Electron Spin-echo Envelope Modulation Spectroscopy Research Articles

Utilizing Electron Spin Echo Envelope Modulation To Distinguish between the Local Secondary Structures of an α-Helix and an Amphipathic 310-Helical Peptide.

Electron spin echo envelope modulation (ESEEM) spectroscopy was used to distinguish between the local secondary structures of an α-helix and a 310-helix. Previously, we have shown that ESEEM spectroscopy in combination with site-directed spin labeling (SDSL) and 2H-labeled amino acids (i) can probe the local secondary structure of α-helices, resulting in an obvious deuterium modulation pattern, where i+4 positions generally show larger 2H ESEEM peak intensities than i+3 positions. Here, we have hypothesized that due to the unique turn periodicities of an α-helix (3.6 residues per turn with a pitch of 5.4 Å) and a 310-helix (3.1 residues per turn with a pitch of 5.8-6.0 Å), the opposite deuterium modulation pattern would be observed for a 310-helix. In this study, 2H-labeled d10-leucine (Leu) was substituted at a specific Leu residue (i) and a nitroxide spin label was positioned 2, 3, and 4 residues away (denoted i+2 to i+4) on an amphipathic model peptide, LRL8. When LRL8 is solubilized in trifluoroethanol (TFE), the peptide adopts an α-helical structure, and alternatively, forms a 310-helical secondary structure when incorporated into liposomes. Larger 2H ESEEM peaks in the FT frequency domain data were observed for the i+4 samples when compared to the i+3 samples for the α-helix whereas the opposite pattern was revealed for the 310-helix. These unique patterns provide pertinent local secondary structural information to distinguish between the α-helical and 310-helical structural motifs for the first time using this ESEEM spectroscopic approach with short data acquisition times (∼30 min) and small sample concentrations (∼100 μM) as well as providing more site-specific secondary structural information compared to other common biophysical approaches, such as CD.

Probing the Structure and Binding of KCNE1 to the Voltage-Gated Potassium Channel KCNQ1 using Pulsed EPR Spectroscopy

Pulsed Electron Paramagnetic Resonance (EPR) spectroscopic techniques coupled with site-directed spin-labeling (SDSL) can provide important structural information on complicated biological systems such as membrane proteins. We are developing EPR techniques such as Double Electron-Electron Resonance (DEER) and Electron Spin Echo Envelope Modulation (ESEEM) for studying membrane proteins. Voltage-gated ion channels are essential for the electrical excitability of neurons, muscles and other excitable cells. KCNE1 is a single transmembrane protein that modulates the activity of voltage gated potassium ion channels (Kv). In the human heart, KCNE1 (E1) interacts with KCNQ1 (Q1) and decreases the rate of channel activation, increases conductance, and generates a slowly activating K+ current critical for cardiac repolarization. Mutations on either KCNE1 or KCNQ1 genes in E1/Q1 complex can lead to long QT syndrome. Despite the biological significance of the Q1/E1 interaction, its exact nature is not fully understood. CW-EPR, ESEEM and DEER measurements will be used to probe the structure and binding of KCNQ1 with KCNE1. ESEEM spectroscopy was used to probe the secondary structure of different regions of 2H labeled E1. DEER was also used to measure distances between the spin labels attached on E1 and Q1 separately in the Q1/E1 complex to determine the interacting sites in detail. These results provide direct evidence of binding of Q1 with E1 and will be very useful for determining the structural model of the Q1/E1 complex.

KvAP Gating States Probed by Electron Spin-Echo Envelope Modulation (ESEEM) Spectroscopy

During a gating event, the voltage gated potassium channel KvAP undergoes a conformational change in the voltage sensor (helices 1-4), causing the opening of the pore domain (helices 5-6). Throughout this conformational change, the extracellular portion of the pore remains relatively immobile. However, the environment surrounding this part of the pore domain may be altered through changes in lipid packing or tertiary contacts within the channel. In this study, model membranes were used to stabilize KvAP in different gating states and the environment surrounding the pore were probed with ESEEM both in the presence and absence of the voltage sensing domain.

