Electron Spin Echo Envelope Modulation Spectra Research Articles

Study of Electron-Nuclear Interactions in Doped Calcium Phosphates by Various Pulsed EPR Spectroscopy Techniques.

Substituted calcium phosphates (CaPs) are vital materials for the treatment of bone diseases and repairing and replacement of defects in human hard tissues. In this paper, we present some applications of the rarely used pulsed electron paramagnetic resonance (EPR) and hyperfine interaction spectroscopy approaches [namely, electron spin-echo envelope modulation (ESEEM) and electron–electron double-resonance detected nuclear magnetic resonance (EDNMR)] to investigate synthetic CaPs (hydroxyapatite, tricalcium, and octacalcium phosphate) doped with various cations (Li+, Na+, Mn2+, Cu2+, Fe3+, and Ba2+). These resonance techniques provide reliable tools to obtain unique information about the presence and localization of impurity centers and values of hyperfine and quadrupole tensors. We show that revealed in CaPs by EPR techniques, radiation-induced stable nitrogen-containing species and carbonate radicals can serve as sensitive paramagnetic probes to follow CaPs’ structural changes caused by cation doping. The most pulsed EPR, ESEEM, and EDNMR spectra can be detected at room temperature, reducing the costs of the measurements and facilitating the usage of pulsed EPR techniques for CaP characterization.

Composition-driven anionic disorder-order transformations triggered single-Eu2+-converted high-color-rendering white-light phosphors

Single-component, single-activator-converted high-color-rendering index (CRI) white-light phosphors have sparked much interest for phosphor-converted white light-emitting diodes. However, manipulating the distribution and locations of single activators to target desired sites in a given host lattice is still a challenge. Herein, we report the remote regulation of the distribution of Eu2+ ions by engineering and controlling the structural ordering in Sr3(Ce1−xLax)(PO4)3:0.05Eu2+ (SCLP:Eu2+) compound, which are designed by using two-color phosphors with different anionic structural ordering, viz., Sr3Ce(PO4)3:Eu2+ (yellow) with disordered state of PO4 tetrahedra, and Sr3La(PO4)3:Eu2+ (blue) with ordered state of PO4 tetrahedra. The successive substitution of Ce by La triggers a pronounced disorder-to-order structural transformation of PO4 tetrahedra within SCLP:Eu2+ compounds and guides remotely the distribution of Eu2+ activators, which is demonstrated by a detailed analysis of synchrotron X-ray diffraction refinement, electron paramagnetic resonance spectra, electron spin-echo envelope modulation spectra, Raman spectra, nuclear magnetic resonance spectra and micro-cathodoluminescence spectrum. Benefitting from the controllable structural ordering transformation, as-prepared samples form a unique disorder-order crystal structure, exhibiting controllable microstructure acting on Eu2+ ions, tunable-color emissions including a high CRI (Ra = 90) white-light, increased structural rigidity and thermal stability. These interesting findings in the local structure-dependent luminescence properties demonstrate that structural ordering engineering may be an effective approach to regulate the migration of Eu2+ activators and improve the thermal stability of phosphors.

Cu(II) EPR Reveals Two Distinct Binding Sites and Oligomerization of Innate Immune Protein Calgranulin C

S100A12 or Calgranulin C is a homodimeric antimicrobial protein of the S100 family of EF-hand calcium-modulated proteins. S100A12 is involved in many diseases such as inflammation, tumor invasion, cancer and neurological disorders such as Alzheimer’s disease. The binding of transition metal ions to the protein is important as the sequestering of the metal ion induces conformational changes in the protein, inhibiting the growth of various pathogenic microorganisms. In this work, we probe the Cu2+ binding properties of Calgranulin C. We demonstrate that the two Cu2+ binding sites in Calgranulin C show different coordination environments in solution. Continuous wave-electron spin resonance (CW-ESR) spectra of Cu2+-bound protein clearly show two distinct components at higher Cu2+:protein ratios, which is indicative of the two different binding environments for the Cu2+ ions. The g|| and A|| values are also different for the two components, indicating that the number of directly coordinated nitrogen in each site differs. Furthermore, we perform CW-ESR titrations to obtain the binding affinity of the Ca2+-loaded protein to Cu2+ ions. We observe a positive cooperativity in binding of the two Cu2+ ions. To further probe the Cu2+ coordination, we also perform electron spin echo envelope modulation (ESEEM) experiment. We perform ESEEM at two different fields where one Cu2+ binding site dominates the other. At both sites we see distinct signatures of Cu2+–histidine coordination. However, we clearly see that the ESEEM spectra corresponding to the two Cu2+ binding sites are significantly different. There is clear change in the intensity of the double quantum peak with respect to the nuclear quadrupole interaction peak at the two different fields. Furthermore, ESEEM along with hyperfine sublevel correlation show that only one of the two Cu2+ binding sites has backbone coordination, confirming our previous observation. Finally, we perform double electron–electron resonance spectroscopy to probe if the difference in binding environment is due to the Cu2+ binding to different sites in the protein. We obtain a distance distribution with a sharp peak at ~ 3 nm and a broad peak at ~ 4 nm. The shorter distance agrees with the Cu2+–Cu2+ distance expected for a dimer from the crystal structure. The longer distance is consistent with the Cu2+–Cu2+ distance when oligomerization occurs.

