Unfolding of cytochrome c immobilized on self-assembled monolayers. An electrochemical study.

The TE process of progressively unfolded bovine cytochrome c (cyt c), immobilized on different self-assembled monolayers, was investigated. Direct electrochemical measurements were performed on cyt c adsorbed on mercaptopyridine (MP) and mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol (MUA/MU) at varying temperature, in the presence of urea as unfolding agent. Under strongly unfolding conditions, a non-native form of cyt c, in which the Met ligand is replaced by a His, was observed on both MP and MUA/MU SAMs. The E\ub0 of the native form, in which the haem is axially coordinated by Met and His, slightly shifts to negative values upon increasing urea concentration. However, the non-native bis-His species shows a much lower E\ub0 value (by approximately 0.4 V) which is by far enthalpic in origin and largely determined by axial ligand swapping. Analysis of the reduction enthalpies and entropies and of the ET rate constants indicate that the nature of the SAM (hydrophilic or anionic) results in changes in the conformational rearrangement of the cyt c under unfolding conditions

Immobilized cytochrome c bound to cardiolipin exhibits peculiar oxidation state-dependent axial heme ligation and catalytically reduces dioxygen

Mitochondrial cytochrome c (cytc) plays an important role in programmed cell death upon binding to cardiolipin (CL), a negatively charged phospholipid of the inner mitochondrial membrane (IMM). Although this binding has been thoroughly investigated in solution, little is known on the nature and reactivity of the adduct (cytc-CL) immobilized at IMM. In this work, we have studied electrochemically cytc-CL immobilized on a hydrophobic self-assembled monolayer (SAM) of decane-1-thiol. This construct would reproduce the motional restriction and the nonpolar environment experienced by cytc-CL at IMM. Surface-enhanced resonance Raman (SERR) studies allowed the axial heme iron ligands to be identified, which were found to be oxidation state dependent and differ from those of cytc-CL in solution. In particular, immobilized cytc-CL experiences an equilibrium between a low-spin (LS) 6c His/His and a high-spin (HS) 5c His/- coordination states. The former prevails in the oxidized and the latter in the reduced form. Axial coordination of the ferric heme thus differs from the (LS) 6c His/Lys and (LS) 6c His/O

Laser assisted bioprinting of laminin on biodegradable PLGA substrates: Effect on neural stem cell adhesion and differentiation.

Laser Assisted Bioprinting (LAB) is recognized to be an enabling and versatile microfabrication technology for regenerative medicine and artificial tissue engineering. Current bioprinting concentrates on a layer-by-layer approach to print cells in consecutive stacks or nets, to recreate specialized tissue functions with a top-down approach. This synthering of proximal cells however reduces the long range correlation of tissue parenchyma and stroma given by natural development, as result of cells mobility and signaling. In this work, laminin, one of the main components of brain extracellular matrix is deposited by LAB on a biodegradable scaffold made of poly(lactic-co-glycolic acid) (PLGA), providing chemical cues for the adhesion and differentiation of neural stem cells NE-4C induced by retinoic acid. Surface roughness and LAB induced aggregates promote the initial adhesion of neuronal stem cells to the PLGA substrate and influence the formation of clusters and interconnection between them. The amount of laminin delivered inside the spot area may be controlled down to sub-monolayer coverage and a positive correlation between the laminin spots and soma of trafficking cells is demonstrated, also by computational modelling. Anisotropic orientation of neurite outgrowth is induced upon differentiation, up to 70% of processes protruding from stem cell clusters. The comparative analysis shows that the topological cue plays a major role in enhabling cluster formation on the scaffold, but the bioprinted laminin spots appear to be regulating the strength of connection between them, opening the way to control the functional morphology of artificial neural tissue constructs

Electrochemical data on redox properties of human Cofilin-2 and its Mutant S3D

The reported data are related to a research paper entitled "Phosphorylated cofilin-2 is more prone to oxidative modifications on Cys39 and favors amyloid fibril formation" [1]. Info about the formation and redox properties of the disulfide bridge of a protein is quite difficult to obtain and only in a few cases was it possible to observe a cyclic voltammetry (CV) signal [2,3]. Human cofilin-2 contains two cysteines (Cys39 and Cys80) which can be oxidized in suitable conditions and form a disulfide bridge [1]. For this purpose, CV measurements were carried out on human cofilin-2 WT and its mutant S3D immobilized on a gold electrode coated by an anionic self-assembled monolayer (SAM), after a pre-oxidation time which was fundamental for observing a CV signal relating to the oxidation/reduction process of the disulfide bridge of the proteins. The data include CV curves obtained with and without electrochemical pre-oxidation and after oxidation with H2O2. In addition, the plot of the cathodic peak current vs. electrochemical pre-oxidation time and the pH dependence of the formal potential (E\ub0\u2019) are reported. The data obtained by CV measurements were used to determine the time required to form the disulfide bridge for the immobilized proteins and, consequently, to observe the CV signal, to calculate the E\ub0\u2019 values and analyse the pH dependence of E\ub0\u2019. The electrochemical data were provided which will be useful for further electrochemical investigations regarding proteins bearing disulfide bridge(s) or cysteines prone to oxidation

Phosphorylated cofilin-2 is more prone to oxidative modifications on Cys39 and favors amyloid fibril formation

Cofilins are small protein of the actin depolymerizing family. Actin polymerization/depolymerization is central to a number of critical cellular physiological tasks making cofilin a key protein for several physiological functions of the cell. Cofilin activity is mainly regulated by phosphorylation on serine residue 3 making this post-translational modification key to the regulation of myofilament integrity. In fact, in this form, the protein segregates in myocardial aggregates in human idiopathic dilated cardiomyopathy. Since myofilament network is an early target of oxidative stress we investigated the molecular changes induced by oxidation on cofilin isoforms and their interplay with the protein phosphorylation state to get insight on whether/how those changes may predispose to early protein aggregation. Using different and complementary approaches we characterized the aggregation properties of cofilin-2 and its phosphomimetic variant (S3D) in response to oxidative stress in silico, in vitro and on isolated cardiomyocytes. We found that the phosphorylated (inactive) form of cofilin-2 is mechanistically linked to the formation of an extended network of fibrillar structures induced by oxidative stress via the formation of a disulfide bond between Cys39 and Cys80. Such phosphorylation-dependent effect is likely controlled by changes in the hydrogen bonding network involving Cys39. We found that the sulfide ion inhibits the formation of such structures. This might represent the mechanism for the protective effect of the therapeutic agent Na2S on ischemic injury

A Bacterial Photosynthetic Enzymatic Unit Modulating Organic Transistors with Light

The photochemical core of every photosynthetic apparatus is the reaction center, a transmembrane enzyme that converts photons into charge-separated states across the biological membrane with an almost unitary quantum yield. A light-responsive organic transistor architecture, which converts light into electrical current by exploiting the efficiency of this biological machinery, is presented. Proper surface tailoring enables the integration of the bacterial reaction center as photoactive element in organic transistors, allowing the transduction of its photogenerated voltage into photomodulation of the output current up to two orders of magnitude. This device architecture, termed light-responsive electrolyte-gated organic transistor, is the prototype of a new generation of low-power hybrid bio-optoelectronic organic devices