Echo Envelope Modulation Spectroscopy Research Articles

Self-assembly of spin-labeled antimicrobial peptides magainin 2 and PGLa in lipid bilayers

The cationic antimicrobial peptides PGLa and magainin 2 (Mag2) are known for their antimicrobial activity and synergistic enhancement in antimicrobial and membrane leakage assays. Further use of peptides in combinatory therapy requires knowledge of the mechanisms of action of both individual peptides and their mixtures. Here, electron paramagnetic resonance (EPR), double electron-electron resonance (DEER, also known as PELDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies were applied to study self-assembly and localization of spin-labeled PGLa and Mag2 in POPE/POPG membranes with a wide range of peptide/lipid ratios (P/L) from ∼1/1500 to 1/50. EPR and DEER data showed that both peptides tend to organize in clusters, which occurs already at the lowest peptide/lipid molar ratio of 1/1500 (0.067 mol%). For individual peptides, these clusters are quite dense with intermolecular distances of the order of ∼2 nm. In the presence of a synergistic peptide partner, these homo-clusters are transformed into lipid-diluted hetero-clusters. These clusters are characterized by a local surface density that is several times higher than expected from a random distribution. ESEEM data indicate a slightly different insertion depth of peptides in hetero-clusters when compared to homo-clusters.

Engineering Synthetic Electron Transfer Chains from Metallopeptide Membranes.

The energetic and geometric features enabling redox chemistry across the copper cupredoxin fold contain key components of electron transfer chains (ETC), which have been extended here by templating the cross-β bilayer assembly of a synthetic nonapeptide, HHQALVFFA-NH2 (K16A), with copper ions. Similar to ETC cupredoxin plastocyanin, these assemblies contain copper sites with blue-shifted (λmax 573 nm) electronic transitions and strongly oxidizing reduction potentials. Electron spin echo envelope modulation and X-ray absorption spectroscopies define square planar Cu(II) sites containing a single His ligand. Restrained molecular dynamics of the cross-β peptide bilayer architecture support metal ion coordination stabilizing the leaflet interface and indicate that the relatively high reduction potential is not simply the result of distorted coordination geometry (entasis). Cyclic voltammetry (CV) supports a charge-hopping mechanism across multiple copper centers placed 10-12 Å apart within the assembled peptide leaflet interface. This metal-templated scaffold accordingly captures the electron shuttle and cupredoxin functionality in a peptide membrane-localized electron transport chain.

Tuning the Type 1 Reduction Potential of Multicopper Oxidases: Uncoupling the Effects of Electrostatics and H-Bonding to Histidine Ligands.

In multicopper oxidases (MCOs), the type 1 (T1) Cu accepts electrons from the substrate and transfers these to the trinuclear Cu cluster (TNC) where O2 is reduced to H2O. The T1 potential in MCOs varies from 340 to 780 mV, a range not explained by the existing literature. This study focused on the ∼350 mV difference in potential of the T1 center in Fet3p and Trametes versicolor laccase (TvL) that have the same 2His1Cys ligand set. A range of spectroscopies performed on the oxidized and reduced T1 sites in these MCOs shows that they have equivalent geometric and electronic structures. However, the two His ligands of the T1 Cu in Fet3p are H-bonded to carboxylate residues, while in TvL they are H-bonded to noncharged groups. Electron spin echo envelope modulation spectroscopy shows that there are significant differences in the second-sphere H-bonding interactions in the two T1 centers. Redox titrations on type 2-depleted derivatives of Fet3p and its D409A and E185A variants reveal that the two carboxylates (D409 and E185) lower the T1 potential by 110 and 255-285 mV, respectively. Density functional theory calculations uncouple the effects of the charge of the carboxylates and their difference in H-bonding interactions with the His ligands on the T1 potential, indicating 90-150 mV for anionic charge and ∼100 mV for a strong H-bond. Finally, this study provides an explanation for the generally low potentials of metallooxidases relative to the wide range of potentials of the organic oxidases in terms of different oxidized states of their TNCs involved in catalytic turnover.

Influence of Polar Mutations on the Electronic and Structural Properties of QA-· in Bacterial Reaction Centers.

