Twisting and subunit rotation in single F(O)(F1)-ATP synthase.
ABSTRACT: F(O)F(1)-ATP synthases are ubiquitous proton- or ion-powered membrane enzymes providing ATP for all kinds of cellular processes. The mechanochemistry of catalysis is driven by two rotary nanomotors coupled within the enzyme. Their different step sizes have been observed by single-molecule microscopy including videomicroscopy of fluctuating nanobeads attached to single enzymes and single-molecule Förster resonance energy transfer. Here we review recent developments of approaches to monitor the step size of subunit rotation and the transient elastic energy storage mechanism in single F(O)F(1)-ATP synthases.
Project description:F-type ATP synthases are extraordinary multisubunit proteins that operate as nanomotors. The Escherichia coli (E. coli) enzyme uses the proton motive force (pmf) across the bacterial plasma membrane to drive rotation of the central rotor subunits within a stator subunit complex. Through this mechanical rotation, the rotor coordinates three nucleotide binding sites that sequentially catalyze the synthesis of ATP. Moreover, the enzyme can hydrolyze ATP to turn the rotor in the opposite direction and generate pmf. The direction of net catalysis, i.e. synthesis or hydrolysis of ATP, depends on the cell's bioenergetic conditions. Different control mechanisms have been found for ATP synthases in mitochondria, chloroplasts and bacteria. This review discusses the auto-inhibitory behavior of subunit ? found in FOF1-ATP synthases of many bacteria. We focus on E. coli FOF1-ATP synthase, with insights into the regulatory mechanism of subunit ? arising from structural and biochemical studies complemented by single-molecule microscopy experiments.
Project description:ATP synthases (ATPases) are enzymes that produce ATP and control the pH in the cell or cellular compartments. While highly conserved over different species, ATPases are structurally well-characterised but the existence and functional significance of many post-translational modifications (PTMs) is not well understood. We combined a range of mass spectrometric techniques to unravel the location and extent of PTMs in the chloroplast ATP synthase (cATPase) purified from spinach leaves. We identified multiple phosphorylation and acetylation sites and found that both modifications stabilise binding of ? and ? subunits. Comparing cross-linking of naturally modified cATPase with the in vitro deacetylated enzyme revealed a major conformational change in the ? subunit in accord with extended and folded forms of the subunit. Locating modified residues within the catalytic head we found that phosphorylated and acetylated residues are primarily on ?/? and ?/? interfaces respectively. By aligning along different interfaces the higher abundance acetylated residues are proximal to the regulatory sites while the lower abundance phosphorylation sites are more densely populated at the catalytic sites. We propose that modifications in the catalytic head, together with the conformational change in subunit ?, work in synergy to fine-tune the enzyme during adverse conditions.
Project description:The epsilon subunit of bacterial and chloroplast F(o)F(1)-ATP synthases modulates their ATP hydrolysis activity. Here, we report the crystal structure of the ATP-bound epsilon subunit from a thermophilic Bacillus PS3 at 1.9-A resolution. The C-terminal two alpha-helices were folded into a hairpin, sitting on the beta sandwich structure, as reported for Escherichia coli. A previously undescribed ATP binding motif, I(L)DXXRA, recognizes ATP together with three arginine and one glutamate residues. The E. coli epsilon subunit binds ATP in a similar manner, as judged on NMR. We also determined solution structures of the C-terminal domain of the PS3 epsilon subunit and relaxation parameters of the whole molecule by NMR. The two helices fold into a hairpin in the presence of ATP but extend in the absence of ATP. The latter structure has more helical regions and is much more flexible than the former. These results suggest that the epsilon C-terminal domain can undergo an arm-like motion in response to an ATP concentration change and thereby contribute to regulation of F(o)F(1)-ATP synthase.