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.

Polaron spin echo envelope modulations in an organic semiconducting polymer

Theoretical treatment of the electron spin echo envelope modulation (ESEEM) spectra from polarons in a semiconducting $\pi$- conjugated polymer is presented. The contact hyperfine coupling and the dipolar interaction between the polaron and proton spins are found to have distinct contributions in the ESEEM spectra. However, since the two contributions are spaced very closely, and the dipolar contribution is dominant, the detection of the contact hyperfine interaction is difficult. To resolve this problem, a recipe of probing the contact hyperfine and dipolar interactions selectively is proposed, and a method for detecting the polaron contact hyperfine interaction is formulated. The ESEEM decay due to the polaron random hopping is analyzed, and the robustness of the method against this decay is verified. Moreover, this decay is linked to the transport properties of polarons, providing an auxiliary probe for the polaron transport.

Aminoxyl Radicals of B/P Frustrated Lewis Pairs: Refinement of the Spin-Hamiltonian Parameters by Field- and Temperature-Dependent Pulsed EPR Spectroscopy.

Q-band and X-band pulsed electron paramagnetic resonance spectroscopic methods (EPR) in the solid state were employed to refine the parameters characterizing the anisotropic interactions present in six nitroxide radicals prepared by N,N addition of NO to various borane-phosphane frustrated Lewis pairs (FLPs). The EPR spectra are characterized by the g-anisotropy as well as by nuclear hyperfine coupling between the unpaired electron and the 11B/10B, 14N and 31P nuclear magnetic moments. It was previously shown that continuous-wave spectra measured at X-band frequency (9.5 GHz) are dominated by the magnetic hyperfine coupling to 14N and 31P, whereas the g-tensor values and the 11B hyperfine coupling parameters cannot be refined with high precision from lineshape fitting. On the other hand, the X-band electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectra are completely dominated by the nuclear hyperfine coupling to the 11B nuclei, allowing a selective determination of their interaction parameters. In the present work this analysis has been further validated by temperature dependent ESEEM measurements. In addition, pulsed EPR data measured in the Q-band (34 GHz) are reported, which present an entirely different situation: the g-tensor components can be measured with much higher precision, and the ESEEM and HYSCORE spectra contain information about all of the 10B, 11B, 14N and 31P hyperfine interaction parameters. Based on these new results, we report here high-accuracy and precision data of the EPR spin Hamiltonian parameters measured on six FLP-NO radical species embedded in their corresponding hydroxylamine host structures. While the ESEEM spectra at Q-band frequency turn out to be very complex (due to the multinuclear contribution to the overall signal) in the HYSCORE experiment the extension over two dimensions renders a better discrimination between the different nuclear species, and the signals arising from hyperfine coupling to 10B, 11B, 14N and 31P nuclei can be individually analyzed.

