Determinants of directionality and efficiency of the ATP synthase Fo motor at atomic resolution

Fo subcomplex of ATP synthase is an membrane-embedded rotary motor that converts proton motive force into mechanical energy. Despite a rapid increase in the number of high-resolution structures, the mechanism of tight coupling between proton transport and motion of the rotary c-ring remains elusive. Here, using extensive all-atom free energy simulations, we show how the motor's directionality naturally arises from the interplay between intra-protein interactions and energetics of protonation of the c-ring. Notably, our calculations reveal that the strictly conserved arginine in the a-subunit (R176) serves as a jack-of-all-trades: it dictates the direction of rotation, controls the protonation state of the proton-release site and separates the two proton-access half-channels. Therefore, arginine is necessary to avoid slippage between the proton flux and the mechanical output and guarantees highly efficient energy conversion. We also provide mechanistic explanations for the reported defective mutations of R176, reconciling the structural information on the Fo motor with previous functional and single-molecule data.

Structure of the molecular bushing of the bacterial flagellar motor

The basal body of the bacterial flagellum is a rotary motor that consists of several rings (C, MS and LP) and a rod. The LP ring acts as a bushing supporting the distal rod for its rapid and stable rotation without much friction. Here, we use electron cryomicroscopy to describe the LP ring structure around the rod, at 3.5 Å resolution, from Salmonella Typhimurium. The structure shows 26-fold rotational symmetry and intricate intersubunit interactions of each subunit with up to six partners, which explains the structural stability. The inner surface is charged both positively and negatively. Positive charges on the P ring (the part of the LP ring that is embedded within the peptidoglycan layer) presumably play important roles in its initial assembly around the rod with a negatively charged surface.

Crystal structures of two camelid nanobodies raised against GldL, a component of the type IX secretion system from Flavobacterium johnsoniae

GldL is an inner-membrane protein that is essential for the function of the type IX secretion system (T9SS) in Flavobacterium johnsoniae. The complex that it forms with GldM is supposed to act as a new rotary motor involved in the gliding motility of the bacterium. In the context of structural studies of GldL to gain information on the assembly and function of the T9SS, two camelid nanobodies were selected, produced and purified. Their interaction with the cytoplasmic domain of GldL was characterized and their crystal structures were solved. These nanobodies will be used as crystallization chaperones to help in the crystallization of the cytoplasmic domain of GldL and could also help to solve the structure of the complex using molecular replacement.

Inertial Piezoelectric Rotary Motor Based on Low Profile Stator with Trapezoidal Waveguides

Results of numerical and experimental investigations of a novel inertial piezoelectric rotary type motor based on a low profile stator with trapezoidal waveguides. The proposed motor has a simple design and is well scalable. Moreover, the proposed design of the motor allows mount it on a printed circuit board and use it in a small-size mobile positioning and actuating systems. The structure of the stator is based on a square type hollowed steel frame with four straight trapezoidal waveguides that are used to transfer vibrations of the stator to the rotation of the rotor. Piezo ceramic plates are glued on both sides of the stator. The thickness of the assembled stator is 0.9 mm, while the total area needed for stator mounting does not exceed 625 mm2. The driving of the rotor is based on the stick-slip principle, which is induced by excitation of the second in-plane bending mode of the four bimorph plates applying two saw tooth waveform signals with a phase difference by π. The numerical and experimental investigation was carried out to validate the operation principle of the motor and to measure the mechanical and electrical characteristics. The maximum angular rotation speed of 1304 RPM was achieved at a resonance frequency of 44.81 kHz when a preload of a 7.35mN was applied.

Site-Directed Cross-Linking Identifies the Stator-Rotor Interaction Surfaces in a Hybrid Bacterial Flagellar Motor

ABSTRACT The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are located in the cytoplasmic membrane and cytoplasm. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been shown by genetic analyses but have not been demonstrated biochemically. Here, we used site-directed photo-cross-linking and disulfide cross-linking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-l-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions in PomA and formed a cross-link with FliG. PomA residue K89 gave the highest yield of cross-links, suggesting that it is the PomA residue nearest to FliG. UV-induced cross-linking stopped motor rotation, and the isolated hook-basal body contained the cross-linked products. pBPA inserted to replace residue R281 or D288 in FliG formed cross-links with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide cross-links with cysteine inserted in place of FliG residues R281 and D288 and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA. IMPORTANCE The bacterial flagellum is a unique organelle that functions as a rotary motor. The interaction between the stator and rotor is indispensable for stator assembly into the motor and the generation of motor torque. However, the interface of the stator-rotor interaction has only been defined by mutational analysis. Here, we detected the stator-rotor interaction using site-directed photo-cross-linking and disulfide cross-linking approaches. We identified several residues in the PomA stator, especially K89, that are in close proximity to the rotor. Moreover, we identified several pairs of stator and rotor residues that interact. This study directly demonstrates the nature of the stator-rotor interaction and suggests how stator units assemble around the rotor and generate torque in the bacterial flagellar motor.

Site-directed crosslinking identifies the stator-rotor interaction surfaces in a hybrid bacterial flagellar motor

The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are embedded in the cytoplasmic membrane. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been suggested by genetic analyses, but have not been demonstrated directly. Here, we used site-directed photo- and disulfide-crosslinking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-L-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions formed a crosslink with FliG. PomA residue K89 gave the highest yield of crosslinks, suggesting that it is the PomA residue nearest to FliG. UV-induced crosslinking stopped motor rotation, and the isolated hook-basal body contained the crosslinked products. pBPA inserted to replace residues R281 or D288 in FliG formed crosslinks with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide crosslinks with cysteine inserted in place of FliG residues R281 and D288, and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA.

Reducing Effect of Vibration on Footing using Different Materials

This paper investigates the reduction of vibration effect between two footings by placing a trench between them. On the first footing (designated as the source footing), on electric-rotary motor is fitted, it has dimensions of (80x80) mm. Beside the source footing a second footing is placed and on this footing the reduction in vibration effects are to be investigated. Both the source footing, nearby footing and trench are placed over compacted gypseous soil in a steel tank having gypsum content with (50%). Tests are performed under dry and soaking conditions. The tests are managed under dynamic load for three frequencies (8,14 and 20) Hz, to install amplitude, velocity, and acceleration of the footing before and after drilling the trench, also to compare between the materials to choose the best material to reduce vibrations. The results showed that all parameters in above for the footing varying in values when change the material used to filling trench.