Biomedical Chemistry: Research and Methods 2021, 4(4), e00160

Proteoliposomes as a method of membrane protein immobilization for SPR-analysis with the human CYP3A4 and CYB5A interaction as an example

L.A. Kaluzhskiy1,*, E.O. Yablokov1, M.S. Kisel2, A.M. Tumilovich2, S.A. Usanov2, T.V. Shkel2,
O.V. Gnedenko1, A.S. Ivanov1

1Institute of Biomedical Chemistry, 10/8 Pogodinskaya str., Moscow, 119121 Russia;
2Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus,
5/2 Acad. Kuprevich Street, Minsk, 220141 Belarus

Keywords:surface plasmon resonance; cytochrome P450; cytochrome b5; proteoliposomes


The whole version of this paper is available in Russian.

Microsomal systems of human cytochrome P450 consist of three components, which are membrane proteins: cytochrome P450 hemoprotein (CYP), NADPH-dependent cytochrome P450 reductase (CPR), and a small regulatory heme-containing protein cytochrome b5 (CYB5A). In the study of the cytochrome P450 system functioning the study of intermolecular interactions both with partner proteins and with possible drug prototypes is of great importance. Surface plasmon resonance (SPR) is a powerful and reliable tool for studying intermolecular interactions. However, there is a problem of immobilization of membrane proteins on the optical chip of the SPR biosensor. It is important to immobilize such proteins in native conditions with respect to the correct orientation of the protein globule to the surface of sensor. Previously, we have developed and described a method involving direct native immobilization of membrane proteins into a planar bilayer lipid membrane on the surface of a biosensor chip. At the same time, one of the commonly used approaches to working with membrane proteins using various methods is the construction of proteoliposomes containing membrane proteins. In this work, using CYP3A4 and CYB5A as protein partners, we evaluated two approaches to the creation of proteoliposomes: incorporation of a membrane protein into liposomes saturated with detergents and incorporation of a membrane protein into the forming proteoliposomes by the mechanism of micellar coalescence. The interaction of CYP3A4 with proteoliposomes obtained by incorporating CYB5A into detergent-saturated liposomes. On the contrary, interaction between CYP3A4 and proteoliposomes containing CYB5A, obtained by the method of micellar coalescence, was not detected. Thus, it was shown that the incorporation of the membrane protein into liposomes saturated with a detergent was a more preferable method for working with an SPR biosensor as compared to the method of proteoliposomes formation by micellar coalescence. Detailed protocols for the creation of proteoliposomes and SPR-analysis can be useful to a wide range of researchers.

Figure 1. Sensorgrams of the CYP3A4 interaction with proteoliposomes containing CYB5A. The figure shows the CYP3A4 concentrations used. A: Proteoliposomes with CYB5A were obtained by transferring a membrane protein to detergent-saturated liposomes. B: Proteoliposomes with CYB5A were obtained by micellar coalescence.

Table 1. Reproducibility of immobilization of liposomes and proteoliposomes obtained by the method of incorporating membrane protein into detergent-saturated liposomes and by the method of micellar coalescence. SD is the standard deviation, CV is the coefficient of variation, expressed as a percentage.

Table 2.

Comparison of kinetic (kon, koff) and equilibrium (Kd) constants of CYP3A4 interaction with CYB5A immobilized in different ways. n / i - no interaction.


The work was performed within the financial support of RFBR, grant № 19-04-00485 «SPR analysis of intermolecular interactions involving membrane proteins embedded in the bilayer lipid membrane» using the equipment of «Human Proteome» Core Facility of the Institute of Biomedical Chemistry supported by MINOBRNAUKI, Agreement 075-15-2019-1502 from 5 September 2019.


  1. Krogh, A., Larsson, B., von Heijne, G., Sonnhammer, E.L. (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol., 305(3), 567–580. DOI
  2. Liszewski, K. (2015) Dissecting the Structure of Membrane Proteins. Genetic Engineering & Biotechnology News, 35(17), 1, 14, 16–17. DOI
  3. Overington, J. P., Al-Lazikani, B., Hopkins, A. L. (2006) How many drug targets are there? Nat. Rev. Drug Discov., 5(12), 993–996. DOI
  4. Davydov, D. R. (2011) Microsomal monooxygenase as a multienzyme system: the role of P450-P450 interactions. Expert Opin. Drug Metab. Toxico.l, 7(5), 543–558. DOI
  5. Korošec, T., Ačimovič, J., Seliškar, M., Kocjan, D., Tacer, K.F., Rozman, D., Urleb, U. (2008) Novel cholesterol biosynthesis inhibitors targeting human lanosterol 14α-demethylase (CYP51). Bioorganic & Medicinal Chemistry, 16(1), 209–221. DOI
  6. Recalde-Gil, A.M., Klein-Júnior, L., Salton, J., Bordignon, S., Cechinel-Filho, V., Matté, C., Henriques, A. (2019) Aromatase (CYP19) inhibition by biflavonoids obtained from the branches of Garcinia gardneriana (Clusiaceae). Zeitschrift für Naturforschung C, 74(9–10), 279–282. DOI
  7. Attard, G., Reid, A.H.M., Olmos, D., de Bono, J.S. (2009) Antitumor activity with CYP17 blockade indicates that castration-resistant prostate cancer frequently remains hormone driven. Cancer Res., 69(12), 4937–4940. DOI
  8. Sligar, S.G., Denisov, I.G. (2021) Nanodiscs: A toolkit for membrane protein science. Protein Sci., 30(2), 297–315. DOI
  9. 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 Involving Cytochromes P450 and Cytochrome b5 Integrated into Lipid Membrane. Biomeditsinskaya Khimiya, 65(5), 374–379. DOI
  10. Scalise, M., Pochini, L., Giangregorio, N., Tonazzi, A., Indiveri, C. (2013) Proteoliposomes as tool for assaying membrane transporter functions and interactions with xenobiotics. Pharmaceutics, 5(3), 472–497. DOI
  11. Rigaud, J.-L., Lévy, D. (2003) Reconstitution of Membrane Proteins into Liposomes. Methods in Enzymology, 372, 65–86. DOI
  12. Gilep, A.A., Guryev, O.L., Usanov, S A., Estabrook, R.W. (2001) Apo-cytochrome b5 as an indicator of changes in heme accessability: preliminary studies with cytochrome P450 3A4. J. Inorg. Biochem., 87(4), 237–244. DOI
  13. Sergeev, G.V., Gilep, A.A., Usanov, S.A. (2014) The role of cytochrome b5 structural domains in interaction with cytochromes P450. Biochemistry (Moscow), 79(5), 406–416. DOI
  14. Zhang, H., Gao, N., Liu, T., Fang, Y., Qi, B., Wen, Q., Zhou, J., Jia, L., Qiao, H. (2015) Effect of Cytochrome b5 Content on the Activity of Polymorphic CYP1A2, 2B6, and 2E1 in Human Liver Microsomes. PLoS One, 10(6), e0128547. DOI
  15. Lee, S.-J., Goldstein, J.A. (2012) Comparison of CYP3A4 and CYP3A5: The Effects of Cytochrome b5 and NADPH-cytochrome P450 Reductase on Testosterone Hydroxylation Activities. Drug Metabolism and Pharmacokinetics, 27(6), 663–667. DOI
  16. Verchère, A., Broutin, I., Picard, M. (2017) Reconstitution of Membrane Proteins in Liposomes. Methods Mol Biol, 1635, 259–282. DOI