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In the present study, the possibility of hepatitis C virus core antigen (HCVcoreAg) detection in buffer solution, using atomic force microscope chip (AFM-chip) with immobilized aptamers, has been demonstrated. The target protein was detected in 1mL of solution at concentrations from 10 -10 М to 10 -13 М. The registration of aptamer/antigen complexes on the chip surface was carried out by atomic force microscopy (AFM). The further mass-spectrometric (MS) identification of AFM-registered objects on the chip surface allowed reliable identification of HCVcoreAg target protein in the complexes. Aptamers, which were designed for therapeutic purposes, have been shown to be effective in HCVcoreAg detection as probe molecules., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Atomic force microscopy (AFM) and photon correlation spectroscopy (PCS) were used for monitoring of the procedure for cytochrome CYP11A1 monomerization in solution without phospholipids. It was shown that the incubation of 100 μM CYP11A1 with 12% Emulgen 913 in 50 mM KP, pH 7.4, for 10 min at T = 22°C leads to dissociation of hemoprotein aggregates to monomers with the monomerization degree of (82 ± 4)%. Following the monomerization procedure, CYP11A1 remained functionally active. AFM was employed to detect and visualize the isolated proteins as well as complexes formed between the components of the cytochrome CYP11A1-dependent steroid hydroxylase system. Both Ad and AdR were present in solution as monomers. The typical heights of the monomeric AdR, Ad and CYP11A1 images were measured by AFM and were found to correspond to the sizes 1.6 ± 0.2 nm, 1.0 ± 0.2 nm and 1.8 ± 0.2 nm, respectively. The binary Ad/AdR and AdR/CYP11A1mon complexes with the heights 2.2 ± 0.2 nm and 2.8 ± 0.2 nm, respectively, were registered by use of AFM. The Ad/CYP11A1mon complex formation reaction was kinetically characterized based on optical biosensor data. In addition, the ternary AdR/Ad/CYP11A1 complexes with a typical height of 4 ± 1 nm were AFM registered.

A real-time optical biosensor study on the interactions between putidaredoxin reductase (PdR), putidaredoxin (Pd), and cytochrome P450cam (P450cam) within the P450cam system was conducted. The binary Pd/P450cam and Pd/PdR complexes were revealed and kinetically characterized. The dominant role of electrostatic interactions in formation of productive electron transfer complexes was demonstrated. It was found that Pd/P450cam complex formation and decay obeys biphasic kinetics in contrast to the monophasic one for complexes formed by other redox partners within the system. Evidence for PdR/P450cam complex formation was obtained. It was found that, in contrast to Pd, which binds only to its redox partners, PdR and P450cam were able to form PdR/PdR and P450cam/P450cam complexes. A ternary PdR/Pd/P450cam complex was also registered. Its lifetime was sufficient to permit up to 60 turnovers to occur. The binding of Pd to P450cam and to PdR within the ternary complex occurred at distinct sites, with Pd serving as a bridge between the two proteins., (Copyright 2001 Academic Press.)

The optical biosensor method was used for the revelation of ternary complexes, formed by the full-length NADPH-cytochrome P450 reductase (d-Fp) and cytochromes P4502B4 (d-2B4) and b5 (d-b5) in the course of their interactions within the reconstituted d-2B4-containing system. Based on the lack of competition between d-b5 and d-Fp for the binding sites on immobilized 2B4 (3) as well as on the analysis of data obtained in the three proteins' dissociation reactions, the possibility of formation of ternary complexes through the interactions between membranous hydrophobic fragments of proteins was substantiated. All the complexes obtained were productive., (Copyright 2000 Academic Press.)

The application of the AFM technique for visualization of membrane proteins and for measuring their dimensions was demonstrated. The AFM images of the microsomal monooxygenase system components-cytochrome P450 2B4 and NADPH-cytochrome P450 reductase-were obtained by using two types of supports-hydrophobic, highly oriented pyrolytic graphite (HOPG) and hydrophilic mica. It was shown that hemo- and flavoprotein monomers and oligomers can be adsorbed to and visualized on HOPG. On the negatively charged mica matrix, flavoprotein oligomers dissociated to monomers while hemoprotein oligomers dissociated into less aggregated particles. The images of cytochrome P450 2B4 and NADPH-cytochrome P450 reductase monomers were about 3 and 5 nm high, respectively, while the images of oligomeric forms of these proteins were about 10 and 8 nm high, respectively. We were able to observe the binary complexes composed of monomeric proteins, cytochrome P450 2B4 and its reductase and to measure the heights of these complexes (7 nm). The method is applicable for visualization of not only individual proteins but also their complexes., (Copyright 1999 Academic Press.)