Electron spin echo envelope modulation of molecular motions of deuterium nuclei

Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy is a powerful technique for the study of hyperfine interactions between an unpaired electron and nearby nuclei in solids, and is employed in quantitative structural studies. Here, we describe the use of ESEEM to study the slow motion of deuterium nuclei using their nuclear quadrupole resonance (NQR) line shapes. Two ESEEM techniques were employed: the conventional three-pulse ESEEM experiment, π/2 – τ – π/2 – T– π/2 – τ – echo, and the four-pulse ESEEM, π/2 – τ – π/2 – T/2 – π – T/2 – π/2 – τ – echo, with the time variable T scanned in both cases. The nitroxide free radical 4-tert-butyliminomethyl-2,2,5,5-tetramethyl(d12)-3-imidazoline-1-oxyl with four deuterated methyl groups was investigated in a glassy ortho-terphenyl matrix over a wide temperature range. It was shown that four-pulse ESEEM allowed measurement of the nearly pure 2H NQR line shape. Between 90K and 120K, the ESEEM spectra change drastically. At low temperatures, four-pulse ESEEM spectra show a Pake-like pattern, which evolves into a single line at higher temperatures, which is typical for NQR of rotating methyl CD3 groups. Comparison with literature data on NQR allows estimation of the reorientation rate, k. At ∼100K, where the spectral changes are most pronounced, k was found to be ∼105s−1. The spectral linewidths for the three-pulse ESEEM were found to decrease similarly with increasing temperature; so the three-pulse technique is also capable to detect motion of this type. The ESEEM approach, along with site-directed spin labeling, may be useful for detection of motional transitions near the spin labels in biological systems, when information on motion is required in a wide temperature range.

Characterization of Water Coordination to Ferrous Nitrosyl Complexes with fac-N2O, cis-N2O2, and N2O3 Donor Ligands.

Electron paramagnetic resonance (EPR) experiments were done on a series of S = (3)/2 ferrous nitrosyl model complexes prepared with chelating ligands that mimic the 2-His-1-carboxylate facial triad iron binding motif of the mononuclear nonheme iron oxidases. These complexes formed a comparative family, {FeNO}(7)(N2Ox)(H2O)3-x with x = 1-3, where the labile coordination sites for the binding of NO and solvent water were fac for x = 1 and cis for x = 2. The continuous-wave EPR spectra of these three complexes were typical of high-spin S = (3)/2 transition-metal ions with resonances near g = 4 and 2. Orientation-selective hyperfine sublevel correlation (HYSCORE) spectra revealed cross peaks arising from the protons of coordinated water in a clean spectral window from g = 3.0 to 2.3. These cross peaks were absent for the {FeNO}(7)(N2O3) complex. HYSCORE spectra were analyzed using a straightforward model for defining the spin Hamiltonian parameters of bound water and showed that, for the {FeNO}(7)(N2O2)(H2O) complex, a single water conformer with an isotropic hyperfine coupling, Aiso = 0.0 ± 0.3 MHz, and a dipolar coupling of T = 4.8 ± 0.2 MHz could account for the data. For the {FeNO}(7)(N2O)(H2O)2 complex, the HYSCORE cross peaks assigned to coordinated water showed more frequency dispersion and were analyzed with discrete orientations and hyperfine couplings for the two water molecules that accounted for the observed orientation-selective contour shapes. The use of three-pulse electron spin echo envelope modulation (ESEEM) data to quantify the number of water ligands coordinated to the {FeNO}(7) centers was explored. For this aspect of the study, HYSCORE spectra were important for defining a spectral window where empirical integration of ESEEM spectra would be the most accurate.

Solid-state EPR strategies for the structural characterization of paramagnetic NO adducts of frustrated Lewis pairs (FLPs).

Anisotropic interactions present in three new nitroxide radicals prepared by N,N addition of NO to various borane-phosphane frustrated Lewis pairs (FLPs) have been characterized by continuous-wave (cw) and pulsed X-band EPR spectroscopies in solid FLP-hydroxylamine matrices at 100 K. Anisotropic g-tensor values and (11)B, (14)N, and (31)P hyperfine coupling tensor components have been extracted from continuous-wave lineshape analyses, electron spin echo envelope modulation (ESEEM), and hyperfine sublevel correlation spectroscopy (HYSCORE) experiments with the help of computer simulation techniques. Suitable fitting constraints are developed on the basis of density functional theory (DFT) calculations. These calculations reveal that different from the situation in standard nitroxide radicals (TEMPO), the g-tensors are non-coincident with any of the nuclear hyperfine interaction tensors. The determination of these interaction parameters turns out to be successful, as the cw- and pulse EPR experiments are highly complementary in informational content. While the continuous-wave lineshape is largely influenced by the anisotropic hyperfine coupling to (14)N and (31)P, the ESEEM and HYSCORE spectra contain important information about the (11)B hyperfine coupling and nuclear electric quadrupolar interaction. The set of cw- and pulsed EPR experiments, with fitting constraints developed by DFT calculations, defines an efficient strategy for the structural analysis of paramagnetic FLP adducts.

