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Investigates the incorporation of Mn(II) into the mesoporous material MCM-41, synthesized under acidic conditions with [Si]/[H+]. Use of tetraethyl-orthosilicon as the silica source and cetyltrimethylammonium chloride or bromide as the structure-directing agent; Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy measurements.

Discusses the local environment of the manganese in MnAPSO-44. Application of magnetic resonance spectroscopic techniques; Probe of weak superhyperfine interactions between paramagnetic center and neighboring nuclear spins; Analysis of dipolar interaction; Spatial distribution of manganese.

We studied the structural evolution during the formation of large-pore cubic Ia3¯d silica-based mesoporous materials, synthesized with Pluroinc P123 and butanol as structure directing agents. We used cryogenic high resolution scanning electron microscopy (cryo-HRSEM) and freeze-fracture-replication (FFR) transmission electron microscopy (TEM). Typically a silica precursor is added to an acid-catalyzed solution of Pluronic P123 and butanol. The latter serves as a cosolute, which can be added either at the beginning of the reaction, or after precipitation and the formation of a hexagonal phase. In this study we focused on the structural evolution from the hexagonal phase to the final cubic phase in the two different reactions. The same structural evolution with different kinetics was detected for both reactions. Cryo-HRSEM and FFR-TEM images revealed that from the hexagonal phase a perforated layer (PL) phase is formed, which later evolves into a bicontinuous structure. The final cubic phase forms within the layers, maintaining their orientation. We suggest a formation mechanism involving cylinder merging for the hexagonal to PL transition. Upon additional polymerization of the silica, the PL phase relaxes into the stable Ia3¯d cubic phase. Another minor mechanism detected involves the direct transition between the hexagonal to the final cubic phase through cylinder branching. [ABSTRACT FROM AUTHOR]

Concanavalin A is a member of the plant hemeagglutinin (or plant lectin) family that contains two metal binding sites; one, called S1, is occupied by Mn2+ and the other, S2, by Ca2+. 55Mn electron—nuclear double resonance (ENDOR) measurements were performed on a single crystal of concanavalin A at W-band (95 GHz, ~3.5 T) to determine the 55Mn nuclear quadrupole interaction in a protein binding site and its relation to structural parameters. Such measurements are easier at a high field because of the high sensitivity for size-limited samples and the reduction of second-order effects on the spectrum which simplifies spectral analysis. The analysis of the 55Mn ENDOR rotation patterns showed that two chemically inequivalent Mn2+ types are present at low temperatures, although the high-resolution X-ray structure reported only one site. Their quadrupole coupling constants, e2Qq/h, are significantly different; 10.7 ± 0.6 MHz for MnA2+ and only -2.7 ± 0.6 MHz for MnB2+. The ENDOR data also refined the hyperfine coupling determined earlier by single-crystal EPR measurements, yielding a small but significant difference between the two: -262.5 MHz for MnA2+ and -263.5 MHz for MnB2+. The principal z-axis for MnA2+ is not aligned with any of the Mn-ligand directions, but is 25° off the Mn-asp10 direction, and its orientation is different than that of the zero-field splitting (ZFS) interaction. Because of the small quadrupole interaction of MnB2+ the orientation dependence was very mild, leading to larger uncertainties in the asymmetry parameter. Nonetheless, there too z is not along the Mn-ligand bonds and is rotated 90° with respect to MnA. These results show, that similar to the ZFS, the quadrupolar interaction is highly sensitive to small differences in the coordination sphere of the Mn2+, and the resolution of the two types is in agreement with the earlier observation of a two-site conformational dynamic detected through the ZFS interaction, which is frozen out at low temperatures and averaged at room temperature. To account for the structural origin of the different e2Qq/h values, the electric field gradient tensor was calculated using the point-charge model. The calculations showed that a relatively small displacement of the oxygen ligand of asp10 can lead to differences on the order observed experimentally. [ABSTRACT FROM AUTHOR]

The article reports on the investigation of the location and coordination shell of the copper (Cu) ions within the zeolite structure which can account for the catalytic activity. High-field electron-nuclear double resonance spectra of Cu ions in zeolite X are presented. Additional valuable constraints for density functional theory calculations can be provided by the determined tensors.