Reaction centers from Rhodobacter sphaeroides with residue M265 mutated from isoleucine to threonine, serine, and asparagine (M265IT, M265IS, and M265IN, respectively) in the QA-· state are studied by high-resolution electron spin echo envelope modulation (ESEEM) and electron nuclear double resonance spectroscopy methods to investigate the structural characteristics of these mutants influencing the redox properties of the QA site. All three mutants decrease the redox midpoint potential (Em) of QA by ∼0.1 V, yet the mechanism for this drop in Em is unclear. In this work, we examine (i) the hydrogen bonding interactions between QA-· and residues histidine M219 and alanine M260, (ii) the electron spin density distribution of the semiquinone, and (iii) the orientations of the ubiquinone methoxy substituents. 13C measurements show no significant contribution of methoxy dihedral angles to the observed decrease in Em for the QA mutants. Instead, 14N three-pulse ESEEM data suggest that electrostatic or hydrogen bond formation between the mutated M265 side chain and His-M219 Nδ may be involved in the observed lowering of the QA midpoint potential. For mutant M265IN, analysis of the proton hyperfine couplings reveals a weakened hydrogen bond network, resulting in an altered QA-· spin density distribution. The magnetic resonance study presented here is most consistent with an electrostatic or structural perturbation of the His-M219 Nδ hydrogen bond in these mutants as a mechanism for the ∼0.1 V decrease in QA Em.

Synthesis of Spin-Labeled Ibuprofen and Its Interaction with Lipid Membranes.

Ibuprofen is a non-steroidal anti-inflammatory drug possessing analgesic and antipyretic activity. Electron paramagnetic resonance (EPR) spectroscopy could be applied to study its interaction with biological membranes and proteins if its spin-labeled analogs were synthesized. Here, a simple sequence of ibuprofen transformations—nitration, esterification, reduction, Sandmeyer reaction, Sonogashira cross-coupling, oxidation and saponification—was developed to attain this goal. The synthesis resulted in spin-labeled ibuprofen (ibuprofen-SL) in which the spin label TEMPOL is attached to the benzene ring. EPR spectra confirmed interaction of ibuprofen-SL with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers. Using 2H electron spin echo envelope modulation (ESEEM) spectroscopy, ibuprofen-SL was found to be embedded into the hydrophobic bilayer interior.

OOP-ESEEM Spectroscopy: Accuracies of Distances of Spin-Correlated Radical Pairs in Biomolecules.

In addition to the commonly used electron–electron double resonance (ELDOR) technique, there are several other electron paramagnetic resonance (EPR) methods by which structure information can be obtained by exploiting the dipolar coupling between two radicals based on its characteristic r −3 dependence. In this contribution, we explore the potential of out-of-phase-electron-spin echo envelope modulation (OOP-ESEEM) spectroscopy to collect accurate distance information in photo-sensitive (bio) molecules. Although the method has already been applied to spin-correlated radical pairs in several classes of light-active proteins, the accuracy of the information obtained has not yet been extensively evaluated. To do this in a system-independent fashion, OOP-ESEEM time traces simulated with different values of the dipolar and exchange couplings were generated and analyzed in a best-possible way. Excellent agreement between calculated and numerically fitted values over a wide range of distances (between 15 and 45 Å) was obtained. Furthermore, the limitations of the method and the dependence on various experimental parameters could be evaluated.

Electron Spin Echo Envelope Modulation Spectroscopy Reveals How Adenosylcobalamin-Dependent Lysine 5,6-Aminomutase Positions the Radical Pair Intermediates and Modulates Their Stabilities for Efficient Catalysis

