José Fernando Lima

Deconvolution of the EPR spectra of vanadium oxide nanotubes

In this work we report results of continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy of vanadium oxide nanotubes. The observed EPR spectra are composed of a weak well-resolved spectrum of isolated V(4+) ions on top of an intense and broad structure-less line shape, attributed to spin-spin exchanged V(4+) clusters. With the purpose to deconvolute the structured weak spectrum from the composed broad line, a new approach based on the Krylov basis diagonalization method (KBDM) is introduced. It is based on the discrimination between broad and sharp components with respect to a selectable threshold and can be executed with few adjustable parameters, without the need of a priori information on the shape and structure of the lines. This makes the method advantageous with respect to other procedures and suitable for fast and routine spectral analysis, which, in conjunction with simulation techniques based on the spin Hamiltonian parameters, can provide a full characterization of the EPR spectrum. Results demonstrate and characterize the coexistence of two V(4+) species in the nanotubes and show good progress toward the goal of obtaining high fidelity deconvoluted spectra from complex signals with overlapping broader line shapes.

Structure and peroxidase activity of ferric Streptomyces clavuligerus orf10-encoded protein P450CLA: UV-visible, CD, MCD and EPR spectroscopic characterization

The present study reports the spectroscopic characterization by UV-visible absorption spectroscopy, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) of the recombinant orf10-encoded P450-camphor like protein (P450CLA)of Streptomyces clavuligerus expressed in Escherichia coli Rosetta in the native form and associated to external ligands containing the β-lactam, oxazole and alkylamine-derived (alcohol) moieties of the clavulamic acid. Considering the diversity of potential applications for the enzyme, the reactivity with tert-butylhydroperoxide (tert-BuOOH) was also characterized. P450CLA presents a covalently bound heme group and exhibited the UV-visible, CD and MCD spectral features of P450CAM including the fingerprint Soret band at 450 nm generated by the ferrous CO-complex. P450CLA was converted to high valence species by tert-BuOOH and promoted homolytic scission of the O-O bond. The radical profile of the reaction was tert-butyloxyl as primary and methyl and butylperoxyl as secondary radicals. The secondary methyl and butylperoxyl radicals resulted respectively from the β-scission of the alkoxyl radical and from the reaction of methyl radical with molecular oxygen.

The Conformational Flexibility of an Internal Fusion Peptide from Sars-Cov Spike Glycoprotein is Modulated by Lipid Membrane Composition

The S2 domain of the spike glycoprotein from SARS-CoV is responsible for driving viral and host cell membrane fusion. An internal fusion peptide (SARSIFP), located near the S2 domain N-terminus, plays a crucial role in the fusion process. Although much information has been obtained in recent years on membrane fusion, many aspects of the molecular mechanisms behind virus-host cell membrane fusion are not totally understood yet. Questions regarding structure, dynamics, and topology of fusion peptides in lipid membranes still remain and are addressed in this work by means of a variety of experimental and computational techniques. The minimum-energy conformation of the peptide was determined by GSA/MD simulations in different conditions. Cluster analysis and free-energy surfaces revealed a conformational promiscuity for SARSIFP, including α-helices, β-strands, and random coil conformations. The distance distribution between the N- and C-termini was also experimentally determined from four-pulse DEER experiments of a doubly labeled derivative. Different membrane mimetics modulated the average peptide end-to-end distance. Moreover, SARSIFP spontaneously adsorbed at the membrane-solvent interface in a random conformation and then forms a helix. Free-energy values and secondary structure content were modulated by the presence of negative membranes, in agreement with our CD and fluorescence data. MD and electron spin resonance (ESR) results indicated a water-exposed C-terminal and a membrane-inserted N-terminal. Spin-labeled lipids and ESR showed that SARSIFP increased lipid packing and head group ordering only in the presence of negatively charged lipids. To our knowledge, this is the first report that provides dynamics, structural and topological information of SARSIFP in model membranes at atomic resolution, which may shed light on the mechanism by which the peptide folds and inserts into membranes.ACKNOLEDGEMENTS: FAPESP, CAPES, CNPq.