Atomic force microscopy revelation of molecular complexes in the multiprotein cytochrome P450 2B4‐containing system

Abstract

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.

Irreversible chemical afm-fishing for the detection of low-copied proteins

“…На рисунка 3 представлены распределения по высотам АСМ-изображений молекул авидина, выловленных в процессе АСМ-НФ Хим при С 0 10 -9 и 10 -13 М. Из рисунка 3 видно, что в случае фишинга белка из раствора с С 0 =10 -9 М молекулы имели характеристическую высоту, соответствующую максимуму плотности распределения по высотам с величиной h max =1,8±0,1 нм. Эти результаты хорошо согласуются со значениями высот других белков со сходной молекулярной массой, таких, как d-b5 (M r =50 кДа [58]; h max =1,8±0,1 нм [38]) и d-Fp (M r =78 кДа; h max =2,3±0,2 нм [38]). Из рисунка 3 видно, что при уменьшении С 0 до 10 -13 М высота выловленных объектов становится меньше, h max =1,2±0,1 нм.…”

“…Высокая чувствительность регистрации может быть достигнута при использовании зонда АСМ, имеющего размер, сопоставимый с размером биологической макромолекулы (1-10 нм). Было показано, что АСМ-зонд позволяет осуществлять регистрацию не только вирусных частиц [30], но и отдельных молекул белков [27,28,[35][36][37][38][39][40][41][42][43].…”

Nanotechnologies in proteomics

“…At the same time, these two technologies present interest from the viewpoint of high spatial resolving capacity of biological molecules -in the order of 1 nm and less. The appearance, in recent years, of increasingly numerous studies dealing with the analysis of single molecules and based on the tunnelling and, especially, on the AFM techniques [109][110][111][112][113][114][115][116][117][118][119][120][121][122][123] is explainable. Indeed, the AFM offers a unique ability to easily detect and visualize proteins and their complexes in nearnative conditions while not requiring protein labelling (in contrast to the earlier mentioned optical microscopy techniques) [114].…”

“…In [118] the imaging of the extracellular surface of the protein porin OmpF with a vertical resolution of 0.1 nm and a lateral resolution of up to 0.6 nm was reported. We have undertaken the task to study the microsomal membrane-bound cytochrome P452B4 monoxygenase system involving three membrane proteins: cytochrome P4502B4, cytochrome P450 reductase and cytochrome b5, in the presence of the detergent Emulgen 913; as a result, the binary and ternary complexes of partner proteins were revealed based on the increase of imaged objects' heights upon complex formation [119].…”

Oligomeric state investigation of flavocytochrome CYP102A1 using afm with standard and supersharp probes

“…К методам, позволяющим определять распределение биомолекул по размерам непосредственно в жидкости, относится атомно-силовая микроскопия (АСМ) [6][7][8][9][10][11][12][13][14]. Этот метод находит все большее применение и позволяет визуализировать отдельные белки и их комплексы в условиях, близких к нативным.…”