Protein structure investigations are usually carried out in vitro under conditions far from their native environment in the cell. Differences between in-cell and in vitro structures of proteins can be generated by crowding effects, local pH changes, specific and nonspecific protein and ligand binding events, and chemical modifications. Double electronelectron resonance (DEER), in conjunction with site-directed spin-labeling, has emerged in the past decade as a powerful technique for exploring protein conformations in frozen solutions. The major challenges facing the application of this methodology to in-cell measurements are the instabilities of the standard nitroxide spin labels in the cell environment and the limited sensitivity at conventional X-band frequencies. We present a new approach for in-cell DEER distance measurement in human cells, based on the use of: (i) reduction resistant Gd3+ chelates as spin labels, (ii) high frequency (94.9 GHz) for sensitivity enhancement, and (iii) hypo-osmotic shock for efficient delivery of the labeled protein into the cell. The proof of concept is demonstrated on doubly labeled ubiquitin in HeLa cells. [ABSTRACT FROM AUTHOR]

ATP-dependent binding of the chaperonin GroEL to its cofactor GroES forms a cavity in which encapsulated substrate proteins can fold in isolation from bulk solution. It has been suggested that folding in the cavity may differ from that in bulk solution owing to steric confinement, interactions with the cavity walls, and differences between the properties of cavity-confined and bulk water. However, experimental data regarding the cavity-confined water are lacking. Here, we report measurements of water density and diffusion dynamics in the vicinity of a spin label attached to a cysteine in the Tyr71 → Cys GroES mutant obtained using two magnetic resonance techniques: electronspin echo envelope modulation and Overhauser dynamic nuclear polarization. Residue 71 in GroES is fully exposed to bulk water in free GroES and to confined water within the cavity of the GroEL—GroES complex. Our data show that water density and translational dynamics in the vicinity of the label do not change upon complex formation, thus indicating that bulk water-exposed and cavity-confined GroES surface water share similar properties. Interestingly, the diffusion dynamics of water near the GroES surface are found to be unusually fast relative to other protein surfaces studied. The implications of these findings for chaperonin-assisted folding mechanisms are discussed. [ABSTRACT FROM AUTHOR]

The RNA helicase DbpA promotes RNA remodeling coupled to ATP hydrolysis. It is unique because of its specificity to hairpin 92 of 23S rRNA (HP92). Although DbpA kinetic pathways leading to ATP hydrolysis and RNA unwinding have been recently elucidated, the molecular (atomic) basis for the coupling of ATP hydrolysis to RNA remodeling remains unclear. This is, in part, due to the lack of detailed structural information on the ATPase site in the presence and absence of RNA in solution. We used high-field pulse ENDOR (electron-nuclear double resonance) spectroscopy to detect and analyze fine conformational changes in the protein's ATPase site in solution. Specifically, we substituted the essential Mg2+ cofactor in the ATPase active site for paramagnetic Mn2+ and determined its close environment with different nucleotides (ADP, ATP, and the ATP analogues ATPγS and AMPPnP) in complex with single- and double-stranded RNA. We monitored the Mn2+ interactions with the nucleotide phosphates through the 31P hyperfine couplings and the coordination by protein residues through 13C hyperfine coupling from 13C-enriched DbpA. We observed that the nucleotide binding site of DbpA adopts different conformational states upon binding of different nucleotides. The ENDOR spectra revealed a clear distinction between hydrolyzable and nonhydrolyzable nucleotides prior to RNA binding. Furthermore, both the 13C and the 31P ENDOR spectra were found to be highly sensitive to changes in the local environment of the Mn2+ ion induced by the hydrolysis. More specifically, ATPγS was efficiently hydrolyzed upon binding of RNA, similar to ATP. Importantly, the Mn2+ cofactor remains bound to a single protein side chain and to one or two nucleotide phosphates in all complexes, whereas the remaining metal coordination positions are occupied by water. The conformational changes in the protein's ATPase active site associated with the different DbpA states occur in remote coordination shells of the Mn2+ ion. Finally, a competitive Mn2+ binding site was found for single-stranded RNA construct. [ABSTRACT FROM AUTHOR]

