Skip to main content
European Commission logo print header
Zawartość zarchiwizowana w dniu 2022-12-23

Optical biosensor studies of protein-protein recognitions in cytochromes P450-containing monooxygenase systems under hydroxylating conditions

Cel

The project is aimed at measuring of kinetic rate constants of complex formation within reconstituted cytochrome P450-containing monooxygenase systems in hydroxylation reaction conditions.

Three systems will be analysed: the cytosolic cytochrome P450cam system, containing only water soluble proteins; the membrane-bound cytochrome P4502B4 system, containing only membrane bound proteins; and the mixed cytochrome P450scc- system, containing both water-soluble and membrane proteins. The complex formation kinetic parameters, as measured for the redox partners of the three systems in conditions of hydroxylation reactions, presents entirely new scientific information; comparison of these kinetic parameters with the inter-protein electron transfer time and with the time required for a complete hydroxylation cycle will make it possible to construct a model (either ternary or shuttle mechanism) describing the operation of the P450cam, the P4502B4 and the P450scc systems.

The registration of binary and ternary complexes between redox partners in these systems and determination of association and dissociation rate constants for the complexes formed will be attained by the optical biosensor technique using an IAsys "resonant mirror" biosensor - Affinity Sensors Division of Labsystems, UK) in real time without protein labelling. The following procedure is proposed. One of the protein partners is immobilized via carboxylate groups of dextran layer of the biosensor's support - which, being coupled with the prism, serves as a wave-guide. The other protein partner is then added to the cuvette in reconstituted-system conditions. With the complex formation process-taking place, the refraction coefficient in the dextran layer is beginning to change; these changes are monitored in real time by the resonant angle shift of the probe laser beam propagating along the wave-guide. Comparison of the complex lifetime with the inter protein electron transfer time allows the conclusion to be drawn as to the functional capability of each particular complex.

Zaproszenie do składania wniosków

Data not available

System finansowania

Data not available

Koordynator

National Institute of Health and Medical Research (INSERM)
Wkład UE
Brak danych
Adres
84 rue du G?n?ral Leclerc
94276 Le Kremlin Bic?tre
Francja

Zobacz na mapie

Koszt całkowity
Brak danych

Uczestnicy (3)