Probing the Secondary Structure of Membrane Protein using Bacterial Expression System and Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy

Membrane proteins possess a variety of functions essential to the survival of organisms. However, due to the inherent hydrophobic property, it is extremely difficult to gain the structural information. Previously, we probed the structure of an α-helical component of a small model membrane peptide, AchR M2δ, by detecting 2H modulation from a 2H-labeled Val side chain that was 3 or 4 residues away from a nitroxide spin label using ESEEM spectroscopy. In this study, for the first time, ESEEM technique was applied to determine the local secondary structure of a biological membrane protein system using bacterial expression method for incorporating the 2H-labeled Val side chain both in micelle and lipid bilayer environment. An integral membrane protein, KCNE1 with known 3D structure, was utilized as a model membrane protein to test the feasibility and validity of this ESSEM technique. The side chain of valine at position 21 (outside domain), 50 (transmembrane domain), and 95 (cytoplasmic domain) was 2H labeled (denoted as ith position). An MTSL nitroxide spin label was positioned at 2, 3, and 4 residues away (denoted i+2 to i+4, respectively) from 2H-labeled Val residue of each domain. Our results indicated that i+3 and i+4 positions have a significant 2H modulation while 2H modulation is absent at i+2 position, which is consistent with the 3.6 residue/turn periodicity of the α-helix. These results are also consistent with the micelle structure of KCNE1. This ESEEM technique was further validated using the protein incorporated into lipid bilayer of DMPC/DPC bicelle and the transmembrane domain was probed to be α-helical as well. This ESEEM technique is very powerful, efficient, and quick to examine the local secondary structure of any protein system without size limitation.

The Topology, in Model Membranes, of the Core Peptide Derived from the T‐Cell Receptor Transmembrane Domain

The T-cell receptor-CD3 complex (TCR-CD3) serves a critical role in protecting organisms from infectious agents. The TCR is a heterodimer composed of α- and β-chains, which are responsible for antigen recognition. Within the transmembrane domain of the α-subunit, a region has been identified to be crucial for the assembly and function of the TCR. This region, termed core peptide (CP), consists of nine amino acids (GLRILLLKV), two of which are charged (lysine and arginine) and are crucial for the interaction with CD3. Earlier studies have shown that a synthetic peptide corresponding to the CP sequence can suppress the immune response in animal models of T-cell-mediated inflammation, by disrupting proper assembly of the TCR. As a step towards the understanding of the source of the CP activity, we focused on CP in egg phosphatidylcholine/cholesterol (9:1, mol/mol) model membranes and determined its secondary structure, oligomerization state, and orientation with respect to the membrane. To achieve this goal, 15-residue segments of TCRα, containing the CP, were synthesized and spin-labeled at different locations with a nitroxide derivative. Electron spin-echo envelope modulation spectroscopy was used to probe the position and orientation of the peptides within the membrane, and double electron-electron resonance measurements were used to probe its conformation and oligomerization state. We found that the peptide is predominantly helical in a membrane environment and tends to form oligomers (mostly dimers) that are parallel to the membrane plane.

Hydrogen bonding to the nitroxide of protein bound spin labels

Deuterium electron spin-echo envelope modulation spectroscopy (2H-ESEEM) (9.3 GHz, 0.33T) and continuous wave high-field (95 GHz, 3.4T) electron paramagnetic resonance (EPR) spectroscopy were applied to disentangle the sources of protons which are hydrogen bonded to nitroxides of protein bound spin labels. For a set of 12 colicin A mutants with nitroxide spin labels site-specifically introduced at residue positions 26, 33, 42, 62, 91, 105, 115, 154, 166, 169, 181 and 192 of the channel forming domain we analysed the heterogeneity in the gxx region of EPR spectra detected at 95 GHz and quantified the fractions of nitroxide populations engaged in H-bonding to water and protein protons. The amplitudes of the narrow and broad components of 2H-ESEEM spectra arising from non-bonded and bonded water (D2O) molecules or deuterated protein components reflect the local H-bonding equilibria. The combination of both methods allowed estimating the fraction of nitroxide populations hydrogen bonded to non-exchangeable protein protons. Hydrogen bonds are known to play a governing role in protein folding and function, and the presented approach extends the information obtainable from site-directed spin labelling in the investigation of complex biomolecular systems and their conformational changes.

