Development of Nanobiosensor Based on a Lipid Biomembrane for Study of its Interaction with Cytochrome b₅ Protein

Abstract

The article proposes an experimental setup of a biosensor with a sensitive element based on a biomembrane made of the phospholipid 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) to study its interaction with the membrane protein cytochrome b₅. The electrophysical characteristics of the biomembrane are being investigated. The created setup makes it possible to register the interaction of a biomembrane (which is a phospholipid bilayer formed in a biosensor) with a membrane protein by measuring the electrical conductivity of the biomembrane. The electrical conductivity of the membrane is determined by the presence of protein and its absence. It is experimentally demonstrated that such a biosensor makes it possible to determine the interaction of cytochrome b₅ with the phospholipid membrane in real time. Using the installation, it is determined that the full-size cytochrome b₅ (d-b₅) protein, containing a hydrophobic domain in the structure, significantly affects the membrane conductivity, leading to the appearance of conduction channels. In the absence of cytochrome b₅ membrane fragment (t-b₅), protein interaction with the lipid layer is practically not observed. The results can be used to create biosensors based on lipid layers to simulate the interaction of proteins with lipid membranes in the human body

The work was carried out with the support of the Ministry of Science and Higher Education of the Russian Federation (agreement no. 075-15-2024-643)

Please cite this article in English as:

Ableev A.N., Shumov I.D., Shumyantseva V.V., et al. Development of nanobiosensor based on a lipid biomembrane for study of its interaction with cytochrome b₅ protein. Herald of the Bauman Moscow State Technical University, Series Natural Sciences, 2026, no. 1 (124), pp. 97--117 (in Russ.). EDN: VOTYNO

References

[1] Khan M.S., Dosoky N.S., Williams J.D. Engineering lipid bilayer membranes for protein studies. Int. J. Mol. Sci., 2013, vol. 14, iss. 11, pp. 21561--21597. DOI: https://doi.org/10.3390/ijms141121561

[2] Robinson M., Filice C.T., McRae D.M., et al. Atomic force microscopy and other scanning probe microscopy methods to study nanoscale domains in model lipid membranes. Adv. Phys. X, 2023, vol. 8, iss. 1, art. 2197623. DOI: https://doi.org/10.1080/23746149.2023.2197623

[3] Singer S.J. Architecture and topography of biologic membranes. Hosp. Pract., 1973, vol. 8, no. 5, pp. 81--90. DOI: https://doi.org/10.1080/21548331.1973.11707910

[4] Carey P.R. Biochemical applications of Raman and Resonance spectroscopies. Academic Press, 1982.

[5] Viles J.H. Imaging amyloid-β membrane interactions: ion-channel pores and lipid-bilayer permeability in Alzheimer’s disease. Angew. Chem. Int. Ed., 2023, vol. 62, no. 25, art. e202215785. DOI: https://doi.org/10.1002/anie.202215785

[6] Rascol E., Devoisselle J.-M., Chopineau J. The relevance of membrane models to understand nanoparticles--cell membrane interactions. Nanoscale, 2016, vol. 8, no. 9, pp. 4780--4798. DOI: https://doi.org/10.1039/C5NR07954C

[7] Broda J., Setzler J., Leifert A., et al. Ligand-lipid and ligand-core affinity control the interaction of gold nanoparticles with artificial lipid bilayers and cell membranes. Nanomed. Nanotechnol. Biol. Med. 2016, vol. 12, no. 5, pp. 1409--1419. DOI: https://doi.org/10.1016/j.nano.2015.12.384

[8] Tuominen E.K.J., Wallace C.J.A., Kinnunen P.K.J. Phospholipid-cytochrome c interaction: evidence for the extended lipid anchorage. J. Biol. Chem., 2002, vol. 277, no. 11, pp. 8822--8826. DOI: https://doi.org/10.1074/jbc.M200056200

[9] Bernabeu A., Contreras L.M., Villalain J. Two-dimensional infrared correlation spectroscopy study of the interaction of oxidized and reduced cytochrome c with phospholipid model membranes. Biochim. Biophys. Acta Biomembr., 2007, vol. 1768, iss. 10, pp. 2409--2420. DOI: https://doi.org/10.1016/j.bbamem.2007.05.002

[10] Archakov A.I., Bachmanova G.I. Cytochrome P450 and active oxygen. Taylor & Francis, 1990.

[11] Belikova N.A., Vladimirov Yu.A., Osipov O.N., et al. Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes. Biochemistry, 2006, vol. 45, iss. 15, pp. 4998--5009. DOI: https://doi.org/10.1021/bi0525573

[12] Cecconi C., Shank E.A., Bustamante C., et al. Direct observation of the three-state folding of a single protein molecule. Science, 2005, vol. 309, pp. 2057--2060. DOI: https://doi.org/10.1126/science.1116702

[13] Kuznetsov V.Yu., Ivanov Yu.D., Archakov A.I. Atomic force microscopy revelation of molecular complexes in the multiprotein cytochrome P450 2B4-containing system. Proteomics, 2004, vol. 4, iss. 8, pp. 2390--2396. DOI: https://doi.org/10.1002/pmic.200300751

