Cristina Paternolli

Bacteriorhodopsin-based Langmuir-Schaefer films for solar energy capture

The photovoltaic (PV) solar cell, converting incident solar radiation directly into electrical energy, today represents the most common power source for the Earth-orbiting spacecraft, and the utilization of organic materials in this context is here explored in comparison with the present state of the art placing emphasis in organic nanotechnology. Poly[3-3(vinylcarbazole)] (PVK) was synthesized by oxidative polymerization with ferric chloride of N-vinylcarbazole. The resulting polymer was then deposited on solid support by using the Langmuir-Schaefer (LS) technique. The pressure-area isotherm of PVK revealed the possibility of compact monolayer formation at the air-water interface. Different layers of PVK were doped with iodine vapors. The cyclic voltammetry investigation of PVK-doped I/sub 2/ showed a distinctive electrochemical behavior. The photoinduced charge transfer across a donor/acceptor (D/A) hybrid interface provided an effective method to study the PV properties of the composite LS films. The results are compared with other approaches within the biological framework, such as bacteriorhodopsin (BR), and organic nanostructured materials.

Electrochemical study of the interaction between cytochrome P450sccK201E and cholesterol

Cytochrome P450sccK201E, mutated form of cytochrome P450scc native recombinant (P450sccNR), was employed to study the enzyme-substrate interaction. The detection of the cholesterol was performed by electrochemical method using cyclic voltammetry (CV) and chronoamperometry measurements. The biochemical analysis was realized to observe the electrochemical responses of the engineerized enzyme to three different forms of cholesterol: free, low-density lipoprotein (LDL) and high-density lipoproteins (HDL). Compared to cytochrome P450sccNR, the cytochrome P450sccK201E displays a different behavior in the interaction with the substrate detection. The results show that the engineerized enzyme can be utilized for the cholesterol detection in biosensor field.

Pollutant sensing layer based on cytochrome P450

Abstract In this work, cytochrome P4502B4 fusion protein was utilized to realize sensing layers for detection of styrene in atmosphere. Glutathione S-transferase (GST) fusion protein was utilized to realize thin films by Langmuir–Blodgett (LB) horizontal transfer technique. The interaction between the styrene and the sensing layer was monitored by spectrophotometric and by gravimetric measurements. A shift of the Soret peak of cytochrome P450 in the presence of the substrate was found when absorbance measurements were performed. Index of spin state equilibrium variation of P4502B4 was calculated in order to verify the interaction between styrene and cytochrome. A saturation trend in mass density was found when quartzes nanobalance was utilized.

Toward bacteriorhodopsin based photocells

Bacteriorhodopsin from purple membrane has unique photochromic and photoelectric properties and a capability of providing light-induced proton pumping. These features make it one of the most promising biological molecules for developing a bioelectronic photocell. In a recent work, we have shown that the BR molecules can be oriented by applying, with the traditional Langmuir–Blodgett technique, an electric field at the air-water interface. This work is aimed at the development of a BR-based photocell. The monolayers deposited by the electric field-assisted LB technique were further characterized by Brewster Microscopy at the air-water interface prior to their transfer onto solid supports. The photoresponse measurements of such a BR-based photocell confirmed that the BR molecules in the film were oriented. For suggested possible applications, molecular electrodes were fabricated by depositing thin films of doped poly(o-anisidine) (POAS) conducting polymer.

Photoreversibility and photostability in films of octopus rhodopsin isolated from octopus photoreceptor membranes

In this work, a new biomaterial resulting from the isolation of octopus rhodopsin (OR) starting from octopus photoreceptor membranes is presented. Mass spectroscopic characterization was employed in order to verify the presence of rhodopsin in the extract. Photoreversibility and photochromic properties were investigated using spectrophotometric measurements and pulsed light. Thin films of OR were realized using the gel-matrix entrapment method in polyvinyl alcohol solution. The results indicate that the photoreversibility and the photostability of the OR in gel-matrices are maintained. Several measurements were performed to test the stability of the resulting biomaterial in time and at room temperature. Preliminary tests demonstrate that the photoreversibility and the photostability are still found after few days from the biomaterial preparation and after the exposure for several hours at room temperature.

