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Atomic force microscopy revelation of molecular complexes in the multiprotein cytochrome P450 2B4‐containing system
Author(s) -
Kuznetsov Vadim Yu.,
Ivanov Yuri D.,
Archakov Alexander I.
Publication year - 2004
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200300751
Subject(s) - cytochrome b5 , chemistry , ternary operation , molecule , monomer , crystallography , cytochrome p450 , enzyme , biochemistry , organic chemistry , polymer , computer science , programming language
The application of atomic force microscopy (AFM) to the identification and visualization of individual molecules and their complexes in a reconstituted monooxygenase P450 2B4 system without the phospholipid was demonstrated. The method employed in this study distinguishes the monomeric proteins from their binary complexes and, also, the binary from the ternary complexes. The AFM images of the full‐length P450 2B4 system's constituent components – cytochrome P450 2B4 (2B4), NADPH‐cytochrome P450 reductase and cytochrome b5 (b5), were obtained on highly‐oriented pyrolitic graphite. The typical heights of the d‐2B4, d‐flavoprotein (Fp) and d‐b5 molecules were measured and found to be 2.2 ± 0.2, 2.3 ± 0.2 and 1.8 ± 0.1 nm, respectively. The measured heights of the binary d‐Fp/d‐2B4 and d‐2B4/d‐b5 complexes were estimated to be 3.4 ± 0.2 and 2.8 ± 0.2 nm, respectively. No formation of d‐Fp/d‐b5 complexes was registered. The ternary d‐Fp/d‐2B4/d‐b5 complexes were visualized and their heights were found to be roughly equal to 4.3 ± 0.3 nm and 6.2 ± 0.3 nm.