Heterogeneous Ordered–Disordered Structure of the Mesodomain in Frozen Sucrose–Water Solutions Revealed by Multiple Electron Paramagnetic Resonance Spectroscopies

The microscopic structure of frozen aqueous sucrose solutions, over concentrations of 0-75% (w/v), is characterized by using multiple continuous-wave and pulsed electron paramagnetic resonance (EPR) spectroscopic and relaxation techniques and the paramagnetic spin probe, TEMPOL. The temperature dependence of the TEMPOL EPR line-shape anisotropy reveals a mobility transition, specified at 205 K in pure water and 255 ± 5 K for >1% (w/v) added sucrose. The transition temperature is >>Tg, where Tg is the homogeneous water glass transition temperature, which shows that TEMPOL resides in the mesoscopic domain (mesodomain) at water-ice crystallite boundaries and that the mesodomain sucrose concentrations are comparable at >1% (w/v) added sucrose. Electron spin-echo envelope modulation (ESEEM) spectroscopy of TEMPOL-(2)H2-sucrose hyperfine interactions also indicates comparable sucrose concentrations in mesodomains at >1% (w/v) added sucrose. Electron spin-echo (ESE) detected longitudinal and phase memory relaxation times (T1 and TM, respectively) at 6 K indicate a general trend of increased mesodomain volume with added sucrose, in three stages: 1-15, 20-50, and >50% (w/v). The calibrated TEMPOL concentrations indicate that the mesodomain volume is less than the predicted maximally freeze-concentrated value [80 (w/w); 120% (w/v)], with transitions at 15-20% and 50% (w/v) starting sucrose. An ordered sucrose hydrate phase, which excludes TEMPOL, and a disordered, amorphous sucrose-water glass phase, in which TEMPOL resides, are proposed to compose a heterogeneous mesodomain. The results show that the ratio of ordered and disordered volume fractions in the mesodomain is exquisitely sensitive to the starting sucrose concentration.

Mn2+–Nucleotide Coordination at the Myosin Active Site As Detected by Pulsed Electron Paramagnetic Resonance

Pulsed electron paramagnetic resonance at the microwave K(a) band (~30 GHz) was used to study the coordination of adenosine nucleotides to Mn(2+) at the active site of myosin ATPase and in solution. We have found that the electron spin echo (ESE) field sweep, electron-nuclear double resonance (ENDOR) and ESE envelope modulation (ESEEM) techniques are not sufficiently specific for reliable differentiation between the solvated and myosin-bound Mn·nucleotide complexes. Therefore, to directly detect binding of the Mn·nucleotide to myosin, we used nonhydrolizable nucleotide analogs, site-directed spin labeling, and pulsed electron-electron double resonance to detect spin probe-manganese dipolar interaction. We found that under substoichiometric conditions, both Mn·AMPPNP and Mn·ADP·AlF(4) form a complex with myosin, and Mn·ADP does not form such a complex. This correlates well with the biological dissociation of Mg·ADP from myosin after the hydrolysis of ATP. The analysis of (31)P ENDOR spectra reveals that in Mn·AMPPNP, Mn·ATP, and Mn·ADP at myosin or in solution, the nucleotide is coordinated to Mn(2+) by two phosphate groups, whereas in Mn·ADP·AlF(4), only one phosphate group is coordinated. The observation of two phosphates and one nitrogen in the coordination sphere of Mn·ADP in solution by ESEEM spectroscopy suggests that a significant population of Mn ions is coordinated by two ADP molecules, one of which is coordinated by phosphates, and the other one, by a nitrogen atom. The developed approach will be generally useful for monitoring the metal-protein binding when such binding does not provide reliable spectroscopic signatures.