Electron spin echo envelope modulation (ESEEM) spectroscopy reveals several interactions that serve to weaken the Co–C5′ bond, through stabilization of the cleaved state, thereby facilitating the 5′-deoxyadenosyl (5′-dAdo•) radical formation in unlabeled and 15N-labeled lysine 5,6-aminomutase. In the first place, we show that the CoII–His133β interaction enhances the spin delocalization from CoII to the α-axial ligand, reducing the 5′-dAdo•–CoII recombination probability in situ and allowing nascent 5′-dAdo• to migrate toward the substrate. Next, using [5′-2H]-deoxyadenosylcobalamin, we show that the C5′ methyl group of 5′-dAdoH is at closest contact with the substrate radical. In the case of 5′-dAdo•, this would allow the 5′-methylene radical to be rapidly quenched by H-transfer from the substrate. Finally, we show that the spin density is transferred noncovalently from the substrate radical to the adenine N7 of 5′-dAdoH. In the one-radical substrate–5′-dAdo•–product H-exchange triad, which is central in the mechanism of action of B12 enzymes, such transfer would promote the forward H-transfer reaction by dynamically lowering the free energy of the substrate radical, thereby facilitating its formation and quenching of 5′-dAdo•. In the reverse H-transfer reaction following the 1,2-rearrangement, the reversibility of the noncovalent spin transfer allows the product radical to restore full radical character to reform 5′-dAdo• and a diamagnetic product. The role of dynamical and reversible spin partitioning as a mechanism for modulating the radical stability is likely common in B12 enzymes. This study leads to a better comprehension about how an enzyme controls the reaction trajectory of 5′-dAdo• exquisitely with specificity. The ESEEM results support the positioning of the interacting partners in the catalytic form of lysine 5,6-aminomutase obtained by modeling.

Solvent accessibility in interdigitated and micellar phases formed by DPPC/Lyso-PPC mixtures: D2O-ESEEM of chain labeled lipids.

Electron spin echo envelope modulation (ESEEM) spectroscopy was used to investigate binary mixtures of single-chain micelle-forming lipids and diacyl bilayer-forming lipids dispersed in D2O at 77 K. Mixtures of dipalmitoylphosphatidylcholine (DPPC) and lyso-palmitoylphosphatidylcholine (Lyso-PPC) over the entire composition range (0-100 mol%) and phosphatidylcholine spin-labeled at selected carbon atom position along the sn-2 chain (n-PCSL) were considered. On increasing the content of the lysolipids incorporated in DPPC, the lipid bilayers are first transformed in interdigitated lamellae and then converted in micelles of Lyso-PPC. In the interdigitated phase, the profile of translamellae water accessibility is rather uniform as all the hydrocarbon segments are equally exposed to the solvent. In Lyso-PPC micelle, water penetrates at any depth of the hydrocarbon region with a tendency to increase toward the chain termini. The extent of water penetration is higher in the interdigitated DPPC/Lyso-PPC dispersions than in Lyso-PPC micelles. The profiles of water permeation revealed directly by D2O-ESEEM are also confirmed by more indirect evaluation of the polarity profiles based on the 14N-hyperfine splitting in the conventional electron paramagnetic resonance spectra of n-PCSL in frozen DPPC/Lyso-PPC mixtures at 77 K. The ESEEM data reveal that H-bonding formation between the -NO group of the spin-label and the D2O molecules is favored in the intergitated phase with respect to the micellar phase and, in any lipid dispersion, the fraction of nitroxides that are singly H-bonded to deuterons is higher than the fraction that are doubly H-bonded. The overall results highlight the differences in the accessibility and properties of the solvent in the hydrocarbon region of lipid bilayers, interdigitated bilayers and micelles.

ESEEM Reveals Bound Substrate Histidine in the ABC Transporter HisQMP2

Localization of substrates in membrane proteins is an important but challenging task. In this paper, we show that deuterium electron spin echo envelope modulation spectroscopy (2H ESEEM) combined with site-directed spin labeling is a powerful tool to localize the substrate, histidine-d5, in the ABC transporter HisQMP2. Based on a homology model and spin label rotamer analyses, we calculated 2H ESEEM spectra for eight possible labeling positions close to the putative substrate-binding site. Experimental 2H ESEEM spectra were determined with spin labels bound either at position 169 of HisM, for which a detectable 2H ESEEM signal was calculated, or with a spin label bound at position 54 of HisQ as a negative control. The agreement between the calculated and experimental ESEEM spectra provides strong evidence for the histidine located in a binding site primarily liganded by residues of HisM as proposed by the homology model.