Double electron-electron resonance (DEER) distance measurements of a protein complex tagged with two Gd3+ chelates developed for rigid positioning of the metal ion are shown to deliver outstandingly accurate distance measurements in the 6 nm range. The accuracy was assessed by comparison with modeled distance distributions based on the three-dimensional molecular structures of the protein and the tag and further comparison with paramagnetic NMR data. The close agreement between the predicted and experimentally measured distances opens new possibilities for investigating the structure of biomolecular assemblies. As an example, we show that the dimer interface of rat ERp29 in solution is the same as that determined previously for human ERp29 in the single crystal. [ABSTRACT FROM AUTHOR]

cd1 nitrite reductase (NIR) is a key enzyme in the denitrification process that reduces nitrite to nitric oxide (NO). It contains a specialized d1-heme cofactor, found only in this class of enzymes, where the substrate, nitrite, binds and is converted to NO. For a long time, it was believed that NO must be released from the ferric d1-heme to avoid enzyme inhibition by the formation of ferrous-nitroso complex, which was considered as a dead-end product. However, recently an enhanced rate of NO dissociation from the ferrous form, not observed in standard b-type hemes, has been reported and attributed to the unique d1-heme structure (Rinaldo, S.; Arcovito, A.; Brunori, M.; Cutruzzol, F. J. Biol. Chem. 2007, 282, 14761-14767). Here, we report on a detailed study of the spatial and electronic structure of the ferrous d1-heme NO complex from Pseudomonas aeruginosa cd1 NIR and two mutants Y10F and H369A/H327A in solution, searching for the unique properties that are responsible for the relatively fast release. There are three residues at the "distal" side of the heme (Tyr10, His327, and His369), and in this work we focus on the identification and characterization of possible H-bonds they can form with the NO, thereby affecting the stability of the complex. For this purpose, we have used high field pulse electron-nuclear double resonance (ENDOR) combined with density functional theory (DFT) calculations. The DFT calculations were essential for assigning and interpreting the ENDOR spectra in terms of geometric structure. We have shown that the NO in the nitrosyl d1-heme complex of cd1 NIR forms H-bonds with Tyr10 and His369, whereas the second conserved histidine, His327, appears to be less involved in NO H-bonding. This is in contrast to the crystal structure that shows that Tyr10 is removed from the NO. We have also observed a larger solvent accessibility to the distal pocket in the mutants as compared to the wild-type. Moreover, it was shown that the H-bonding network within the active site is dynamic and that a change in the protonation state of one of the residues does affect the strength and position of the H-bonds formed by the others. In the Y10F mutant, His369 is closer to the NO, whereas mutation of both distal histidines displaces Tyr10, removing its H-bond. The implications of the H-bonding network found in terms of the complex stability and catalysis are discussed. [ABSTRACT FROM AUTHOR]

Methods for measuring nanometer-scale distances between specific sites in proteins are essential for analysis of their structure and function. In this work we introduce Gd3+ spin labeling for nanometer- range distance measurements in proteins by high-field pulse electron paramagnetic resonance (EPR). To evaluate the performance of such measurements, we carried out four-pulse double-electron electron resonance (DEER) measurements on two proteins, p75ICD and ΤC4, labeled at strategically selected sites with either two nitroxides or two Gd3+ spin labels. In analogy to conventional site-directed spin labeling using nitroxides, Gd3+ tags that are derivatives of dipicolinic acid were covalently attached to cysteine thiol groups. Measurements were carried out on X-band (~9.5 GHz, 0.35 T) and W-band (95 GHz, 3.5 T) spectrometers for the nitroxide-labeled proteins and at W-band for the Gd3+-labeled proteins. In the protein p75ICD, the orientations of the two nitroxides were found to be practically uncorrelated, and therefore the distance distribution could as readily be obtained at W-band as at X-band. The measured Gd3+-Gd3+ distance distribution had a maximum at 2.9 nm, as compared to 2.5 nm for the nitroxides. In the protein rcl4, however, the orientations of the nitroxides were correlated, and the W-band measurements exhibited strong orientation selection that prevented a straightforward extraction of the distance distribution. The X-band measurements gave a nitroxide-nitroxide distance distribution with a maximum at 2.5 nm, and the W-band measurements gave a Gd3+-Gd3+ distance distribution with a maximum at 3.4 nm. The Gd3+-Gd3+ distance distributions obtained are in good agreement with expectations from structural models that take into account the flexibility of the tags and their tethers to the cysteine residues. These results show that Gd3+ labeling is a viable technique for distance measurements at high fields that features an order of magnitude sensitivity improvement, in terms of protein quantity, over X-band pulse EPR measurements using nitroxide spin labels. Its advantage over W-band distance measurements using nitroxides stems from an intrinsic absence of orientation selection. [ABSTRACT FROM AUTHOR]