Enhancement of Electron Spin Echo Envelope Modulation Spectroscopic Methods to Investigate the Secondary Structure of Membrane Proteins

This paper reports on a significant improvement of a new structural biology approach designed to probe the secondary structure of membrane proteins using the pulsed EPR technique of electron spin echo envelope modulation (ESEEM) spectroscopy. Previously, we showed that we could characterize an α-helical secondary structure with ESEEM spectroscopy using a (2)H-labeled Val side chain coupled with site-directed spin-labeling (SDSL). In order to further develop this new approach, molecular dynamic (MD) simulations were conducted on several different hydrophobic residues that are commonly found in membrane proteins. (2)H-SL distance distributions from the MD results indicated that (2)H-labeled Leu was a very strong candidate to significantly improve this ESEEM approach. In order to test this hypothesis, the secondary structure of the α-helical M2δ peptide of the acetylcholine receptor (AChR) incorporated into a bicelle was investigated with (2)H-labeled Leu d(10) at position 10 (i) and nitroxide spin labels positioned 1, 2, 3, and 4 residues away (denoted i+1 to i+4) with ESEEM spectroscopy. The ESEEM data reveal a unique pattern that is characteristic of an α-helix (3.6 residues per turn). Strong (2)H modulation was detected for the i+3 and i+4 samples, but not for the i+2 sample. The (2)H modulation depth observed for (2)H-labeled d(10) Leu was significantly enhanced (×4) when compared to previous ESEEM measurements that used (2)H-labeled d(8) Val. Computational studies indicate that deuterium nuclei on the Leu side chain are closer to the spin label when compared to Val. The enhancement of (2)H modulation and the corresponding Fourier Transform (FT) peak intensity for (2)H-labeled Leu significantly reduces the ESEEM data acquisition time for Leu when compared to Val. This research demonstrates that a different (2)H-labeled amino acid residue can be used as an efficient ESEEM probe further substantiating this important biophysical technique. Finally, this new method can provide pertinent qualitative structural information on membrane proteins in a short time (few minutes) at low sample concentrations (~50 μM).

ESEEM of industrial quartz powders: insights into crystal chemistry of Al defects

A set of raw industrial materials, that is, pure quartz and quartz-rich mixtures, were investigated through electron paramagnetic resonance and electron spin echo-envelope modulation spectroscopies, with the aim of evaluating the effective role played by defect centres and of assessing whether they can be used to monitor changes in the physical properties of quartz powders with reference to their health effects. The obtained results point to two interactions of the Al defect centres with H+, hosted in sites within the channels parallel and perpendicular to the c axis of quartz, respectively. These two Al/H+ (hAl) centres exhibit a weak chemical bond, and their relative amounts appear to be modified/controlled by the thermo-mechanical processes underwent by powders. Indeed, a mechanically promoted inter-conversion between the two kinds of site is suggested. As a consequence, the hAl centres are effective in monitoring even modest activations of powders, through thermal or mechanical processes, and they are also supposed to play a specific, relevant role in quartz reactivity during the considered industrial processes.

Ligation of D1-His332 and D1-Asp170 to the Manganese Cluster of Photosystem II from Synechocystis Assessed by Multifrequency Pulse EPR Spectroscopy

Multifrequency electron spin-echo envelope modulation (ESEEM) spectroscopy is used to ascertain the nature of the bonding interactions of various active site amino acids with the Mn ions that compose the oxygen-evolving cluster (OEC) in photosystem II (PSII) from the cyanobacterium Synechocystis sp. PCC 6803 poised in the S(2) state. Spectra of natural isotopic abundance PSII ((14)N-PSII), uniformly (15)N-labeled PSII ((15)N-PSII), and (15)N-PSII containing (14)N-histidine ((14)N-His/(15)N-PSII) are compared. These complementary data sets allow for a precise determination of the spin Hamiltonian parameters of the postulated histidine nitrogen interaction with the Mn ions of the OEC. These results are compared to those from a similar study on PSII isolated from spinach. Upon mutation of His332 of the D1 polypeptide to a glutamate residue, all isotopically sensitive spectral features vanish. Additional K(a)- and Q-band ESEEM experiments on the D1-D170H site-directed mutant give no indication of new (14)N-based interactions.