Utilization of 13C-labeled amino acids to probe the α-helical local secondary structure of a membrane peptide using electron spin echo envelope modulation (ESEEM) spectroscopy

Electron spin echo envelope modulation (ESEEM) spectroscopy in combination with site-directed spin labeling (SDSL) has been established as a valuable biophysical technique to provide site-specific local secondary structure of membrane proteins. This pulsed electron paramagnetic resonance (EPR) method can successfully distinguish between α-helices, β-sheets, and 310-helices by strategically using 2H-labeled amino acids and SDSL. In this study, we have explored the use of 13C-labeled residues as the NMR active nuclei for this approach for the first time. 13C-labeled d5-valine (Val) or 13C-labeled d6-leucine (Leu) were substituted at a specific Val or Leu residue (i), and a nitroxide spin label was positioned 2 or 3 residues away (denoted i-2 and i-3) on the acetylcholine receptor M2δ (AChR M2δ) in a lipid bilayer. The 13C ESEEM peaks in the FT frequency domain data were observed for the i-3 samples, and no 13C peaks were observed in the i-2 samples. The resulting spectra were indicative of the α-helical local secondary structure of AChR M2δ in bicelles. This study provides more versatility and alternative options when using this ESEEM approach to study the more challenging recombinant membrane protein secondary structures.

Investigating the Secondary Structure of Membrane Peptides Utilizing Multiple 2H-Labeled Hydrophobic Amino Acids via Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy.

An electron spin echo envelope modulation (ESEEM) approach was used to probe local secondary structures of membrane proteins and peptides. This ESEEM method detects dipolar couplings between 2H-labeled nuclei on the side chains of an amino acid (Leu or Val) and a strategically placed nitroxide spin-label in the proximity up to 8 Å. ESEEM spectra patterns for different samples correlate directly to the periodic structural feature of different secondary structures. Since this pattern can be affected by the side chain length and flexibility of the 2H-labeled amino acid used in the experiment, it is important to examine several different hydrophobic amino acids (d3 Ala, d8 Val, d8 Phe) utilizing this ESEEM approach. In this work, a series of ESEEM data were collected on the AChR M2δ membrane peptide to build a reference for the future application of this approach for various biological systems. The results indicate that, despite the relative intensity and signal-to-noise level, all amino acids share a similar ESEEM modulation pattern for α-helical structures. Thus, all commercially available 2H-labeled hydrophobic amino acids can be utilized as probes for the further application of this ESEEM approach. Also, the ESEEM signal intensities increase as the side chain length gets longer or less rigid. In addition, longer side chain amino acids had a larger 2H ESEEM FT peak centered at the 2H Larmor frequency for the i ± 4 sample when compared to the corresponding i ± 3 sample. For shorter side chain amino acids, the 2H ESEEM FT peak intensity ratio between i ± 4 and i ± 3 was not well-defined.

Electron paramagnetic resonance of a 10B-containing heterocyclic radical

Electron paramagnetic resonance measurements for a 10B-containing heterocyclic phenanthrenedione radical, (C6F5)2B(O2C14H8), were made at X-band in 9:1 toluene:dichloromethane from 10 to 293 K and in toluene from 180 to 293 K. In well-deoxygenated 0.1 mM toluene solution at room temperature hyperfine couplings to 10B, four pairs of protons and five pairs of fluorines contribute to a continuous wave spectrum with many resolved lines. Hyperfine couplings were adjusted to provide the best fit for spectra of the radical enriched in 10B and the analogous radical synthesized with 10,11B in natural abundance, resulting in small refinements of the hyperfine coupling constants previously reported for the natural abundance sample. Electron spin relaxation rates at temperatures between 15 and 293 K were similar for samples containing 10B and natural isotope abundance. Analysis of electron spin echo envelope modulation and hyperfine correlation spectroscopy data at 80 K found Axx = −7.5 ± 0.3, Ayy = −8.5 ± 0.3, and Azz = −10.8 ± 0.3 MHz for 11B, which indicates small spin density on the boron. The spin echo and hyperfine spectroscopy data for the 10B –containing radical are consistent with the factor of 2.99 smaller hyperfine values for 10B than for 11B.