Probing the Secondary Structure of Membrane Proteins with the Pulsed EPR ESEEM Technique

A novel approach is being developed to probe the secondary structure of membrane proteins and peptides qualitatively utilizing the three-pulse Electron Spin Echo Envelope Modulation (ESEEM) pulse sequence. In order to demonstrate the practicality of this biophysical technique, the M2delta subunit of AChR (α-helical), KAGAKI (β-sheet) and VRL8 (310-helix) peptides were incorporated into phospholipids bicelles to probe their secondary structure with ESSEM spectroscopy. Utilizing site-directed spin-labeling (SDSL) coupled with deuterated amino acid labeling of the peptides, the corresponding ESEEM spectra reveal characteristic patterns for α-helix, β-sheet and 310-helical structures. This ESSEM secondary structural approach can be used with different deuterated amino acids and provide pertinent qualitative structural information on membrane proteins in a short period of time (10 minutes) with small amounts of sample (30 μg).

Measuring the orientation of taurine in the active site of the non-heme Fe(II)/α-ketoglutarate-dependent taurine hydroxylase (TauD) using electron spin echo envelope modulation (ESEEM) spectroscopy.

The position and orientation of taurine near the non-heme Fe(II) center of the α-ketoglutarate (α-KG)-dependent taurine hydroxylase (TauD) was measured using Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy. TauD solutions containing Fe(II), α-KG, and natural abundance taurine or specifically deuterated taurine were prepared anaerobically and treated with nitric oxide (NO) to make an S = 3/2 {FeNO}(7) complex that is suitable for robust analysis with EPR spectroscopy. Using ratios of ESEEM spectra collected for TauD samples having natural abundance taurine or deuterated taurine, (1)H and (14)N modulations were filtered out of the spectra and interactions with specific deuterons on taurine could be studied separately. The Hamiltonian parameters used to calculate the amplitudes and line shapes of frequency spectra containing isolated deuterium ESEEM were obtained with global optimization algorithms. Additional statistical analysis was performed to validate the interpretation of the optimized parameters. The strongest (2)H hyperfine coupling was to a deuteron on the C1 position of taurine and was characterized by an effective dipolar distance of 3.90 ± 0.25 Å from the {FeNO}(7) paramagnetic center. The principal axes of this C1-(2)H hyperfine coupling and nuclear quadrupole interaction tensors were found to make angles of 26 ± 5 and 52 ± 17°, respectively, with the principal axis of the {FeNO}(7) zero-field splitting tensor. These results are discussed within the context of the orientation of substrate taurine prior to the initiation of hydrogen abstraction.

Determining α-Helical and β-Sheet Secondary Structures via Pulsed Electron Spin Resonance Spectroscopy

A new method has been developed to determine α-helical and β-sheet secondary structural components of aqueous and membrane-bound proteins using pulsed electron paramagnetic resonance (EPR) spectroscopy. The three-pulse electron spin echo envelope modulation (ESEEM) technique was used to detect weakly coupled (2)H-labeled nuclei on side chains in the proximity of a strategically placed nitroxide spin-label up to 8 Å away. Changes in the ESEEM spectra for different samples correlate directly to periodic structural differences between α-helical and β-sheet motifs. These distinct trends were demonstrated with α-helical (M2δ subunit of the acetylcholine receptor) and β-sheet (ubiquitin) peptides in biologically relevant sample environments.

Rabi oscillation and electron-spin-echo envelope modulation of the photoexcited triplet spin system in silicon

We report on a pulsed electron paramagnetic resonance (EPR) study of the photoexcited triplet state ($S=1$) of oxygen-vacancy centers in silicon. Rabi oscillations between the triplet sublevels are observed using coherent manipulation with a resonant microwave pulse. The Hahn echo and stimulated echo decay profiles are superimposed with strong modulations known as electron-spin-echo envelope modulation (ESEEM). The ESEEM spectra reveal a weak but anisotropic hyperfine coupling between the triplet electron spin and a ${}^{29}$Si nuclear spin ($I=1/2$) residing at a nearby lattice site, that cannot be resolved in conventional field-swept EPR spectra.

