[Atomic force microscopy fishing of gp120 on immobilized aptamer and its mass spectrometry identification].

A method of atomic force microscopy-based fishing (AFM fishing) has been developed for protein detection in the analyte solution using a chip with an immobilized aptamer. This method is based on the biospecific fishing of a target protein from a bulk solution onto the small AFM chip area with the immobilized aptamer to this protein used as the molecular probe. Such aptamer-based approach allows to increase an AFM image contrast compared to the antibody-based approach. Mass spectrometry analysis used after the biospecific fishing to identify the target protein on the AFM chip has proved complex formation. Use of the AFM chip with the immobilized aptamer avoids interference of the antibody and target protein peaks in a mass spectrum.

[Atomic force microscopy monitoring of temperature dependence of cytochrome BM3 oligomeric state].

The change in temperature is one of the factors affecting the activity of enzymes. In this work thermal denaturation and aggregation of cytochrome P450 BM3 were studied by atomic force microscopy. To determine specific temperature transitions the fluorescence analysis was used. In the low melting temperature range, 10-33 degrees C, a decrease in the fluorescence intensity of aromatic residues was observed with an increase in the fluorescence intensity of flavin groups. Protein melting in this range indicated three narrow S-shaped cooperative transitions at temperatures 16, 22 and 29 degrees C. Atomic force microscopy analysis in this temperature range showed that the shape of BM3 molecules remained globular in the form of compact objects (heights h < 7 nm, lateral dimensions d < 50 nm), but protein oligomeric state changed. The first two transitions were accompanied by a decrease in the degree of oligomerization and the third one was accompanied by its increase.

[Oligomeric state investigation of flavocytochrome CYP102A1 using AFM with standard and supersharp probes].

Atomic force microscopy with two types of probes - standard (radius of curvature R approximately 10 nm) and supersharp (R approximately 2 nm)- was used to determine CYP102A1oligomeric state. CYP102A1 images were obtained in a liquid, air and vacuum environment using the standard probes, also a ratio of monomers to oligomers (alpha) of CYP102A1 were determined as alpha=0.48:0.52. At the same time use of standard probes did not allow to resolve the structure of these oligomers. Supersharp probes allowed to obtain the data about the monomers to oligomers ratio, and also about the dimers/trimers/tetramers ratio in air and vacuum. So, a ratio alpha of CYP102A1 in liquid can be determined by the standard probes, and an oligomeric state of protein can be specified by the supersharp probes.

[Atomic force microscopy visualization and measurement of the activity and physicochemical properties of single monomeric and oligomeric enzymes].

An approach to measure the activity of single oligomers of the heme-containing enzyme the cytochrome P450 CYP102A1 (CYP102A1) by atomic force microscopy (AFM) has been developed. It was found that the amplitude of fluctuations of the height of single CYP102A1 molecules performing the catalytic cycle is twice as great as the amplitude of fluctuations of the height of the same enzymes in the inactive state. It was shown that the amplitude of height fluctuations of a CYP102A1 protein globule depends on temperature, the maximum of this dependence being observed at 22 degrees C. The activity of a single CYP102A1 molecule in the unit amplitude of height fluctuations of a protein globule reduced to the unit time was 5+/-2 Ac. The elasticity of a single protein molecule was measured from the deformation of this molecule by the action of an AFM probe. The use of AFM probes of different geometry made i t possible t o determine t he integral andlocal Young's modulus for the monomers of the protein putidaredoxin reductase from the cytochrome P450 CYP101 (P450cam)-containing monooxigenase system, which were 37+/-117 and 1+/-3 MPa, respectively.