Localization of Guest Molecules in Nanopores by Pulsed EPR Spectroscopy

The localization of guest molecules at the molecular scale in mesoporous host materials is crucial for applications in heterogeneous catalysis, chromatography, drug delivery, and in different biomedical applications. Here, we present for the first time the precise localization of different guest molecules inside the mesoporous organosilica material UKON2a with a pore size of 6 nm. We exploited paramagnetic probe molecules 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (4-amino-TEMPO) and 4-carboxy-TEMPO in combination with a deuteration strategy. Applying a complementary set of different pulsed electron paramagnetic resonance methods, we obtained information about the dimensionality of the spatial distribution and local concentration via double electron–electron resonance experiments, orientation of the guest molecules with respect to the pore walls via electron spin echo envelope modulation spectroscopy, and about the distance between guest molecules and pore walls via electron nuclear double resonance spe...

Structural Effects of Ammonia Binding to the Mn4CaO5 Cluster of Photosystem II.

The Mn4CaO5 oxygen-evolving complex (OEC) of photosystem II catalyzes the light-driven oxidation of two substrate waters to molecular oxygen. ELDOR-detected NMR along with computational studies indicated that ammonia, a substrate analogue, binds as a terminal ligand to the Mn4A ion trans to the O5 μ4 oxido bridge. Results from electron spin echo envelope modulation (ESEEM) spectroscopy confirmed this and showed that ammonia hydrogen bonds to the carboxylate side chain of D1-Asp61. Here we further probe the environment of OEC with an emphasis on the proximity of exchangeable protons, comparing ammonia-bound and unbound forms. Our ESEEM and electron nuclear double resonance (ENDOR) results indicate that ammonia substitutes for the W1 terminal water ligand without significantly altering the electronic structure of the OEC.

Alamethicin self-assembling in lipid membranes: concentration dependence from pulsed EPR of spin labels.

The antimicrobial action of the peptide antibiotic alamethicin (Alm) is commonly related to peptide self-assembling resulting in the formation of voltage-dependent channels in bacterial membranes, which induces ion permeation. To obtain a deeper insight into the mechanism of channel formation, it is useful to know the dependence of self-assembling on peptide concentration. With this aim, we studied Alm F50/5 spin-labeled analogs in a model 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane, for peptide-to-lipid (P/L) ratios varying between 1/1500 and 1/100. Pulsed electron-electron double resonance (PELDOR) spectroscopy reveals that even at the lowest concentration investigated, the Alm molecules assemble into dimers. Moreover, under these conditions, electron spin echo envelope modulation (ESEEM) spectroscopy of D2O-hydrated membranes shows an abrupt change from the in-plane to the trans-membrane orientation of the peptide. Therefore, we hypothesize that dimer formation and peptide reorientation are concurrent processes and represent the initial step of peptide self-assembling. By increasing peptide concentration, higher oligomers are formed. A simple kinetic model of equilibrium among monomers, dimers, and pentamers allows for satisfactorily describing the experimental PELDOR data. The inter-label distances in the oligomers obtained from PELDOR experiments become better resolved with increasing P/L ratio, thus suggesting that the supramolecular organization of the higher-order oligomers becomes more defined.

Pulse EPR and ENDOR Study of Manganese Doped [(CH3)2NH2][Zn(HCOO)3] Hybrid Perovskite Framework

We present a pulse electron paramagnetic resonance (EPR) and electron–nuclear double resonance (ENDOR) study of a manganese-doped [(CH3)2NH2][Zn(HCOO)3] dense metal–organic framework which exhibits a structural phase transition at 163 K. The echo-detected field sweep Mn2+ EPR spectrum of the low-temperature phase is in a perfect agreement with the previous continuous-wave EPR results, while the spectrum of the disordered phase reveals a significant EPR transition-dependent relaxation. The 1H ENDOR pattern indicates several protons in the vicinity of the Mn2+ ion. The experimental ENDOR spectrum is successfully simulated using the proton hyperfine tensors calculated by the density functional theory. A multifrequency electron spin echo envelope modulation (ESEEM) spectroscopy shows a peculiar signal which is unaffected by the external magnetic field. The modulation depth of this signal starts to decrease above 40 K, coinciding with the temperature at which the methyl groups of the (CH3)2NH2+ cations start t...