Theoretical Calculation of the Magnetic Resonance Parameters of Trigonal-Prismatic Tris(o-aminobenzenethiol)technetium and -rhenium Complexes

Spin Hamiltonian parameters for the neutral trigonal-prismatic Tc(abt)(3) and Re(abt)(3) chelates (abt = o-aminobenzenethiol) are calculated using relativistic density functional theory at the all-electron level. The small magnitude of the calculated g shifts and metal hyperfine interactions is in excellent agreement with previous experimental predictions based upon a ligand-centered ground-state magnetic orbital. The theoretical (14)N ligand hyperfine and quadrupole couplings also reproduce the nuclear frequencies measured by electron spin-echo envelope modulation spectroscopy. The nuclear quadrupole interaction of (187)Re is predicted to be 2 orders of magnitude larger than that of (99)Tc, in agreement with empirical simulation of the continuous-wave electron paramagnetic resonance spectrum. The spectrum of Tc(abt)(3) at high solute concentrations contains a central resonance not predicted for the isolated complex by theoretical calculations. The absence of this resonance at low solute concentrations provides evidence of intermolecular interactions in these systems.

Electron Spin-Echo Envelope Modulation (ESEEM) Reveals Water and Phosphate Interactions with the KcsA Potassium Channel,

Electron spin-echo envelope modulation (ESEEM) spectroscopy is a well-established technique for the study of naturally occurring paramagnetic metal centers. The technique has been used to study copper complexes, hemes, enzyme mechanisms, micellar water content, and water permeation profiles in membranes, among other applications. In the present study, we combine ESEEM spectroscopy with site-directed spin labeling (SDSL) and X-ray crystallography in order to evaluate the technique's potential as a structural tool to describe the native environment of membrane proteins. Using the KcsA potassium channel as a model system, we demonstrate that deuterium ESEEM can detect water permeation along the lipid-exposed surface of the KcsA outer helix. We further demonstrate that (31)P ESEEM is able to identify channel residues that interact with the phosphate headgroup of the lipid bilayer. In combination with X-ray crystallography, the (31)P data may be used to define the phosphate interaction surface of the protein. The results presented here establish ESEEM as a highly informative technique for SDSL studies of membrane proteins.

Direct Evidence for Nitrogen Ligation to the High Stability Semiquinone Intermediate in Escherichia coli Nitrate Reductase A

The membrane-bound heterotrimeric nitrate reductase A (NarGHI) catalyzes the oxidation of quinols in the cytoplasmic membrane of Escherichia coli and reduces nitrate to nitrite in the cytoplasm. The enzyme strongly stabilizes a menasemiquinone intermediate at a quinol oxidation site (Q(D)) located in the vicinity of the distal heme b(D). Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. (14)N and (15)N ESEEM and HYSCORE measurements carried out at X-band ( approximately 9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with (15)N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant A(iso)((14)N) approximately 0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using (14)N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, approximately 3.4 GHz), the (14)N nuclear quadrupolar parameters of the interacting nitrogen nucleus (kappa = 0.49, eta = 0.50) were determined and correspond to those of a histidine N(delta), assigned to the heme b(D) ligand His-66 residue. Moreover S-band (15)N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T((15)N) = 0.16 MHz). A distance of approximately 2.2 Abetween the carbonyl oxygen and the nitrogen could then be calculated. Mechanistic implications of these results are discussed in the context of the peculiar properties of the menasemiquinone intermediate stabilized at the Q(D) site of NarGHI.

