Monolayer engineering of P450scc cytochrome for bioelectronics
Paola Ghisellini1,*, Sergio Paddeu2, Victor
Erokhin2, Cristina Paternolli1, Ilaria Chiossone3,
Manoj K. Ram3 and Claudio Nicolini1
1 Institute
of Biophysics, Faculty of Medicine and Surgery, University of Genoa,
Corso Europa 30, Italy.
2 Fondazione EL.B.A.
- Corso Europa 30, 16132 - Genova, Italy.
3 Polo Nazionale Bioelettronica,
Via Roma, 28 - 57030 Marciana (LI), Italy.
Cytochromes P450 are a class of enzymes which play an important role catalyzing
several biochemical reactions in nature, such as steroid oxidations (1)
and cellular detoxification of xenobiotic compounds. In this work a kind
of cytochromes which catalyze the key stereogenic reaction of cholesterol
side cleavage to form pregnenolone (2) were utilized, namely P450scc. Cytocrome
P450scc was expressed in E.coli bacteria in order to provide high purity
and yield of the protein (3). The purification procedure was carried out
by using two different detergents, namely sodium cholate and n-octyl-[beta]-D-glucopyranoside,
in order to optimize the thin film formation.
P450 thin films were fabricated by the Langmuir-Blodgett (horizontal)
(4) and the layer by layer (5) techniques. The films were structurally
and functionally characterized in order to utilize them toward the development
of bioelectronic devices, namely biosensors and diagnostic kits. [pi]-A
isotherms and Brewster angle microscopic measurements allowed one to study
the Langmuir film formation and its morphology at the air/water interface,
whereas UV/Vis spectroscopic and X-ray synchrotron radiation measurements
provided evidence about the functionality and the molecular organization
respectively in films transferred onto solid substrates. Langmuir monolayer
formation was also optimized by the selection of the proper subphase composition.
It was possible to assess that the P450 formed stable Langmuir
films and that the molecules were closely packed overcoming the surface
pressure of 20 mN/m. Surface density of the transferred layers was estimated
from the gravimetric measurements with quartz nanobalance. The comparison
of these results with the protein sizes allowed to make a conclusion about
the close packing of the protein molecules in the layer. Two types of samples
were realized for the investigation of the film structure and function.
The first one was a direct overlapping of P450 monolayers, while the second
one was a heterostructure, where protein monolayers were alternated with
polymer and fatty acid monolayers. Alternation with conducting polymers,
provide direct access to the protein molecules. Conversely, the alternation
with fatty acid monolayers was carried out in order to organize more oriented
structures for further investigation with X-ray diffraction techniques.
It was shown the possibilty of the realization of stable homogeneous
layers of cytochrome P450scc. It was also demonstrated the increased thermal
stability of the secondary structure of this protein in the film. The functionality
of the protein in the film was studied by means of CO-differential spectroscopy
and electrochemical measurements.
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