Crystals of Zn2+/Mn2+ yeast enolase with the inhibitor PhAH (phosphonoacetohydroxamate) were grown under conditions with a slight preference for binding of Zn2+ at the higher affinity site, site I. The structure of the Zn2+/Mn2+-PhAH complex was solved at a resolution of 1.54 Å, and the two catalytic metal binding sites, I and II, show only subtle displacement compared to that of the corresponding complex with the native Mg2+ ions. Low-temperature echo-detected high-field (W-band, 95 GHz) EPR (electron paramagnetic resonance) and ¹H ENDOR (electron-nuclear double resonance) were carried out on a single crystal, and rotation patterns were acquired in two perpendicular planes. Analysis of the rotation patterns resolved a total of six Mn2+ sites, four symmetry-related sites of one type and two out of the four of the other type. The observation of two chemically inequivalent Mn2+ sites shows that Mn2+ ions populate both sites I and II and the zero-field splitting (ZFS) tensors of the Mn2+ in the two sites were determined. The Mn2+ site with the larger D value was assigned to site I based on the ¹H ENDOR spectra, which identified the relevant water ligands. This assignment is consistent with the seemingly larger deviation of site I from octahedral symmetry, compared to that of site II. The ENDOR results gave the coordinates of the protons of two water ligands, and adding them to the crystal structure revealed their involvement in a network of H bonds stabilizing the binding of the metal ions and PhAH. Although specific hyperfine interactions with the inhibitor were not determined, the spectroscopic properties of the Mn2+ in the two sites were consistent with the crystal structure. Density functional theory (DFT) calculations carried out on a cluster representing the catalytic site, with Mn2+ in site I and Zn2+ in site II, and vice versa, gave overestimated D values on the order of the experimental ones, although the larger D value was found for Mn2+ in site II rather than in site I. This discrepancy was attributed to the high sensitivity of the ZFS parameters to the MnO bond lengths and orientations, such that small, but significant, differences between the optimized and crystal structures alter the ZFS considerably, well above the difference between the two sites. [ABSTRACT FROM AUTHOR]

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 °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 HCI and TMOS at 35 °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. [ABSTRACT FROM AUTHOR]

Binuclear, mixed valence copper complexes with a [Cu+1.5, Cu+1.5] redox state and S = 1/2 can be stabilized with rigid azacryptand ligands. In this system the unpaired electron is delocalized equally over the two copper ions, and it is one of the very few synthetic models for the electron mediating CuA site of nitrous oxide reductase and cytochrome c oxidase. The spatial and electronic structures of the copper complex in frozen solution were obtained from the magnetic interactions, namely the g-tensor and the 6365Cu, 14N, 2H, and 1H hyperfine couplings, in combination with density functional theory (DFT) calculations. The magnetic interactions were determined from continuous wave (CW) electron paramagnetic resonance (EPR), pulsed electron nuclear double resonance (ENDOR), two-dimensional TRIPLE, and hyperline sublevel correlation spectroscopy (HYSCORE) carried out at W-band or/and X-band frequencies. The DFT calculated g and Cu hyperfine values were in good agreement with the experimental values showing that the structure in solution is indeed close to that of the optimized structure. Then, the DFT calculated hypertine parameters were used as guidelines and starting points in the simulations of the various experimental ENDOR spectra. A satisfactory agreement with the experimental results was obtained for the 14N hyperfine and quadrupole interactions. For 1H the DFT calculations gave good predictions for the hyperfine tensor orientations and signs, and they were also successful in reproducing trends in the magnitude of the various proton hyperfine couplings. These, in turn, were very useful for ENDOR signals assignments and served as constraints on the simulation parameters. [ABSTRACT FROM AUTHOR]

