Robert Ishmukhametov

Abstract:

F₁F_o ATP synthase functions as a biological generator and makes a major contribution to cellular energy production. Proton flow generates rotation in the F_o motor that is transferred to the F₁ motor to catalyze ATP production, with flexible F₁/F_o coupling required for efficient catalysis. F₁F_o ATP synthase can also operate in reverse, hydrolyzing ATP and pumping protons, and in bacteria this function can be regulated by an inhibitory ε subunit. Here we present cryo-EM data showing E. coli F₁F_o ATP synthase in different rotational and inhibited sub-states, observed following incubation with 10 mM MgATP. Our structures demonstrate how structural transitions within the inhibitory ε subunit induce torsional movement in the central stalk, thereby enabling its rotation within the F_ο motor. This highlights the importance of the central rotor for flexible coupling of the F₁ and F_o motors and provides further insight into the regulatory mechanism mediated by subunit ε.

Abstract:

The article describes a complex study of detonation nanodiamonds (DND) aqueous dispersions. In this research, DND sample was characterised by means of IR, NMR spectroscopy, TEM, thermogravimetric analysis, size distribution, and ζ-potentials. It was shown that DND sample includes several surface groups, mainly hydroxylic, carboxylic, and carbonyl ones. Dynamic light scattering results revealed that in the concentration range C = 0.002–0.3 wt%, DND nanoparticles size is equal to 55 ± 5 nm. It was demonstrated that DND possessed weak antiradical activity, had an inhibitory effect on F1F0-ATPase activity, almost did not affect platelet aggregation, formed a stronger complex with human serum albumin (HSA) in subdomain IB (digitoxin, Kb = 20.0 ± 2.4 l·g−1) and a less strong complex in subdomain IIA (warfarin, Kb = 3.7 ± 0.1 l·g−1), inhibited the esterase activity of HSA, DND dispersions (C = 0.0012–0.15 wt%) revealed genotoxic effect towards PBMCs, did not affect cellular proliferation in the experiment with HEK293 cell line, did not reveal cytotoxic effect up to 0.01 wt%. Using DFT and MD approaches allowed us to perform a simulation of interaction between DND nanoparticle and water molecules.

Abstract:

Archaea swim using the archaellum (archaeal flagellum), a reversible rotary motor consisting of a torque-generating motor and a helical filament, which acts as a propeller. Unlike the bacterial flagellar motor (BFM), ATP (adenosine-5′-triphosphate) hydrolysis probably drives both motor rotation and filamentous assembly in the archaellum. However, direct evidence is still lacking due to the lack of a versatile model system. Here, we present a membrane-permeabilized ghost system that enables the manipulation of intracellular contents, analogous to the triton model in eukaryotic flagella and gliding Mycoplasma. We observed high nucleotide selectivity for ATP driving motor rotation, negative cooperativity in ATP hydrolysis, and the energetic requirement for at least 12 ATP molecules to be hydrolyzed per revolution of the motor. The response regulator CheY increased motor switching from counterclockwise (CCW) to clockwise (CW) rotation. Finally, we constructed the torque–speed curve at various [ATP]s and discuss rotary models in which the archaellum has characteristics of both the BFM and F1-ATPase. Because archaea share similar cell division and chemotaxis machinery with other domains of life, our ghost model will be an important tool for the exploration of the universality, diversity, and evolution of biomolecular machinery.

Abstract:

F1Fo ATP synthase functions as a biological rotary generator that makes a major contribution to cellular energy production. It comprises two molecular motors coupled together by a central and a peripheral stalk. Proton flow through the Fo motor generates rotation of the central stalk, inducing conformational changes in the F1 motor that catalyzes ATP production. Here we present nine cryo-EM structures of E. coli ATP synthase to 3.1–3.4 Å resolution, in four discrete rotational sub-states, which provide a comprehensive structural model for this widely studied bacterial molecular machine. We observe torsional flexing of the entire complex and a rotational sub-step of Fo associated with long-range conformational changes that indicates how this flexibility accommodates the mismatch between the 3- and 10-fold symmetries of the F1 and Fo motors. We also identify density likely corresponding to lipid molecules that may contribute to the rotor/stator interaction within the Fo motor.

Abstract:

ATP synthase is an essential enzyme found in all known forms of life, generating the majority of cellular energy via a rotary catalytic mechanism. Here, we describe the in-depth methods for expression, purification, and functional assessment of E. coli ATP synthase.