Surface Immobilized His-tagged Azurin as a Model Interface for the Investigation of Vectorial Electron Transfer in Biological Systems

A model system for the electrochemical investigation of vectorial electron transfer in biological systems was designed, assembled and characterized. Gold electrodes, functionalized with a -OCH3 terminated, aromatic self-assembled monolayer, were used as a substrate for the adsorption of variants of copper- containing, redox metalloprotein azurin. The engineered azurin bears a polyhistidine tag at its C-terminus. Thanks to the presence of the solvent exposed tag, which chelates Cu2+ ions in solution, we introduced an exogenous redox centre. The different reduction potentials of the two redox centres and their positioning with respect to the surface are such that electron transfer from the exogenous copper centre and the electrode is mediated by the native azurin active site, closely paralleling electron transfer processes in naturally occurring multicentre metalloprotein

Pre-amyloid oligomers budding:a metastatic mechanism of proteotoxicity

The pathological hallmark of misfolded protein diseases and aging is the accumulation of proteotoxic aggregates. However, the mechanisms of proteotoxicity and the dynamic changes in fiber formation and dissemination remain unclear, preventing a cure. Here we adopted a reductionist approach and used atomic force microscopy to define the temporal and spatial changes of amyloid aggregates, their modes of dissemination and the biochemical changes that may influence their growth. We show that pre-amyloid oligomers (PAO) mature to form linear and circular protofibrils, and amyloid fibers, and those can break reforming PAO that can migrate invading neighbor structures. Simulating the effect of immunotherapy modifies the dynamics of PAO formation. Anti-fibers as well as anti-PAO antibodies fragment the amyloid fibers, however the fragmentation using anti-fibers antibodies favored the migration of PAO. In conclusion, we provide evidence for the mechanisms of misfolded protein maturation and propagation and the effects of interventions on the resolution and dissemination of amyloid pathology

Thermodynamics and Kinetics of Electron Transfer of 2 Electrode-Immobilized Small Laccase from Streptomyces coelicolor

The thermodynamic and kinetic properties for the heterogeneous electron transfer (ET) were measured for the electrode-immobilized small laccase (SLAC) from Streptomyces coelicolor subjected to different electrostatic and covalent protein-electrode linkages, using cyclic voltammetry. Once immobilized electrostatically onto a gold electrode using mixed carboxyl- and hydroxy-terminated alkane-thiolate SAMs or covalently exploiting the same SAM subjected to N-hydroxysuccin-imide+1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS-EDC) chemistry, the SLAC-electrode electron flow occurs through the T1 center. The E°’ values (from +0.2 to +0.1 V vs. SHE at pH 7.0) are lower by more than 0.2 V compared to the protein either in solution or immobilized with different anchoring strategies using uncharged SAMs. For the present electrostatic and covalent binding, this effect can respectively be ascribed to the negative charge of the SAM surfaces and to deletion of the positive charge of Lys/Arg residues due to amide bond formation which both selectively stabilize the more positively charged oxidized SLAC. Observation of enthalpy/entropy compensation within the series indicates that the immobilized proteins experience different reduction-induced solvent reorganization effects. The E°’ values for the covalently attached SLAC are sensitive to three acid base equilibria, with apparent pKa values of pKa1ox =5.1, pKa1red=7.5, pKa2ox=8.4, pKa2red=10.9, pKa2ox=8.9, pKa2red=11.3 possibly involving one residue close to the T1 center and two residues (Lys and/or Arg) along with moderate protein unfolding, respectively. Therefore, the E°’ value of immobilized SLAC turns out to be particularly sensitive to the anchoring mode and me-30 dium conditions

Assessing the functional and structural stability of the Met80Ala mutant of cytochrome c in dimethylsulfoxide

The Met80Ala variant of yeast cytochrome c is known to possess electrocatalytic properties that are absent in the wild type form and that make it a promising candidate for biocatalysis and bi-osensing. The versatility of an enzyme is enhanced by the stability in mixed aqueous/organic solvents that would allow poorly water-soluble substrates to be targeted. In this work, we have evaluated the effect of dimethylsulfoxide (DMSO) on the functionality of the Met80Ala cyto-chrome c mutant, by investigating the thermodynamics and kinetics of electron transfer in mixed water/DMSO solutions up to 50% DMSO v/v. In parallel, we have monitored spectroscop-ically the retention of the main structural features in the same medium, focusing on both the overall protein structure and the heme center. We found that the organic solvent exerts only minor effects on the redox and structural properties of the mutant mostly as a result of the mod-ification of the dielectric constant of the solvent. This would warrant proper functionality of this variant also under these potentially hostile experimental conditions, that differ from the physi-ological milieu of cytochrome c

Insights into heme degradation and hydrogen peroxide induced dimerization of human neuroglobin.

In this work, we investigated the H2O2-induced oligomerization of wild-type human neuroglobin (hNgb) and of some selected variants (C46AC55A, Y44A, Y44F, Y44AC46AC55A, Y44AC46AC55A) to clarify how the process is affected by the Cys46/Cys55 disulfide bond and the distal H-bonding network and to figure out the molecular determinants of the H2O2-induced formation of amyloid type structures and hNgb aggregates. It turns out that hydrogen peroxide exerts a twofold effect on hNgb, inducing both heme breakdown and protein dimerization/polymerization. The enhanced resistance to the oxidizing effect of H2O2 of the disulfide free variants indicates that both effects are strictly influenced by the heme accessibility for H2O2. Most importantly, the H2O2-induced neuroglobin dimerization/polymerization turns out to be triggered by tyrosyl radicals resulting from the oxidizing action of Compound I ([Por•Fe(IV)=O]+). Peptide mapping indicates that the H2O2-induced dimerization/polymerization of hNgb mainly involves Tyr44, which forms covalent bonds with all the other tyrosine residues, with a minor contribution from Tyr88. The presented findings contribute further important pieces of information in the quest of identifying all capabilities of human neuroglobin and ultimately its physiological task