Arthur Schweiger

Electron paramagnetic resonance evidence for binding of Cu(2+) to the C-terminal domain of the murine prion protein.

Transmissible spongiform encephalopathies in mammals are believed to be caused by scrapie form of prion protein (PrP(Sc)), an abnormal, oligomeric isoform of the monomeric cellular prion protein (PrP(C)). One of the proposed functions of PrP(C) in vivo is a Cu(II) binding activity. Previous studies revealed that Cu(2+) binds to the unstructured N-terminal PrP(C) segment (residues 23-120) through conserved histidine residues. Here we analyzed the Cu(II) binding properties of full-length murine PrP(C) (mPrP), of its isolated C-terminal domain mPrP(121-231) and of the N-terminal fragment mPrP(58-91) in the range of pH 3-8 with electron paramagnetic resonance spectroscopy. We find that the C-terminal domain, both in its isolated form and in the context of the full-length protein, is capable of interacting with Cu(2+). Three Cu(II) coordination types are observed for the C-terminal domain. The N-terminal segment mPrP(58-91) binds Cu(2+) only at pH values above 5.0, whereas both mPrP(121-231) and mPrP(23-231) already show identical Cu(II) coordination in the pH range 3-5. As the Cu(2+)-binding N-terminal segment 58-91 is not required for prion propagation, our results open the possibility that Cu(2+) ions bound to the C-terminal domain are involved in the replication of prions, and provide the basis for further analytical studies on the specificity of Cu(II) binding by PrP.

Single crystal ESR and ENDOR of bis-(salicylaldoximato) Cu(II): Bis-(Salicylaldoximato)Ni(II): Copper, nitrogen and proton hyperfine data and structure of the internal H-bond

Abstract An investigation of the ESR and ENDOR spectra of the complex Cusal 2 substituted into single crystals of Nisal 2 is reported. An analysis of the spectra is presented yielding g tensor, the hyperfine interaction tensors of 63 Cu, 14 N and four protons and the quadrupole tensor of 14 N, as well as the orientation of the principal axes systems of these quantities. Interpretation of the hyperfine pattern of the set of two perfectly equivalent 14 N nuclei is shown to require a detailed analysis of the second order perturbation terms expressed in the coupled spin basis. For certain orientations of the crystal the 14 N hyperfine pattern shows up to sixteen lines. Analysis of this phenomenon is achieved by exact diagonalization of the energy matrix and is traced back to noncrossing of energy levels. From the magnetic data bonding parameters of Cusal 2 in an extended Maki—McGarvey picture and spin densities on Cu and 14 N are derived. Cusal 2 features an intramolecular hydrogen bridge of type OH…O. Based on some assumptions concerning spin densities, the experimentally determined magnetic constants and a seven-center dipole—dipole interaction model, the structure of the H-bond is inferred to be bent and nonsymmetric, with the angle ∠ OH…O lying in the range 130–143°.

Theory and applications of generalized operator transforms for diagonalization of spin Hamiltonians

Abstract A generalized transform formalism for vector operators is devised for diagonalization of a rather wide class of spin hamiltonians. The operator technique leads to equations for transformation matrices, for which analytical solutions are given. These allow analytical formulation of the transformed electron Zeeman term, the sum of the magnetic hyperfine and nuclear Zeeman term, the electric quadrupole term and the electronic and nuclear Zeeman coupling terms. The angular dependence of energy eigenvalues, frequencies and line strengths of ESR and ENDOR transitions to first order will be expressed as compact bilinear and quadratic forms of the columns of the matrix relating the molecular coordinate system to the laboratory system. Thereby the explicit calculation of rotation matrices may be completely avoided, though the latter formally express the operator transforms. The generalized operator transform is also carried out for the off-diagonal blocks originating from hyperfine interaction terms. This allows the second order energy terms to be expressed explicitly as compact hermitean forms of a simple structure, in particular the explicit structure of mixing terms between hyperfine interactions of different (sets of) nuclei is obtained. The relation to the conventional Bleaney transform is discussed and the analogy to the generalized operator transform is worked out. Furthermore, a set of practical formulae is collected for experiments with angularly dependent ENDOR (ESR) crystal spectra. A comparison of exact (numerical) diagonalization with the numerical accuracy of the generalized transform method is given. The latter is shown to approximate exact transition frequencies and line strength for any situation with high precision. Finally two examples are discussed, which illustrate the generalized transform treatment of the second order terms.

