The AFM experiments were carried out using the direct surface adsorption method25 (link). Muscovite mica sheets (SPI, USA) were used as AFM substrates with hydrophilic surface. For AFM sample preparation, a freshly cleaved mica sheet was immersed into 800 µL of 0.1 μM solution of HRP protein in deionized ultrapure water, which was either irradiated or not irradiated (control experiment) in KEMF. The AFM substrate was incubated in the protein solution for 10 min at room temperature in a shaker at 600 rpm. During the incubation, the protein macromolecules adsorbed onto the mica surface. After the incubation, each substrate was rinsed with ultrapure water and dried in air.
The protein concentration used in AFM experiments was selected according to the results of preliminary experimental series on the AFM visualization of HRP adsorbed onto mica substrates from the protein solutions with a concentration ranging from 10−9 M to 10−6 М. Results obtained in this experimental series are described in the Supplementary Information.
Mica surface with adsorbed protein macromolecules was visualized by AFM. This method allows one to reliably measure the heights of single macromolecules with high (0.1 nm) resolution25 (link),43 (link). At the same time, lateral sizes of the resulting AFM images of the studied macromolecules can exceed their real sizes due to the effect of the AFM cantilever’s curvature radius (i.e., the effect of convolution of the probe and the objects under study)25 (link),43 (link). For this reason, in our present study, only the height of AFM images was used as a criterion for the determination of an increase in HRP macromolecules’ sizes (i.e., for the determination of HRP aggregation). All AFM measurements were carried out in tapping mode in air employing a Titanium multimode atomic force microscope (NT-MDT, Russia; this equipment pertains to the equipment of “Human Proteome” Core Facility of the Institute of Biomedical Chemistry, supported by Ministry of Education and Science of Russian Federation, agreement 14.621.21.0017, unique project ID RFMEFI62117X0017) with NSG10 cantilevers (“TipsNano”, Zelenograd, Russia; from 140 to 390 kHz resonant frequency, from 3.1 to 37.6 N/m force constant, tip curvature radius <10 nm). The calibration of the microscope by height was carried out on a TGZ1 calibration grating (NT-MDT, Russia; step height 21.4 ± 1.5 nm). The total number of imaged objects in each sample was no less than 200, and the number of frames for each sample was no less than 10. All samples were analyzed in at least three technical replicates. The density of the distribution of the AFM visualized objects with height ρ(h) was calculated as ρ(h) = (Nh/N)*100%, where Nh is the number of imaged proteins with height h, and N is the total number of imaged proteins. Control experiments were performed with use of protein-free ultrapure water instead of protein solution; in these experiments, no objects with >0.5 nm height were registered.
AFM operation, obtaining AFM images, their treatment (flattening correction etc.) and exporting the obtained data in ASCII format were performed using a standard NOVA Px software (NT-MDT, Moscow, Zelenograd, Russia) supplied with the atomic force microscope.
The number of the visualized particles in the obtained AFM images was calculated automatically using a specialized AFM data processing software developed in Institute of Biomedical Chemistry (Rospatent registration no. 2010613458).
The protein concentration used in AFM experiments was selected according to the results of preliminary experimental series on the AFM visualization of HRP adsorbed onto mica substrates from the protein solutions with a concentration ranging from 10−9 M to 10−6 М. Results obtained in this experimental series are described in the Supplementary Information.
Mica surface with adsorbed protein macromolecules was visualized by AFM. This method allows one to reliably measure the heights of single macromolecules with high (0.1 nm) resolution25 (link),43 (link). At the same time, lateral sizes of the resulting AFM images of the studied macromolecules can exceed their real sizes due to the effect of the AFM cantilever’s curvature radius (i.e., the effect of convolution of the probe and the objects under study)25 (link),43 (link). For this reason, in our present study, only the height of AFM images was used as a criterion for the determination of an increase in HRP macromolecules’ sizes (i.e., for the determination of HRP aggregation). All AFM measurements were carried out in tapping mode in air employing a Titanium multimode atomic force microscope (NT-MDT, Russia; this equipment pertains to the equipment of “Human Proteome” Core Facility of the Institute of Biomedical Chemistry, supported by Ministry of Education and Science of Russian Federation, agreement 14.621.21.0017, unique project ID RFMEFI62117X0017) with NSG10 cantilevers (“TipsNano”, Zelenograd, Russia; from 140 to 390 kHz resonant frequency, from 3.1 to 37.6 N/m force constant, tip curvature radius <10 nm). The calibration of the microscope by height was carried out on a TGZ1 calibration grating (NT-MDT, Russia; step height 21.4 ± 1.5 nm). The total number of imaged objects in each sample was no less than 200, and the number of frames for each sample was no less than 10. All samples were analyzed in at least three technical replicates. The density of the distribution of the AFM visualized objects with height ρ(h) was calculated as ρ(h) = (Nh/N)*100%, where Nh is the number of imaged proteins with height h, and N is the total number of imaged proteins. Control experiments were performed with use of protein-free ultrapure water instead of protein solution; in these experiments, no objects with >0.5 nm height were registered.
AFM operation, obtaining AFM images, their treatment (flattening correction etc.) and exporting the obtained data in ASCII format were performed using a standard NOVA Px software (NT-MDT, Moscow, Zelenograd, Russia) supplied with the atomic force microscope.
The number of the visualized particles in the obtained AFM images was calculated automatically using a specialized AFM data processing software developed in Institute of Biomedical Chemistry (Rospatent registration no. 2010613458).
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