Probing the Secondary Structure of Membrane Proteins with the Pulsed EPR ESSEM Technique

Liu, Lishan; Miami University, Chemistry and Biochemistry; Mayo, Daniel; Miami University, Chemistry and Biochemistry; Sahu, Indra; Miami University, Chemistry and Biochemistry; McCarrick, Robert; Miami University, Chemistry and Biochemistry; Troxel, Kaylee, Miami University, Chemistry and Biochemistry; Lorigan, Gary; Miami University, Chemistry and BiochemistryA novel approach has been applied to probe secondary structures of membrane proteins and peptides qualitatively utilizing three-pulse Electron Spin Echo Envelope Modulation (ESEEM) spectroscope. The a-helical M2delta subunit of the acetylcholine receptor incorporated into phospholipids bicelles has been used as a model peptide. In order to demonstrate the practicality of this method, a cysteine mutated nitroxide spin label is positioned 1, 2, 3 and 4 residues away from two types of deuterated Leu side chains (denoted i+1 to i+4). The characteristic periodicity of the a-helical structure gives rise to a unique pattern in the ESEEM spectra. By using Leu instead of Val, only i+2 and i+3 become critical to distinguish alpha helical structure. Also, due to the longer side chain of Leu when compared to Val, 2H modulation depth is enhanced. Molecular dynamic simulations provide distance distributions that are consistent with the experimental data. The ESSEM technique can be applied to different deuterated amino acids and provide pertinent qualitative structural information on membrane proteins in a short time with small amounts of sample.

Membrane Protein Secondary Structure Determination via Pulsed EPR ESEEM

Membrane proteins are involved in a variety of pertinent roles within the cell such as ion channels, communication, and catalysts for reactions in the membrane. Limited structural information exists for membrane proteins and new approaches need to be developed. A new method has been developed to determine the secondary structure of membrane proteins using pulsed electron paramagnetic resonance (EPR) spectroscopy. The three-pulse electron spin echo envelope modulation (ESEEM) can be used to detect the dipolar coupling between a deuterium-labeled residue and a spin label attached up to 4 residues away (i + 4) and correlate the strength of this coupling with the radial distance between the spin and deuterium labels. In this case, i represents the location of the 2H-labeled residue and 4 represents the location of the spin label 4 residues away. Unique periodic structural difference between an α-helix and a β-sheet can be easily observed in the corresponding ESEEM spectra for i + 2 and i + 3 samples. We have demonstrated these distinct trends with model α-helical (M2δ subunit of the acetylcholine receptor) and β-sheet (first 17-residues of ubiquitin) peptides. Circular Dichorism spectroscopy further confirms the secondary structure of the labeled peptide constructs to ensure the validity of the method. The new ESEEM approach provides site-specific secondary structural information both qualitatively and quantitatively on membrane proteins that otherwise might not have been structurally elucidated.

Pulse electron spin resonance investigation of bismuth-doped silicon: Relaxation and electron spin echo envelope modulation

Donors in silicon represent good candidates for the realization of several innovative devices exploiting classical as well as quantum information processing functionalities. Although most of the current work in the field is focused on the technological donors (P, As), the more exotic Bi offers interesting properties due to its nuclear spin and electronic structure. We present a detailed pulsed electron paramagnetic resonance study of the spin-spin and spin-lattice relaxation rates along with a full angular analysis of electron spin echo envelope modulation (ESEEM) spectra in bismuth-doped silicon samples having two different donor concentrations. The measurements were performed up to $40$ K. ESEEM spectra provide information on the donor wave function via the leading superhyperfine interactions between donor nuclei and nearby $^{29}\mathrm{Si}$ coordination shells. The assignment of the most intense contributions was made on the basis of the angular variation of ESEEM resonances and elementary considerations on the deep donor states.