Project description:F1F0 ATP synthases are bidirectional molecular motors that translocate protons across the cell membrane by either synthesizing or hydrolyzing ATP. Alkaliphile ATP synthases are highly adapted, performing oxidative phosphorylation at high pH against an inverted pH gradient (acidin/alkalineout). Unlike mesophilic ATP synthases, alkaliphilic enzymes have tightly regulated ATP hydrolysis activity, which can be relieved in the presence of lauryldimethylamine oxide. Here, we characterized the rotary dynamics of the Caldalkalibacillus thermarum TA2.A1 F1 ATPase (TA2F1) with two forms of single molecule analysis, a magnetic bead duplex and a gold nanoparticle. TA2F1 rotated in a counterclockwise direction in both systems, adhering to Michaelis-Menten kinetics with a maximum rotation rate (Vmax) of 112.4 revolutions/s. TA2F1 displayed 120° unitary steps coupled with ATP hydrolysis. Torque measurements revealed the highest torque (52.4 piconewtons) derived from an F1 molecule using fluctuation theorem. The implications of high torque in terms of extreme environment adaptation are discussed.
Project description:Designer particles that are embued with nanomachinery for autonomous motion have great potential for biomedical applications; however, their development is highly demanding with respect to biodegradability/compatibility. Previously, biodegradable propulsive machinery based on enzymes has been presented. However, enzymes are highly susceptible to proteolysis and deactivation in biological milieu. Biodegradable hybrid nanomotors powered by catalytic inorganic nanoparticles provide a proteolytically stable alternative to those based upon enzymes. Herein we describe the assembly of hybrid biodegradable nanomotors capable of transducing chemical energy into motion. Such nanomotors are constructed through a process of compartmentalized synthesis of inorganic MnO2 nanoparticles (MnPs) within the cavity of organic stomatocytes. We show that the nanomotors remain active in cellular environments and do not compromise cell viability. Effective tumor penetration of hybrid nanomotors is also demonstrated in proof-of-principle experiments. Overall, this work represents a new prospect for engineering of nanomotors that can retain their functionality within biological contexts.
Project description:The bacterial ATP synthase (F(O)F(1)) of Escherichia coli has been the prominent model system for genetics, biochemical and more recently single-molecule studies on F-type ATP synthases. With 22 total polypeptide chains (total mass of ?529?kDa), E. coli F(O)F(1) represents nature's smallest rotary motor, composed of a membrane-embedded proton transporter (F(O)) and a peripheral catalytic complex (F(1)). The ATPase activity of isolated F(1) is fully expressed by the ?(3)?(3)? 'core', whereas single ? and ? subunits are required for structural and functional coupling of E. coli F(1) to F(O). In contrast to mitochondrial F(1)-ATPases that have been determined to atomic resolution, the bacterial homologues have proven very difficult to crystallize. In this paper, we describe a biochemical strategy that led us to improve the crystallogenesis of the E. coli F(1)-ATPase catalytic core. Destabilizing the compact conformation of ?'s C-terminal domain with a phosphomimetic mutation (?S65D) dramatically increased crystallization success and reproducibility, yielding crystals of E. coli F(1) that diffract to ?3.15?Å resolution.
Project description:Nanomedicine results from nanotechnology where molecular scale minute precise nanomotors can be used to treat disease conditions. Many such biological nanomotors are found and operate in living systems which could be used for therapeutic purposes. The question is how to build nanomachines that are compatible with living systems and can safely operate inside the body? Here we propose that it is of paramount importance to have a workable base model for the development of nanomotors in nanomedicine usage. The base model must placate not only the basic requirements of size, number, and speed but also must have the provisions of molecular modulations. Universal occurrence and catalytic site molecular modulation capabilities are of vital importance for being a perfect base model. In this review we will provide a detailed discussion on ATP synthase as one of the most suitable base models in the development of nanomotors. We will also describe how the capabilities of molecular modulation can improve catalytic and motor function of the enzyme to generate a catalytically improved and controllable ATP synthase which in turn will help in building a superior nanomotor. For comparison, several other biological nanomotors will be described as well as their applications for nanotechnology.
