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a b s t r a c tInteractions of the presynaptic protein a-synuclein with membranes are involved in its physiological action as well as in the pathological misfolding and aggregation related to Parkinsons's disease.We studied the conformation and orientation of a-synuclein bound to model vesicular membranes using multiparametric response polarity-sensitive fluorescent probes together with CD and EPR measurements. At low lipid to a-synuclein ratio the protein binds membranes through its N-terminal domain. When lipids are in excess, the a-helical content and the role of the C-terminus in binding increase. Highly rigid membranes also induce a greater a-helical content and a lower polarity of the protein microenvironment.

You can't top the CopperTop: Tetramolecular G-quadruplexes modified with terminal pyridine ligands exhibit metal-triggered stabilization as monitored by thermal denaturation studies, circular dichroism, and nondenaturing gel electrophoresis. Formation of the square-planar Cu(II) (pyridine)4 complex was confirmed by EPR measurements. The metal complexation is fully reversible by removal of the transition metal with ethylenediaminetetraacetic acid (edta).

We present here the results of a CW-ESR investigation of a double spin labeled α-cyclodextrin-based [2]rotaxane that is characterized by the presence of nitroxide labels both at the wheel and at the dumbbell. This was accomplished by synthesizing a spin labeled α-CD (the wheel) that was mechanically blocked on a thread containing a nitroxide unit by a Cu(I) catalyzed azide-alkyne cycloaddition (CuAAC). Both ESI-MS analysis and NMR spectroscopy were used to provide evidence of the threading processes. Because of the unsymmetrical structure of both the wheel and the axle, two different geometrical isomers could be predicted on the basis of the orientation of the CD along the thread. By combining molecular dynamic calculations and information extracted from the CW-ESR spectra, we were able to determine the geometrical nature of the isomer that was isolated as the only species. The ESR spectra showed J-coupling between the two mechanically connected nitroxide units and were analyzed by a model assuming three main molecular states. The intramolecular noncovalent nature of spin exchange was confirmed by reversibly switching the magnetic interaction on-off by changing the pH of the solution in the presence of a competing macrocyclic host.

PpoA is a fungal dioxygenase that produces hydroxylated fatty acids involved in the regulation of the life cycle and secondary metabolism of Aspergillus nidulans. It was recently proposed that this novel enzyme employs two different heme domains to catalyze two separate reactions: within a heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid [(8R)-HPODE]; in the second reaction step (8R)-HPODE is isomerized within a P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. In the present study, pulsed EPR methods were applied to find spectroscopic evidence for the reaction mechanism, thought to involve paramagnetic intermediates. We observe EPR resonances of two distinct heme centers with g-values typical for Fe(III) S = 5/2 high-spin (HS) and Fe(III) S = 1/2 low-spin (LS) hemes. 14N ENDOR spectroscopy on the S = 5/2 signal reveals resonances consistent with an axial histidine ligation. Reaction of PpoA with the substrate leads to the formation of an amino acid radical on the early millisecond time scale concomitant to a substantial reduction of the S = 5/2 heme signal. High-frequency EPR (95- and 180-GHz) unambiguously identifies the new radical as a tyrosyl, based on g-values and hyperfine couplings from spectral simulations. The radical displays enhanced T 1-spin–lattice relaxation due to the proximity of the heme centers. Further, EPR distance measurements revealed that the radical is distributed among the monomeric subunits of the tetrameric enzyme at a distance of approximately 5 nm. The identification of three active paramagnetic centers involved in the reaction of PpoA supports the previously proposed reaction mechanism based on radical chemistry.