Stability and Cu(II) Binding of Prion Protein Variants Related to Inherited Human Prion Diseases

All inherited forms of human prion diseases are linked with mutations in the prion protein (PrP) gene. Here we have investigated the stability and Cu(II) binding properties of three recombinant variants of murine full-length PrP(23-231)-containing destabilizing point mutations that are associated with human Gerstmann-Sträussler-Scheinker disease (F198S), Creutzfeld-Jakob disease (E200K), and fatal familial insomnia (D178N) by electron paramagnetic resonance and circular dichroism spectroscopy. Furthermore, we analyzed the variants H140S, H177S, and H187S of the isolated C-terminal domain of murine PrP, mPrP(121-231), to test a role of the histidine residues in Cu(II) binding. The F198S and E200K variants of PrP(23-231) differed in Cu(II) binding from the wild-type mPrP(23-231). However, circular dichroism spectroscopy indicated that the variants and the wild type did not undergo conformational changes in the presence of Cu(II). The D178N variant showed a high tendency to aggregate at pH 7.4 both with and without Cu(II). At lower pH values, it showed the same Cu(II) binding behavior as the wild type. The analysis allowed for a better location of the Cu(II) binding sites in the C-terminal part of the protein. Our present data indicate that hereditary forms of prion diseases cannot be rationalized on the basis of altered Cu(II) binding or mutation-induced protein destabilization alone.

Electron-spin resonance probe based on a 100 μm planar microcoil

We describe the fabrication and the performance of a microcoil-based probe for electron spin resonance (ESR) spectroscopy on micrometer sized samples. The probe consists of a 100 μm planar microcoil fabricated on a glass substrate, tuned and matched at 1.4 GHz (L band) using miniaturized ceramic capacitors. We performed continuous wave ESR experiments on samples having a volume between (100 μm)3 and (10 μm)3. At 300 K, we achieved a spin sensitivity of about 1010 spins/G Hz1/2, which is comparable to that of commercial ESR spectrometers operating at 9 GHz (X band). The results reported in this article suggest that microcoil-based probes might represent a valid alternative to conventional microwave cavities for ESR studies of sample of the order of (100 μm)3 and smaller.

The continuous wave electron paramagnetic resonance experiment revisited.

When the modulation frequency used in continuous wave electron paramagnetic resonance (cw EPR) spectroscopy exceeds the linewidth, modulation sidebands appear in the spectrum. It is shown theoretically and experimentally that these sidebands are actually multiple photon transitions, sigma(+)+kxpi, where one microwave (mw) sigma(+) photon is absorbed from the mw radiation field and an arbitrary number k of radio frequency (rf) pi photons are absorbed from or emitted to the modulation rf field. Furthermore, it is demonstrated that both the derivative shape of the lines in standard cw EPR spectra and the distortions due to overmodulation are caused by the unresolved sideband pattern of these lines. The single-photon transition does not even give a contribution to the first-harmonic cw EPR signal. Multiple photon transitions are described semiclassically in a toggling frame and their existence is proven using second quantization. With the toggling frame approach and perturbation theory an effective Hamiltonian for an arbitrary sideband transition is derived. Based on the effective Hamiltonians an expression for the steady-state density operator in the singly rotating frame is derived, completely describing all sidebands in all modulation frequency harmonics of the cw EPR signal. The relative intensities of the sidebands are found to depend in a very sensitive way on the actual rf amplitude and the saturation of single sidebands is shown to depend strongly on the effective field amplitude of the multiple photon transitions. By comparison with the analogous solutions for frequency-modulation EPR it is shown that the field-modulation and the frequency-modulation technique are not equivalent. The experimental data fully verify the theoretical predictions with respect to intensities and lineshapes.