The kinetics of hybridization of 11-meric and 14-meric oligonucleotides, dTGGGAAGAGGG (ODN-11) and dTGGGAAGAGG GTCA (ODN-14), with 14-meric oligonucleotide dpTGACCCTCT TCCCA (p14) attached to the surface of a cuvette was studied by the resonant mirror method. The treatment of the experimental curves with exponential equations leads to the following values for association (kas) and dissociation (kdis) rate constants at 25 degrees C: kas = 219 +/- 39 and 183 +/- 162 M-1 s-1, kdis = (2.0 +/- 0.4) x 10(-3) and (4 +/- 1) x 10(-4) s-1 for the duplexes (p14) x (ODN-11) and p14 x (ODN-14), respectively. The oligonucleotide dTGCCTTGAATGGGAA GAGGGTCA (ODN-23), which forms a hairpin structure, does not associate with p14. The data were compared with the results of melting curve detection and temperature-jump experiments. The association rate constants for ODN-11 and ODN-14 are much slower than those values in homogeneous aqueous solution. The dissociation rate constants have the same magnitude values as estimated by using association constants measured from melting curves but differ from the values estimated in temperature-jump experiments.

The formation of individual complexes between the components of cholesterol side chain cleavage system-cytochrome P450scc, adrenodoxin (Ad) and adrenodoxin reductase (AdR) was kinetically characterized and their association and dissociation rate constants were measured by optical biosensor. The dominant role of interprotein electrostatic interactions in productive complex formation was demonstrated. Despite of the fact that P450scc and AdR complete for the binding with the same or closely placed negatively charged groups on the surface of immobilized Ad, the formation of the AdR/P450scc/Ad ternary complex upon AdR immobilization on dextran was registered. It is shown, that Ad does not bind to AdR immobilized via amino groups AdRim but it is possible only after the preliminary binding of P450scc to AdRim. The life time of such ternary complex, about 15 s, is sufficient for the realization of 5-8 catalytic cycles.

The optical biosensor study of interaction between microsomal proteins-NADPH-cytochrome P450 reductase, cytochrome P450 2B4, and cytochrome b5-was carried out in the monomeric reconstituted system in the absence of phospholipids. The formation of individual complexes was kinetically characterized and their association and dissociation rate constants were determined. The association rate constants for the complexes formed were found to be close to the diffusiion limit-(0.5-4) x 10(6) M-1 s-1-while their dissociation rate constants did not exceed 0.5 s-1. It was shown that the interprotein electron transfer can occur both through complex formation and due to random collision. The dominant role of hydrophobic membraneous protein fragments in formation of productive electron transfer complexes was demonstrated., (Copyright 1999 Academic Press.)

The real-time interactions of membrane proteins - cytochrome P450 2B4, NADPH cytochrome P450 reductase and cytochrome b5 - were studied by use of an optical biosensor system. The association and dissociation rate constants for the individual complexes were measured and the affinities of the redox partners for each other were estimated. The association rate constants of these complexes were found to be close to the diffusion limit and their dissociation rate constants were in the order of 1s-1. A dominant role of the interaction of the membraneous hydrophobic fragments in the formation of productive electron transferring complexes between the proteins was demonstrated.

The secondary structure of microsomal epoxide hydrolase was determined by Raman spectroscopy and the effect of the membrane microenvironment studied. The ratios of the four secondary structure contents, alpha-helix: beta-strand:turn:undefined, were found to be 47:24:17:11 and 58:17:15:10 for the solubilized and the membrane-bound epoxide hydrolase, respectively. Based on the spectral analysis in the 2800-2900 cm-1 range, it was concluded that the protein studied produces the disordering effect on the lipid dimyristoylphosphatidylcholine bilayer at 16 degrees C.