Development of immobilization techniques of cytochrome P450-GST fusion protein

Abstract Glutathione S-transferase (GST) fusion cytochrome P4502B4 enzyme obtained by genetic engineering was used in order to optimize the immobilization of the proteins on solid supports. Langmuir–Schaefer, ‘layer-by-layer (LbL)’ and self-assembling techniques were used to form thin films on solid surfaces. In particular, it was studied the possibility to realize alternated structures stabilized by binding affinity between GST-fusion protein and glutathione (GSH) using ‘LbL’ techniques. The characterization of the films was performed by means of π-A isotherms, Brewster angle microscopy and spectrophotometry. Preliminary analysis of the P4502B4 films functionality was realized monitoring the spin-state of the cytochrome P450 by spectrophotometric measurements.

P450scc mutant nanostructuring for optimal assembly

Molecular modeling and protein engineering were synergically employed to improve the fabrication of cytochrome P450scc mutant nanostructures for biodevice assembly. The optimization of protein three-dimensional structure by molecular modeling was performed using two models: in vacuum and simulating the presence of a polar solvent. Calculations were performed on a model to predict a P450scc mutant which could improve the process of molecules immobilization onto solid supports. Engineerized cytochrome P450scc thin films were prepared and characterized by various biophysical techniques such as /spl pi/-A isotherms, surface potential measurements, Brewster angle microscopy, UV-vis spectroscopy, circular dichroism, nanogravimetry, and electrochemical analysis. This paper takes into consideration biomolecules modified by protein engineering that represent a new and powerful approach for obtaining synthetic simpler artificial structures with new or improved properties (i.e., specificity, stability, sensitivity, etc.) useful for biosensors development.

Spin state transitions in Langmuir-Blodgett films of recombinant cytochrome P450scc and adrenodoxin

Abstract Langmuir–Blodgett (LB) films of recombinant cytochrome P450scc, of P450scc–adrenodoxin (Adx) complex and alternated layers of Adx and P450scc have been obtained. Spectral properties of these proteins in thin films were investigated by UV–Vis absorption spectroscopy. It has been found that cytochrome P450scc exists in LB films only in low-spin state while before the deposition it was in high-spin state. The data suggest that transferring the hemoprotein or its complex with redox partner results in the modification of the spin state by a conformational transition. In order to investigate further the P450scc and Adx interaction, the mass density of the films formed from these molecules has been studied by nanogravimetric measurements. Comparative study between nanogravimetric and spectral characterisation was performed. The results indicate that the protein–protein interaction is disrupted, when the complex is organised in thin film.

Site-directed mutations (Asp405Ile and Glu124Ile) in cytochrome P450scc: effect on adrenodoxin binding.

Cytochrome P450scc, mitochondrial adrenodoxin (Adx), and adrenodoxin reductase (AdR) are an essential components in a steroid hydroxylation system. In particular, mytochondrial cytochrome P450scc enzyme catalyses the first step in steroid hormones biosynthesis, represented by the conversion of cholesterol to pregnenolone. In order to study the effect of single mutations on the Adx binding a model of bovine cytochrome P450scc, previously optimized by molecular modeling, was utilized. It was hypothesized by molecular docking that two residues (Asp405 and Glu124) are involved in Adx binding. By site‐directed mutagenesis, two mutants of cytochrome P450scc (Asp405Ile and Glu124Ile) expressed in Escherichia coli, were realized by replacing with isoleucines. The site‐directed mutations effect on Adx binding was evaluated by differential spectral titration. The apparent dissociation constant values for Asp405Ile and Glu124Ile cytochrome P450scc show that the mutated residues seem to be at the interaction domain with Adx or at least close to it, as predicted by molecular modeling study. Finally, the engineered enzymes were characterized by biochemical and biophysical techniques such as circular dichroism (CD), UV/Vis spectroscopy, and electrochemical analysis.