation on the molecular level, with emphasis on the early stages of the reaction, when the interaction between silica precursors and the Pluronic micelles occurs. This was achieved using in situ X-band electron paramagnetic resonance (EPR) spectroscopy, in combination with electron spin−echo envelope modulation (ESEEM) experiments of Pluronic spin probes with different PEO and PPO chain lengths. In these Pluronic spin probes, the nitroxide spin label is located at the end of the PEO chain, which places them at different regions of the micelles. In the Pluronic micelles, the PPO chains define a hydrophobic region that is referenced as the core, whereas the more-hydrophilic PEO chains form the corona region. In the ESEEM experiments, the reaction was conducted in D2O and it was quenched at different times by rapid freezing to 77 K. The 2H modulation depth (k(2H)) was followed, as a function of the reaction time. Four different systems, which were designed to probe the evolution of the reaction at three different regions of the Pluronic micelles, were examined. By comparing the ESEEM results with the in situ continuous wave (CW) EPR measurements of the different spin probes, four stages were detected. The first occurs within the first 5 min and is characterized by a large increase in the D2O/OD density in the vicinity of all spin probes used, whether located in the core, the core/corona interface, or the corona/water interface. This was attributed to fast hydrolysis of the TMOS where hydrolyzed TMOS and water penetrate into the corona from the original location of the hydrophobic TMOS within the core. The second stage, which lasts ∼1 h, is characterized by a moderate reduction in the D2O/OD density at the core/corona interface, attributed to silica polymerization. During the third stage (∼1 h), the D2O/OD density decrease continues but is felt mainly in the corona region. The results show that the silica polymerization propagates outward from the core/corona interface.

This study focuses on the formation mechanism of the bicontinuous cubic Ia3 j d mesoporous material KIT-6, both on the molecular and on the mesoscopic levels. KIT-6 is synthesized with Pluronic P123 (PEO 20 PPO 70 PEO 20 ), low acid concentration, and n-butanol at 40 °C. Through in situ EPR measurements on a series of spin-labeled Pluronic molecules introduced at minute quantities into the reaction mixture, changes in the hydrophobicity and the mobility of the polymer chains during the reaction were observed. In addition, to learn more on the functionality of the butanol in this synthesis, freeze-quench electron spin-echo envelope modulation (ESEEM) measurements on reaction mixtures in D 2 O and in butanol-d 10 were preformed. The above experiments gave information on variations in the butanol location and content in the micellar structures during the formation of KIT-6. The evolution of the solution nanostructures was determined by cryo-TEM. Five main stages were resolved: the first two occurred during the first 140 min of the reaction, where condensation of the silica oligomers takes place at the micellar/water interface; this induces depletion of water and butanol molecules from the core-corona interface and reduces the mobility of the ends of the Pluronic chains located at the corona-water interface. This in turn leads to a transition from spheroidal micelles to threadlike micelles and to their aggregation toward the end of the second stage. During the third stage, precipitation (140-160 min), reorganization in the micellar structure, and a change in the relative sizes of core and corona take place. The fourth stage, that ends around 6 h, involves the formation of a hexagonal phase, through accelerated condensation of silica oligomers in the corona, accompanied by extensive depletion of water and butanol molecules. The presence of butanol in the micelle corona is essential in the last stage, 6-24 h, where the cubic phase is formed. We show that the addition of butanol to the reaction mixture of SBA-15 after the formation of the hexagonal phase leads to the formation of the cubic phase.

Metal organic frameworks (MOFs) have unique properties that make them excellent candidates for many high-tech applications. Nevertheless, their nonconducting character is an obstacle to their practical utilization in electronic and energy systems. Using the familiar HKUST-1 MOF as a model, we present a new method of imparting electrical conductivity to otherwise nonconducting MOFs by preparing MOF nanoparticles within the conducting matrix of mesoporous activated carbon (AC). This composite material was studied by X-ray diffraction (XRD), scanning electron microscopy (SEM), gas adsorption measurements, and electron paramagnetic resonance (EPR) spectroscopy. We show that MOF nanoparticles grown within the carbon matrix maintain their crystalline characteristics and their surface area. Surprisingly, as a result of the composition process, EPR measurements revealed a copper signal that had not yet been achieved. For the first time, we could analyze the complex EPR response of HKUST-1. We demonstrate the high conductivity of the MOF composite and discuss various factors that are responsible for these results. Finally, we present an optional application for using the conductive MOF composite as a high-performance electrode for pseudocapacitors.

The interactions between proteins and their specific DNA sequences are the basis of many cellular processes. Hence, developing methods to build an atomic level picture of these interactions helps improve our understanding of key cellular mechanisms. CueR is an Escherichia coli copper‐sensing transcription regulator. The inhibition of copper‐sensing transcription regulators can kill pathogens, without harming the host. Several spectroscopic studies and crystallographic data have suggested that changes in the conformation of both the DNA and the protein control transcription. However, due to the inadequate resolution of these methods, the exact number of active conformations of CueR has not been determined. Resolving the structure of CueR in its active state is highly important for the development of specific inhibitors. Herein, the potential of double‐histidine (dHis)‐based CuII spin labeling for the identification of various conformational states of CueR during transcription is shown.