Probing the Structure of Membrane Proteins Using Pulsed EPR ESEEM Spectroscopy

Current methods used for determining the secondary structure of membrane proteins require large sample sizes and have long data acquisition times. Therefore using pulsed EPR spectroscopic technique of Electron Spin Echo Envelope Modulation (ESEEM) is advantageous due to the requirement of less sample, short data acquisition, and high sensitivity. Using this technique, we can determine short to medium range distances (up to 8 A) between a site-specific nitroxide spin label (MTSL) and a nearby NMR-active isotopic labeled residue for a variety of peptides and proteins which ultimately determine the difference between an α-helical and β-sheet secondary structure. The information can be obtained using a three-pulse ESEEM sequence which allows for the detection of the fundamental nuclear spin transitions. It is possible to calculate the radial distance between a paramagnetic electron and a weakly coupled nucleus because the modulation depth produced by a weakly dipolar-coupled nucleus is inversely proportional to the radial distance. This new method is applied to two different membrane peptides, M2δ AChR, and KIGAKI, which have α-helical and β-sheet structural components respectively. A nitroxide spin probe is attached to a specific Cys residue at a given position I, and a 2H isotopic labeled residue is attached to a nearby residue (i + 3). The corresponding data shows that an α-helical structure will yield a very large 2H peak, whereas a β-sheet will yield a much smaller 2H peak, thus a difference can be observed between the two secondary structures. The ESEEM technique requires less protein sample (about 75 μg) and data acquisition only takes about 1 hour which makes it a simple, powerful, and effective technique in the determination of the secondary structure of a membrane protein.

ESEEM and multi-frequency EPR study on Mn2+ luminescence centres in leucophanite

The location and coordination of Mn2+ paramagnetic luminescent centres in three natural samples of leucophanite (ideally, NaCaBeSi2O6F) have been investigated by electron paramagnetic resonance (EPR) and electron spin-echo envelope modulation (ESEEM) spectroscopy. Continuous wave (cw)-EPR was carried out at multiple frequencies (9, 94 and 188 GHz) which allowed the precise determination of the spin Hamiltonian parameters. The simulation of the cw-EPR spectra shows that the signal originates from one manganese located in a single distorted site. This distortion was close to axial symmetry with a slight rhombicity and was consistent with an eight coordinated Mn2+ substituting for Ca2+. Spectral differences between the samples could be attributed mainly to D-strain effect. Three-pulse ESEEM spectroscopy was also used to investigate the coordination environment of the manganese and showed weak coupling between the manganese and beryllium (9Be), sodium (23Na) and fluorine (19F) nuclei in the second and subsequent coordination spheres. The Mn ligand distances estimated from the ESEEM data are in good agreement with bond distances estimated from X-ray diffraction data. To our best knowledge, only few ESEEM studies have been reported so far in Earth science and this is believed to be the first detailed ESEEM study of Mn2+ in a natural mineral system.

Self-Aggregation and Orientation of the Ion Channel-Forming Zervamicin IIA in the Membranes of ePC Vesicles Studied by cw EPR and ESEEM Spectroscopy

The ion channel-forming peptide antibiotic zervamicin A was studied in egg phosphocholin lipid membranes of large multilamellar vesicles (LMV) at 77 K. Continuous wave electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) methods combined with site-specific electron spin labeling were used to study the aggregation and immersion depth of two analog molecules, i.e., each monolabeled either at the N- or C-terminal end of the helical molecule. Analysis of the shape of the EPR spectra indicates that zervamicin molecules form aggregates in which the dipolar interaction between the spin labels at the N-terminus is substantially larger than that between the labels at the C-terminus. The ESEEM method was used to study the interaction between the nitroxide radical spin labels of the zervamicin molecules and deuterium nuclei in LMV, which were prepared using a D2O buffer. It is established that the largest amplitude of deuterium modulation of the unpaired electron is observed for zervamicin molecules labeled at the N-terminus. Based on the analysis of the Fourier parameters of the deuterium modulated spectrum, a model of the immersion depth of the terminal ends of the zervamicin molecule in a lipid bilayer is formulated. All of the spin labels at the N-terminus are grouped at the lipid–water interface, whereas 60% of labels at the C-terminus are located at the lipid–water interface and 40% are more deeply inserted into the lipid bilayer.