Project description:Mitochondrial and chloroplast ATP synthases are key enzymes in plant metabolism, providing cells with ATP, the universal energy currency. ATP synthases use a transmembrane electrochemical proton gradient to drive synthesis of ATP. The enzyme complexes function as miniature rotary engines, ensuring energy coupling with very high efficiency. Although our understanding of the structure and functioning of the synthase has made enormous progress in recent years, our understanding of regulatory mechanisms is still rather preliminary. Here we report a role for 14-3-3 proteins in the regulation of ATP synthases. These 14-3-3 proteins are highly conserved phosphoserine/phosphothreonine-binding proteins that regulate a wide range of enzymes in plants, animals, and yeast. Recently, the presence of 14-3-3 proteins in chloroplasts was illustrated, and we show here that plant mitochondria harbor 14-3-3s within the inner mitochondrial-membrane compartment. There, the 14-3-3 proteins were found to be associated with the ATP synthases, in a phosphorylation-dependent manner, through direct interaction with the F(1) beta-subunit. The activity of the ATP synthases in both organelles is drastically reduced by recombinant 14-3-3. The rapid reduction in chloroplast ATPase activity during dark adaptation was prevented by a phosphopeptide containing the 14-3-3 interaction motif, demonstrating a role for endogenous 14-3-3 in the down-regulation of the CF(o)F(1) activity. We conclude that regulation of the ATP synthases by 14-3-3 represents a mechanism for plant adaptation to environmental changes such as light/dark transitions, anoxia in roots, and fluctuations in nutrient supply.
Project description:In oxidative phosphorylation, ATP synthases interconvert two forms of free energy: they are driven by the proton-motive force across an energy-transducing membrane to synthesize ATP and displace the ADP/ATP ratio from equilibrium. For thermodynamically efficient energy conversion they must be reversible catalysts. However, in many species ATP synthases are unidirectional catalysts (their rates of ATP hydrolysis are negligible), and in others mechanisms have evolved to regulate or minimize hydrolysis. Unidirectional catalysis by Paracoccus denitrificans ATP synthase has been attributed to its unique ? subunit, which is structurally analogous to the mammalian inhibitor protein IF1 Here, we used homologous recombination to delete the ? subunit from the P. denitrificans genome, and compared ATP synthesis and hydrolysis by the wild-type and knockout enzymes in inverted membrane vesicles and the F1-ATPase subcomplex. ATP synthesis was not affected by loss of the ? subunit, and the rate of ATP hydrolysis increased by less than twofold, remaining negligible in comparison with the rates of the Escherichia coli and mammalian enzymes. Therefore, deleting the P. denitrificans ? subunit is not sufficient to activate ATP hydrolysis. We close by considering our conclusions in the light of reversible catalysis and regulation in ATP synthase enzymes.
Project description:The Mycobacteria are a genus of Actinobacteria that include human pathogens such as <i>Mycobacterium tuberculosis</i> (TB). Active TB disease can spread by airborne transmission to healthcare workers and to their community. The HHMI SEA-PHAGES program has contributed to discovering bacteriophages that are able to infect <i>M. smegmatis</i> MC<sup>2</sup> 155, a close relative of <i>M. tuberculosis</i>. This collection of diverse Mycobacteriophages is an excellent resource for trialling bacteriophage-sourced enzymes in novel applications. Herein we measured the ability Mycobacteriophage endolysins to lyse their host strain when functionally fused to biodegradable polyhydroxyalkanoate (PHA) nanobeads. PHA nanobeads facilitate both the expression and the application of enzymes to surfaces and have been demonstrated to stabilize a wide array of proteins for practical applications whilst eliminating the challenges of traditional protein purification. We selected two Lysin A and six Lysin B homologs to be functionally fused to the polyhydroxyalkanoate synthase C (PhaC). Expression of these constructs resulted in functional lysins displayed on the surface of PHA nanobeads. The lysins thus directionally displayed on nanobeads lysed up to 79% of the <i>M. smegmatis</i> MC<sup>2</sup> 155 population using 80 mg/mL of nanobeads in pure culture. In order to determine whether the nanobeads would be effective as a protective layer in PPE we adapted a fabric-based test and observed a maximum of 1 log loss of the cell population after 5 h of exposure on a textile (91% cell lysis). Lysin B enzymes performed better than the Lysin A enzymes as a protective barrier on textiles surface assays. These results suggest that bacterial endolysins are efficient in their action when displayed on PHA nanobeads and can cause significant population mortality in as little as 45 min. Our results provide the proof-of-principle that Mycobacteriophage endolysins can be used on functionalized nanobeads where they can protect surfaces such as personal protective equipment (PPE) that routinely come into contact with aerosolised bacteria.