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.

Dynamical Transitions at Low Temperatures in the Nearest Hydration Shell of Phospholipid Bilayers.

For the so-called dynamical transition from harmonic to anharmonic (or diffusive) motions in biological systems, the presence of hydration water is important. To explain the molecular mechanism of this transition, the information on molecular motions in the nearest hydration shell would be helpful. In this work, to study molecular motions in the nearest hydration shell of spin-labeled model biological membranes, a pulsed version of electron paramagnetic resonance, electron spin echo envelope modulation (ESEEM) spectroscopy, is used. For hydration by deuterium water, the 2H ESEEM frequency spectra resemble the solid-state 2H NMR line shape that is widely used for structural and dynamical studies. Two types of model membranes were investigated and compared: bilayers consisting of unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and bilayers consisting of fully saturated lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The lipid chain packing for the POPC bilayer is known to be more defective than that for the DPPC bilayer. For both the POPC and the DPPC bilayers, the 2H ESEEM NMR-like spectra showed a sharp narrowing between 180 and 190 K. From the other side, in both bilayers at 188 K, an inflection was observed for the temperature dependence of molecular motions detected by the spin relaxation of spin labels in the bilayer interior. It was concluded that dynamical transition in the bilayer interior is accompanied by an onset of isotropic water molecular dynamics in the nearest hydration shell of the bilayer with a rate of ∼105 s-1. Also, the 2H ESEEM NMR-like spectra in the POPC bilayer showed slight changes above 100 K that could be ascribed to another dynamical transition resulting in the appearance of restricted orientational motion of water molecules. These data also are interrelated with spin relaxation of spin labels in the POPC bilayer interior and support the hypothesis ascribing the transition at 100 K to excessive lipid chain flexibility.

Probing the Local Secondary Structure of Human Vimentin with Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy.

Previously, an electron spin echo envelope modulation (ESEEM) spectroscopic approach was established to probe the local secondary structure of membrane proteins and peptides utilizing site-directed spin-labeling (SDSL). In this method, the side chain of one amino acid residue is selectively 2H-labeled and a nitroxide spin label is strategically placed 1, 2, 3, or 4 amino acids away from the 2H-labeled amino acid (denoted as i ± 1 to i ± 4, i represents the 2H-labeled amino acid). ESEEM can detect the dipolar coupling between the nitroxide spin label and 2H atoms on the amino acid side chain. Due to the periodicity of different secondary structures, different ESEEM patterns can be revealed to probe the structure. For an α-helical structural component, a 2H ESEEM signal can be detected for i ± 3 and i ± 4 samples, but not for i ± 1 or i ± 2 samples. Several 2H-labeled hydrophobic amino acids have been demonstrated in model system that can be utilized to identify local secondary structures via this ESEEM approach in an extremely efficient fashion. In this study, the ESEEM approach was used to investigate the rod 2B region of the full-length intermediate filament protein human vimentin. Consistent with previous EPR and X-ray crystallography results, our ESEEM results indicated helical structural components within this region. Thus, this ESEEM approach is able to identify α-helical structural components despite the coiled-coil nature of the vimentin structure. The data show that the human vimentin rod 2B adapted a typical α-helical structure around residue Leu309. This result is consistent with the X-ray data from fragmented protein segments and continuous wave EPR data on the full-length vimentin. Finally, the ESEEM data suggested that a local secondary structure slightly different from a typical α-helix was adopted around residue 340.

Chemical systems modeling the d1 Mo(V) states of molybdenum enzymes

This review focuses on the synthesis, properties and electron paramagnetic resonance (EPR), electron spin echo envelope modulation (ESEEM) and electron-nuclear double resonance (ENDOR) spectroscopy of mononuclear d1 oxo- and sulfido-Mo(V) complexes relevant to the understanding of the EPR-active Mo(V) forms of pterin-containing molybdenum enzymes.