[14] Lv Z., Banerjee S., Zagorski K., et al. Supported lipid bilayers for atomic force microscopy studies. In: Lyubchenko Y. (eds). Nanoscale Imaging. Methods in Molecular Biology, vol. 1814. New York, NY, Humana Press, 2018, pp. 129--143. DOI: https://doi.org/10.1007/978-1-4939-8591-3_8

[15] Mingeot-Leclercq M.P., Deleu M., Brasseur R., et al. Atomic force microscopy of supported lipid bilayers. Nat. Protoc., 2008, vol. 3, pp. 1654--1659. DOI: https://doi.org/10.1038/nprot.2008.149

[16] Wu J., Yamashita T., Hamilton A.D., et al. Single-molecule nanopore dielectrophoretic trapping of a-synuclein with lipid membranes. Cell Rep. Phys. Sci., 2023, vol. 4, art. 101243. DOI: https://doi.org/10.1016/j.xcrp.2022.101243

[17] Zhang H., Gao N., Liu T., et al. Effect of cytochrome b₅ content on the activity of polymorphic CYP1A2, 2B6, and 2E1 in human liver microsomes. PLoS ONE, 2015, vol. 10, no. 6, art. e0128547. DOI: https://doi.org/10.1371/journal.pone.0128547

[18] Zhang M., Le Clair S.V., Huang R., et al. Insights into the role of substrates on the interaction between cytochrome b₅ and cytochrome P450 2B4 by NMR. Sci. Rep., 2015, vol. 5, art. 8392. DOI: https://doi.org/10.1038/srep08392

[19] Simonov A.N., Holien J.K., Yeung J.C.I., et al. Mechanistic scrutiny identifies a kinetic role for cytochrome b₅ regulation of human cytochrome P450c17 (CYP17A1, P450 17A1). PLoS ONE, 2015, vol. 10, no. 11, art. e0141252. DOI: https://doi.org/10.1371/journal.pone.0141252

[20] Kleinfeld A.M., Lukacovic M.F. Energy-transfer study of cytochrome b₅ using the anthroyloxy fatty acid membrane probes. Biochemistry, 1985, vol. 24, iss. 8, pp. 1883--1890. DOI: https://doi.org/10.1021/bi00329a012

[21] Scott E.E., Wolf C.R., Otyepka M., et al. The role of protein-protein and protein-membrane interactions on P450 function. Drug Metab. Dispos., 2016, vol. 44, no. 4, pp. 576--590. DOI: https://doi.org/10.1124/dmd.115.068569

[22] Mustafa G., Nandekar P.P., Bruce N.J., et al. Differing membrane interactions of two highly similar drug-metabolizing cytochrome P450 isoforms: CYP 2C9 and CYP 2C19 Int. J. Mol. Sci., 2019, vol. 20, iss. 18, art. 4328. DOI: https://doi.org/10.3390/ijms20184328

[23] Spatz L., Strittmatter P. A form of cytochrome b₅ that contains an additional hydrophobic sequence of 40 amino acid residues. Proc. Natl. Acad. Sci. USA, 1971, vol. 68, no. 5, pp. 1042--1046. DOI: https://doi.org/10.1073/pnas.68.5.1042

[24] Omura T., Takesue S. A new method for simultaneous purification of cytochrome b₅ and NADH-cytochrome b₅ reductase from rat liver microsomes. J. Biochem., 1970, vol. 67, iss. 2, pp. 249--257. DOI: https://doi.org/10.1093/oxfordjournals.jbchem.a129248

[25] Bachmanova G.I., Kanaeva I.P., Stepanova N.V., et al. [Role of cytochrome b₅ in microsomal soluble reconstituted monooxygenase system]. Abstracts of 9th ICCP450, Zurich, 1995, p. 232.

[26] Johnson M.E., Simon S., Kauffman J.W., et al. A synthetic lecithin containing branched-chain fatty acids: physical properties and membrane studies. Biochim. Biophys. Acta Biomembr., 1973, vol. 291, iss. 3, pp. 587--591. DOI: https://doi.org/10.1016/0005-2736(73)90463-X

[27] Wang S., Larson R.G. Water channel formation and ion transport in linear and branched lipid bilayers. Phys. Chem. Chem. Phys., 2014, vol. 16, no. 16, pp. 7251--7262. DOI: https://doi.org/10.1039/C3CP55116D

[28] Tristram-Nagle S., Kim D.J., Akhunzada N., et al. Structure and water permeability of fully hydrated diphytanoylPC. Chem. Phys. Lipids, 2010, vol. 163, iss. 6, pp. 630--637. DOI: https://doi.org/10.1016/j.chemphyslip.2010.04.011

[29] Vorobyov I., Olson T.E., Kim J.H., et al. Ion-induced defect permeation of lipid membranes. Biophys. J., 2014, vol. 106, iss. 3, pp. 586--597. DOI: http://dx.doi.org/10.1016/j.bpj.2013.12.027

[30] Xiang T.X., Anderson B.D. Influence of a transmembrane protein on the permeability of small molecules across lipid membranes. J. Membrane Biol., 2000, vol. 173, iss. 3, pp. 187--201. DOI: https://doi.org/10.1007/s002320001019