The complexes of copper with histidine exhibit a wide variety of coordination modes in aqueous solution. This stems from the three potential coordination sites of the histidine molecule and the existence of mono- and bis-complexes. The present work concentrates on the determination of the carboxylate binding mode, via the 13C hyperfine coupling of the carboxyl, in a number of copper complexes in frozen solutions. These are then used as references for the determination of the coordination mode of two zeolite encapsulated complexes. The 13C hyperfine coupling (sign and magnitude) was determined by a variety of advanced pulsed EPR and electron-nuclear double resonance (ENDOR) techniques carried out at conventional and high magnetic fields. These showed that while the carboxyl 13C isotropic hyperfine coupling of an equatorially coordinated carboxylate is negative with a magnitude of 3-4 MHz, that of a free carboxylate is small (∼1 MHZ) and positive. To rationalize the experimentally determined ligand hyperfine couplings (¹H and 13C) and further understand their dependence on the coordination mode and degree of protonation, density functional theory (DFT) calculations were carried out on a number of model complexes, representing the different Cu-histidine complexes studied experimentally. The exchange-correlation functional used for the calculation of the EPA parameters was B3LYP with triple-ζ plus polarization (TZP) quality basis sets. While the polarization agreement between the magnitudes of the calculated and experimental values varied among the various nuclei, sometimes exhibiting deviations of up to 40%, an excellent agreement was found for the sign prediction. This shows the unique advantage of combining high field ENDOR, by which the sign of the hyperfine can often be determined, with DFT predictions for structure determination. [ABSTRACT FROM AUTHOR]

Examines the electron-mediating copper centers in proteins. Structure of the nearest ligands of copper sites; Electron-nuclear double resonance spectra of the proteins; Relationship between the structure of the copper sites and the electron transfer characteristics.

Reports on the use of high-field pulsed electron paramagnetic resonance and electron-nuclear double resonance techniques to determine coordinates of ligand protons of a high-spin metal center in a protein single crystal. Use of the protein concanavalin A which contains a Mn[sup 2+] ion; Identification of the water protions involved in H-bonding to neighboring residues.

Investigates the effect of axial ligand mutation on the Cu[sub A] site in the water soluble fragment of subunit II of Thermus thermophilus cytochrome c oxidase ba[sub 3]. Replacement of methionine ligand with glutamate and glutamine; Preparation of M160T0 and M160T9; Changes in spin density in the Cu[sub A] site; Effect of M160Q and M160E mutations.

Explores the elucidation of structure and location of vanadyl V(IV) ions in heteropolyacid catalysts H[sub 4]PVMo[sub 11]O[sub 40] via hyperfine sublevel correlation spectroscopy and pulsed electron nuclear double resonance at W- and X-band frequencies. Heteropolyacids of the Keggin type; Catalysts for the selective oxidation of alkanes and aldehydes.

Characterizes the geometry and framework interactions of zeolite-encapsulated copper(II)-histidine (CuHis) complexes. Diagram of possible models of His coordination in CuHis complexes; Intrazeolite chemistry that affects CuHis structure; Experimental parameters that control their formation.

Examines the structure of nitrogen monoxide adsorption sites involving sodium cations in a sodium zeolite using pulsed electron nuclear double-resonance spectroscopy. Determination of coupling tensors of ligand nuclei in paramagnetic complexes; Measurement of the acidity of Lewis acid sites in zeolites; Spin density distribution; Coordination geometry.

The article examines the complexes of the gadolinium as a potential spin labels for high field (HF) pulsed electron paramagnetic resonance (EPR) distance measurements. According to the authors, the study showed that gadolinium complexes have a potential of being used as spin labels for HF in biological systems. They feature high sensitivity and affording high effective values which allowed for very high repetition rates due to short spin called lattice relaxation times.