Matrix effects on copper(II)phthalocyanine complexes. A combined continuous wave and pulse EPR and DFT study

The effect of the electron withdrawing or donating character of groups located at the periphery of the phthalocyanine ligand, as well as the influence of polar and nonpolar solvents are of importance for the redox chemistry of metal phthalocyanines. Continuous wave and pulse electron paramagnetic resonance and pulse electron nuclear double resonance spectroscopy at X- and Q-band are applied to investigate the electronic structure of the complexes Cu(II)phthalocyanine (CuPc), copper(II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc(t)), and copper(II) 1,2,3,4,8,9,10,11,15,16,17,18,22,23,24,25-hexadecafluoro-29H,31H-phthalocyanine (CuPc(F)) in various matrices. Isotope substitutions are used to determine the g values, the copper hyperfine couplings and the hyperfine interactions with the 14N, 1H and 19F nuclei of the macrocycle and the surrounding matrix molecules. Simulations and interpretations of the spectra are shown and discussed, and a qualitative analysis of the data using previous theoretical models is given. Density functional computations facilitate the interpretation of the EPR parameters. The experimental g, copper and nitrogen hyperfine and nuclear quadrupole values are found to be sensitive to changes of the solvent and the structure of the macrocycle. To elucidate the electronic, structural and bonding properties the changes in the g principal values are related to data from UV/Vis spectroscopy and to density functional theory (DFT) computations. The analysis of the EPR data indicates that the in-plane metal-ligand sigma bonding is more covalent for CuPc(t) in toluene than in sulfuric acid. Furthermore, the out-of-plane pi bonding is found to be less covalent in the case of a polar sulfuric acid environment than with nonpolar toluene or H2Pc environment, whereby the covalency of this bonding is increased upon addition of tert-butyl groups. No contribution from in-plane pi bonding is found.

Multiple quantum coherence in HYSCORE spectra

The implementation of matched pulses in two-dimensional electron spin echo envelope modulation experiments results in a drastic increase in intensity of peaks representing nuclear multiple quantum coherence. For a spin system consisting of one electron spin S=12 and two nuclei with spin I=12 the nuclear coherence created by the sequence (π/2)−τ−(π/2) is calculated. The transfer amplitudes between different kinds of nuclear coherences of a microwave π pulse are given. A new matched HYSCORE pulse sequence, called SMART HYSCORE, is introduced with only one microwave pulse for the generation and detection of nuclear coherence, which does not suffer from blind spots. This sequence is applied to a single crystal and a disordered system, and it is demonstrated how the resolution of weakly coupled nuclei can be increased. Furthermore we show that information about the relative sign of hyperfine couplings and about the relative orientation of the hyperfine tensors can be derived from the ridge patterns in a HYSCOR...

Characterization of the MCRred2 form of methyl-coenzyme M reductase: a pulse EPR and ENDOR study

Methyl-coenzyme M reductase (MCR), which catalyses the reduction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (H-S-CoB) to CH4 and CoM-S-S-CoB, contains the nickel porphinoid F430 as prosthetic group. The active enzyme exhibits the Ni(I)-derived axial EPR signal MCRred1 both in the absence and presence of the substrates. When the enzyme is competitively inhibited by coenzyme M (HS-CoM) the MCRred1 signal is partially converted into the rhombic EPR signal MCRred2. To obtain deeper insight into the geometric and electronic structure of the red2 form, pulse EPR and ENDOR spectroscopy at X- and Q-band microwave frequencies was used. Hyperfine interactions of the four pyrrole nitrogens were determined from ENDOR and HYSCORE data, which revealed two sets of nitrogens with hyperfine couplings differing by about a factor of two. In addition, ENDOR data enabled observation of two nearly isotropic 1H hyperfine interactions. Both the nitrogen and proton data indicate that the substrate analogue coenzyme M is axially coordinated to Ni(I) in the MCRred2 state.

Transition probabilities in electron—nuclear double- and multiple-resonance spectroscopy with non-coherent and coherent radio-frequency fields

Abstract Within the framework of a generalized operator transform technique the problem of the calculation of (time-proportional) transition probabilities of ESR, ENDOR and electron—nuclear multiple-resonance (EN m MR) transitions to first-order Rayleigh—Schrodinger perturbation theory is treated. Fermis golden rule is extended to spin systems subject (besides the microwave field) to several (weak) coherent or non-coherent radio-frequency fields (monochromatic or narrowband modulated). It is shown that for recently introduced experimental techniques using either circularly polarized or polarization-modulated radio-frequency fields (CP ENDOR and PM ENDOR, respectively), both modulu s and phase of the transition matrix elements for each radio-frequency field are required. Based on the first-order basis functions, analytical expressions for ESR and ENDOR transition probabilities for a general spin hamiltonian and various modulation schemes are given, which explicitly contain both zeroth- and first-order (“hyperfine enhancement”) contributions. The accuracy of the analytical first-order expressions is tested for two spin systems and found to be in excellent agreement with exact numerical calculations.