Probing the structure of membrane proteins with electron spin echo envelope modulation spectroscopy

A new approach has been developed to probe the structural properties of membrane peptides and proteins using the pulsed electron paramagnetic resonance technique of electron spin echo envelope modulation (ESEEM) spectroscopy and the α-helical M2δ subunit of the acetylcholine receptor incorporated into phospholipid bicelles. To demonstrate the practicality of this method, a cysteine-mutated nitroxide spin label (SL) is positioned 1, 2, 3, and 4 residues away from a fully deuterated Val side chain (denoted i + 1 to i + 4). The characteristic periodicity of the α-helical structure gives rise to a unique pattern in the ESEEM spectra. In the i + 1 and i + 2 samples, the ²H nuclei are too far away to be detected. However, with the 3.6 residue per turn pattern of an α-helix, the i + 3 and i + 4 samples reveal a strong signal from the ²H nuclei of the Val side chain. Modeling studies verify these data suggesting that the closest ²H-labeled Val to SL distance would in fact be expected in the i + 3 and i + 4 samples. This technique is very advantageous, because it provides pertinent qualitative structural information on an inherently difficult system like membrane proteins in a short period of time (minutes) with small amounts of protein (μg).

Electrically Detected Electron-Spin-Echo Envelope Modulation: A Highly Sensitive Technique for Resolving Complex Interface Structures

We show that the electrical detection of electron-spin-echo envelope modulation (ESEEM) is a highly sensitive tool to study interfaces. Taking the Si/SiO2 interface defects in phosphorus-doped crystalline silicon as an example, we find that the main features of the observed echo modulation pattern allow us to develop a microscopic model for the dangling-bond-like P(b0) center by comparison with the results of ab initio calculations. The ESEEM spectrum is found to be far more sensitive to the defect characteristics than the spectrally resolved hyperfine splitting itself.

Utilizing Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy to Probe the Structure of Membrane Proteins

New approaches are needed to more efficiently probe the structural properties of membrane proteins. A new approach has been developed to probe the structural properties of membrane peptides and proteins using the pulsed Electron Paramagnetic Resonance (EPR) technique of Electron Spin Echo Envelope Modulation (ESEEM). This technique can measure short-range distances between a nitroxide spin label and a 2H nucleus out to approximately 8Å. For this study a model membrane peptide M2δ, was constructed by solid phase peptide synthesis and inserted into a DMPC/DHPC bicelle membrane. We report for the first time, the direct detection of 2H modulation between a 2H-labeled d8 Val residue and a nitroxide spin label three and four residues away that is characteristic of an alpha-helical secondary structure. Simulations of the ESEEM data reveal a distance of approximately 6.4 +/- 0.5Å that agrees well with molecular modeling studies. ESEEM spectra in this work yielded high-quality data in less than an hour with as little as 35μg of protein sample.

ESR Spectroscopy Suggests Unequal Contributions of the Three Histidine Residues to Cu(II) Binding in Amyloid- β at Physiological PH

We provide precise information about the contribution of each of the three histidine residues, His6, His13, and His14, to Cu(II) binding in the equimolar Cu(II)-Aβ(1-16) complex at physiological pH by employing electron spin resonance spectroscopy. Based on our experimental results, we propose probable Cu(II)-coordination environments for the major and the minor component of the Cu(II)-Aβ(1-16) complex. Three-pulse electron spin-echo envelope modulation (ESEEM) experiments conducted on nonlabeled and 15N-labeled Cu(II)-Aβ(1-16) complexes at a pH of 7.4 reveal that the contributions of the three residues to the Cu(II) coordination are in the order of His14 ≈ His6 > His13 in the major component of the Cu(II)-Aβ(1-16) complex. Also, the ESEEM spectra of diethylenetriamine (DETA)-Cu(II)-Aβ(1-16) complexes suggest that the relative Cu(II)-binding affinities of the three histidine residues are in essentially the same order as in the major component. The corresponding hyperfine sublevel correlation spectra show that multiple histidine residues coordinate to Cu(II) and suggest the presence of axially bound water in the Cu(II)-coordination environment. In addition, a broader interpretation of our results suggests a significant contribution from the simultaneous coordination of His13 and His14 in the major component, which has been underappreciated, and indicates the possibility of intermolecular bridges through His13 in the minor component. Our strategy and experimental results are meaningful in that the subtle difference in the three histidine residues at physiological pH is traced by 15N-labeling and introduction of DETA, a tridendate ligand, without any side chain modification, amino acid residue replacement, or pH change, each of which might lead to an alteration in the peptide structure or the coordination environment.