Evidence that D1-His332 in Photosystem II fromThermosynechococcus elongatusInteracts with the S3-State and not with the S2-State

Oxygen evolution by Photosystem II (PSII) is catalyzed by a Mn(4)Ca cluster. Thus far, from the crystallographic three-dimensional (3D) structures, seven amino acid residues have been identified as possible ligands of the Mn(4)Ca cluster. Among them, there is only one histidine, His332, which belongs to the D1 polypeptide. The relationships of the D1-His332 amino acid with kinetics and thermodynamic properties of the Mn(4)Ca cluster in the S(2)- and S(3)-states of the catalytic cycle were investigated in purified PSII from Thermosynechococcus elongatus. This was done by examining site-directed D1-His332Gln and D1-His332Ser mutants by a variety of spectroscopic techniques such as time-resolved UV-visible absorption change spectroscopy, cw- and pulse-EPR, thermoluminescence, and measurement of substrate water exchange. Both mutants grew photo-autotrophically and active PSII could be purified. On the basis of the parameters assessed in this work, the D1-His332(Gln, Ser) mutations had no effect in the S(2)-state. Electron spin-echo envelope modulation (ESEEM) spectroscopy also showed that possible interactions between the nuclear spin of the nitrogen(s) of D1-His332 with the electronic spin S = 1/2 of the Mn(4)Ca cluster in the S(2)-state were not detectable and that the D1-His332Ser mutation did not affect the detected hyperfine couplings. In contrast, the following changes were observed in the S(3)-state of the D1-His332 mutants: (1) The redox potential of the S(3)/S(2) couple was slightly increased by < or = 20 meV, (2) The S(3)-EPR spectrum was slightly modified, (3) The D1-His332Gln mutation resulted in a approximately 3 fold decrease of the slow (tightly bound) exchange rate and a approximately 2 fold increase of the fast exchange rate of the water substrate molecules. All these results suggest that the D1-His332 would be more involved in S(3) than in S(2). This could be one element of the conformational changes put forward in the S(2) to S(3) transition.

Electron spin resonance (ESR) and electron spin echo envelope modulation (ESEEM) studies on the ultramarine analogs obtained from zeolite A with various alkaline cations at different temperatures

Selected pigment samples prepared from mixture of zeolite A, sulfur and relatively low amount of alkaline carbonates (Na, Li, K, and Cs) by thermal treatment at 500 °C or 800 °C were characterized by means of UV–vis, ESR, ED-ESR and ESEEM spectroscopy in order to explain the influence of various cations on their coloration and properties. The samples maintain the original structure of the parent zeolite. The different colors result from the nature of applied cations as well as from the synthesis temperature. It is reflected in UV–vis spectra showing various intensity of the absorption bands assigned to S 2 - and S 3 - anion-radical chromophores. The ESR spectra recorded at 10 K indicated five components attributed to S 3 - . The best quality single-component rhombic spectrum with g x = 2.0011, g y = 2.0510 and g z = 2.0355, appears in the samples prepared with Na and K cations at 800 °C indicating a local homogeneity of the structure around the radicals. The g-factors are analyzed in terms of molecular orbital theory. The other samples (particularly those prepared at 500 °C) are less ordered which is reflected in a strong ESR line broadening along g-factor z-axis. This disorder is explained as due to a distribution of the trisulfur molecule bend angle. The ESEEM spectra of the pigments containing Li and Cs prepared at 500 °C indicate the peaks assigned to 7Li and 133Cs, while for the analogous samples obtained by heating at 800 °C these spectra lack such peaks. This is explained as an effect of a transition from a static disorder to a dynamic disorder visible as a transformation of static ESR spectrum into broad dynamically averaged homogeneous lines.

Studying Supramolecular Assemblies by ESEEM Spectroscopy: Inclusion Complexes of Cyclodextrins

Electron spin-echo envelope modulation (ESEEM) spectroscopy was used to investigate intramolecular and intermolecular complexes of cyclodextrins (CDs) with a nitroxide group. The interaction with solvent molecules (D(2)O) was followed through the (2)H modulation depth. Competition experiments with adamantane-type guests were used to confirm complexation. The shielding of the nitroxide group from solvent upon inclusion into a CD cavity made this technique more sensitive to complexation than cw EPR spectroscopy. ESEEM analysis of a series of CDs mono and bis spin-labeled on the primary rim of the cavity showed that only one compound formed a self-inclusion complex. This suggests that significant linker length/flexibility is required for formation of inclusion complexes in functionalized CDs. DEER (double electron-electron resonance) experiments confirmed that the self-inclusion complex of the spin-labeled CD was intra- rather than intermolecular.