SPR analysis of protein-protein interactions with P450 cytochromes and cytochrome b5 integrated into lipid membrane

Kaluzhskiy L.A.1, Ershov P.V.1 , Kurpedinov K.S.2, Sonina D.S.2, Yablokov E.O.1, Shkel T.V.3, Haidukevich I.V.3, Sergeev G.V.3, Usanov S.A.3, Ivanov A.S.1

1. Institute of Biomedical Chemistry, Moscow, Russia
2. Mendeleev University of Chemical Technology of Russia, Moscow, Russia
3. Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, Belarus
Section: Experimental Study
DOI: 10.18097/PBMC20196505374      PubMed Id: 31666408
Year: 2019  Volume: 65  Issue: 5  Pages: 374-379
Identification of new protein-protein interactions (PPI) and characterization of quantitative parameters of complex formation represent one of central tasks of protein interactomics. This work is a logical continuation of the cycle of our previous works devoted to the study of PPIs among the components of cytochrome P450-dependent monooxygenase system. Using an optical biosensor of Surface Plasmon Resonance (SPR biosensor), a comparative analysis on the determination of kinetic and equilibrium parameters of complex formation between the membrane-bound hemoprotein cytochrome b5 with cytochrome P450s was performed using two different protocols for protein immobilization: 1) covalent non-oriented one on to the carboxymethyl dextran chip type CM and 2) non-covalent oriented immobilization in the lipid environment on the chip type L1 with internal control of liposomes surface distribution. In the second protocol it was shown that the complex formation was characterized by 2.5 times higher affinity due to an decrease in rate dissociation constants. The appropriateness of using both experimental models is discussed.
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Keywords: cytochrome b5, cytochrome P450, liposomes, protein-protein interactions, surface plasmon resonance, biosensor, optical chip CM5 and L1

Kaluzhskiy, L. A., Ershov, P. V., Kurpedinov, K. S., Sonina, D. S., Yablokov, E. O., Shkel, T. V., Haidukevich, I. V., Sergeev, G. V., Usanov, S. A., Ivanov, A. S. (2019). SPR analysis of protein-protein interactions with P450 cytochromes and cytochrome b5 integrated into lipid membrane. Biomeditsinskaya Khimiya, 65(5), 374-379.
This paper is also available as the English translation: 10.1134/S1990750820020067
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