AFM Study of Membrane Proteins, Cytochrome P450 2B4, and NADPH–Cytochrome P450 Reductase and Their Complex Formation

Kiselyova, Olga I.; Яминский, И.В.; Ivanov, Yuri D.; Kanaeva, Irina P.; Kuznetsov, Vadim Yu.; Archakov, Alexander I.

doi:10.1006/abbi.1999.1412

Cited by 64 publications

(60 citation statements)

References 15 publications

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“…Morphology of Protein Homo-oligomers-AFM images of mammalian drug-metabolizing P450s dried on mica (46) and in lipid nanodiscs (47) (and references therein) have been reported, as have peptides and other proteins (27)(28)(29)(30)(31). Our high resolution images of steroidogenic P450c17 and P450arom (Fig.…”

Section: Protein-lipid and Protein-protein Interactions Studied By Qcsupporting

confidence: 54%

Organization of Cytochrome P450 Enzymes Involved in Sex Steroid Synthesis

Praporski

Nguyen

et al. 2009

Journal of Biological Chemistry

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Mounting evidence underscores the importance of proteinprotein interactions in the functional regulation of drug-metabolizing P450s, but few studies have been conducted in membrane environments, and none have examined P450s catalyzing sex steroid synthesis. Here we report specific protein-protein interactions for full-length, human, wild type steroidogenic cytochrome P450 (P450, CYP) enzymes: 17␣-hydroxylase/ 17,20-lyase (P450c17, CYP17) and aromatase (P450arom, CYP19), as well as their electron donor NADPH-cytochrome P450 oxidoreductase (CPR). Fluorescence resonance energy transfer (FRET) 3 in live cells, coupled with quartz crystal microbalance (QCM), and atomic force microscopy (AFM) studies on phosphatidyl choline ؎ cholesterol (mammalian) biomimetic membranes were used to investigate steroidogenic P450 interactions. The FRET results in living cells demonstrated that both P450c17 and P450arom homodimerize but do not heterodimerize, although they each heterodimerize with CPR. The lack of heteroassociation between P450c17 and P450arom was confirmed by QCM, wherein neither enzyme bound a membrane saturated with the other. In contrast, the CPR bound readily to either P450c17-or P450arom-saturated surfaces. Interestingly, N-terminally modified P450arom was stably incorporated and gave similar results to the wild type, although saturation was achieved with much less protein, suggesting that the putative transmembrane domain is not required for membrane association but for orientation. In fact, all of the proteins were remarkably stable in the membrane, such that high resolution AFM images were obtained, further supporting the formation of P450c17, P450arom, and CPR homodimers and oligomers in lipid bilayers. This unique combination of in vivo and in vitro studies has provided strong evidence for homodimerization and perhaps some higher order interactions for both P450c17 and P450arom.

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Section: Protein-lipid and Protein-protein Interactions Studied By Qcsupporting

confidence: 54%

Organization of Cytochrome P450 Enzymes Involved in Sex Steroid Synthesis

Praporski

Nguyen

et al. 2009

Journal of Biological Chemistry

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“…To analyze the obtained results, Monte Carlo method (Hammersley and Handscomb, 1975) was used to simulate proteins deposition on carbon nanotubes. The P450 proteins are globular proteins, and their 3-D structure may be included in a sphere of 8 nm (Locuson and Tracy, 2006), as confirmed by direct AFM imaging on P450 samples (Kiselyova et al, 1999). Although the different isoforms of the P450 cytochrome exhibit similar sizes, we need to take into account a more precise estimation for the protein diameter.…”

Section: Size Analysis On Sem Imagesmentioning

confidence: 97%

Multi-panel drugs detection in human serum for personalized therapy

Carrara

Cavallini

Erokhin

et al. 2011

Biosensors and Bioelectronics

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a b s t r a c tThis work focuses on P450 biosensors based on multiwalled carbon nanotubes (MWCNT) and different cytochrome isoforms: 3A4, 2B4, 2C9. The proposed biosensors exhibit enhanced sensitivities and decreased detection limits thanks to carbon nanotubes. The MWCNT structuring improves the sensitivity from 5.1 to 20.5 nA/mM mm 2 in case of CYP2B4-mediated Benzphetamine detection, from 0.26 to 0.63 nA/M mm 2 in case of CYP3A4-mediated Cyclophosphamide detection, and from 0.11 to 0.25 nA/M mm 2 in case of CYP2C9-mediated Naproxen detection. By using MWCNT, the limit of detection was enhanced from 59 to 12 M in case of Cyclophosphamide and from to 187 to 82 M in case of Naproxen. This makes possible the drug detection in human serum within the pharmacological range. In the paper, a new mathematical model is also proposed to succeed in discriminating different drug contributions in a mixture containing both Cyclophosphamide and Dextromethorphan or combining Naproxen and Flurbiprofen. Data analysis shows variations in reduction peaks that are dependent on the drug ratio, and that are consistent with competitive kinetics of substrates. This new approach enables multiple drug detection and opens the way to possible applications in personalized therapy.

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“…Из уравнения 1.2 следует, что F может достигать величины порядка 10 8 при следующих условиях: высота слоя выловленных молекул h=5 нм, что соответствует высоте молекулы белка [35]; объем раствора аналита V=1 мл; площадь поверхности АСМ-чипа S=1 мм 2 . Это означает, что в случае необратимого фишинга из 1 мл раствора аналита с концентрацией 10 -17 М концентрирование молекул с F=10 8 приведёт к увеличению объёмной концентрации 10 -17 М до приповерхностной концентрации 10 -9 М, что значительно облегчит детекцию белков.…”

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“…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].…”

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

IuD

et al. 2014

BIOMED KHIM

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The atomic-force microscopy-based method of irreversible chemical AFM-fishing (AFM-IF ) has been developed for the detection of proteins at ultra-low concentrations in solution. Using this method, a very low concentration of horseradish peroxidase (HRP) protein (10 17 M) was detected in solution. A theoretical model that allows the description of obtained experimental data, is proposed. This model takes into consideration both the transport of the protein from the bulk solution onto the AFM-chip surface and its irreversible binding to the activated area.

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AFM Study of Membrane Proteins, Cytochrome P450 2B4, and NADPH–Cytochrome P450 Reductase and Their Complex Formation

Cited by 64 publications

References 15 publications

Organization of Cytochrome P450 Enzymes Involved in Sex Steroid Synthesis

Organization of Cytochrome P450 Enzymes Involved in Sex Steroid Synthesis

Multi-panel drugs detection in human serum for personalized therapy

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

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