scholarly journals Covalent modification of the non-catalytic sites of the H + -ATPase from chloroplasts with 2-azido-[α- 32 P]ATP and its effect on ATP synthesis and ATP hydrolysis

2001 ◽  
Vol 1510 (1-2) ◽  
pp. 378-400 ◽  
Author(s):  
Franziska E Possmayer ◽  
Aloysius F Hartog ◽  
Jan A Berden ◽  
Peter Gräber
Keyword(s):  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Covalent Modification ◽  
Catalytic Sites

scholarly journals A New Method for Dispersing Pristine Carbon Nanotubes Using Regularly Arranged S-Layer Proteins

Nanomaterials ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1346
Author(s):  
Andreas Breitwieser ◽  
Uwe B. Sleytr ◽  
Dietmar Pum
Keyword(s):  
Carbon Nanotubes ◽  
Covalent Modification ◽  
Silica Layer ◽  
Medical Sciences ◽  
Lysinibacillus Sphaericus ◽  
Wide Range ◽  
Catalytic Sites ◽  
Multi Walled Carbon Nanotubes ◽  
Pristine Mwnts ◽  
Walled Carbon Nanotubes

Homogeneous and stable dispersions of functionalized carbon nanotubes (CNTs) in aqueous solutions are imperative for a wide range of applications, especially in life and medical sciences. Various covalent and non-covalent approaches were published to separate the bundles into individual tubes. In this context, this work demonstrates the non-covalent modification and dispersion of pristine multi-walled carbon nanotubes (MWNTs) using two S-layer proteins, namely, SbpA from Lysinibacillus sphaericus CCM2177 and SbsB from Geobacillus stearothermophilus PV72/p2. Both the S-layer proteins coated the MWNTs completely. Furthermore, it was shown that SbpA can form caps at the ends of MWNTs. Reassembly experiments involving a mixture of both S-layer proteins in the same solution showed that the MWNTs were primarily coated with SbsB, whereas SbpA formed self-assembled layers. The dispersibility of the pristine nanotubes coated with SbpA was determined by zeta potential measurements (−24.4 +/− 0.6 mV, pH = 7). Finally, the SbpA-coated MWNTs were silicified with tetramethoxysilane (TMOS) using a mild biogenic approach. As expected, the thickness of the silica layer could be controlled by the reaction time and was 6.3 +/− 1.25 nm after 5 min and 25.0 +/− 5.9 nm after 15 min. Since S-layer proteins have already demonstrated their capability to bind (bio)molecules in dense packing or to act as catalytic sites in biomineralization processes, the successful coating of pristine MWNTs has great potential in the development of new materials, such as biosensor architectures.

scholarly journals Aerobic Growth of Escherichia coli Is Reduced, and ATP Synthesis Is Selectively Inhibited when Five C-terminal Residues Are Deleted from the ϵ Subunit of ATP Synthase

2015 ◽  
Vol 290 (34) ◽  
pp. 21032-21041 ◽  
Author(s):  
Naman B. Shah ◽  
Thomas M. Duncan
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Intrinsic Rate ◽  
Synthesis Rate ◽  
Final Segment ◽  
Rotary Catalysis

F-type ATP synthases are rotary nanomotor enzymes involved in cellular energy metabolism in eukaryotes and eubacteria. The ATP synthase from Gram-positive and -negative model bacteria can be autoinhibited by the C-terminal domain of its ϵ subunit (ϵCTD), but the importance of ϵ inhibition in vivo is unclear. Functional rotation is thought to be blocked by insertion of the latter half of the ϵCTD into the central cavity of the catalytic complex (F1). In the inhibited state of the Escherichia coli enzyme, the final segment of ϵCTD is deeply buried but has few specific interactions with other subunits. This region of the ϵCTD is variable or absent in other bacteria that exhibit strong ϵ-inhibition in vitro. Here, genetically deleting the last five residues of the ϵCTD (ϵΔ5) caused a greater defect in respiratory growth than did the complete absence of the ϵCTD. Isolated membranes with ϵΔ5 generated proton-motive force by respiration as effectively as with wild-type ϵ but showed a nearly 3-fold decrease in ATP synthesis rate. In contrast, the ϵΔ5 truncation did not change the intrinsic rate of ATP hydrolysis with membranes. Further, the ϵΔ5 subunit retained high affinity for isolated F1 but reduced the maximal inhibition of F1-ATPase by ϵ from >90% to ∼20%. The results suggest that the ϵCTD has distinct regulatory interactions with F1 when rotary catalysis operates in opposite directions for the hydrolysis or synthesis of ATP.

scholarly journals Purification and Biochemical Characterization of the F1Fo-ATP Synthase from Thermoalkaliphilic Bacillus sp. Strain TA2.A1

2003 ◽  
Vol 185 (15) ◽  
pp. 4442-4449 ◽  
Author(s):  
Gregory M. Cook ◽  
Stefanie Keis ◽  
Hugh W. Morgan ◽  
Christoph von Ballmoos ◽  
Ulrich Matthey ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Biochemical Characterization ◽  
Atp Hydrolysis ◽  
Sodium Dodecyl ◽  
Electrical Potential ◽  
Atp Synthesis ◽  
Intrinsic Property ◽  
Protein Sequencing ◽  
Hydrolysis Activity

ABSTRACT We describe here purification and biochemical characterization of the F1Fo-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F1Fo-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F1 subunits α, β, γ, δ, and ε and Fo subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F1Fo holoenzyme or the isolated F1 moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F1. After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Δψ). A transmembrane proton gradient or sodium ion gradient in the absence of Δψ was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na+/H+ antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na+ ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N′-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.

scholarly journals Structure of a bacterial ATP synthase

2018 ◽  
Author(s):  
Hui Guo ◽  
Toshiharu Suzuki ◽  
John L. Rubinstein
Keyword(s):  
Atp Synthase ◽  
Biochemical Analysis ◽  
Atp Hydrolysis ◽  
Genetic Manipulation ◽  
Proton Translocation ◽  
Atp Synthesis ◽  
Mitochondrial Complex ◽  
Rotational States ◽  
Membrane Region ◽  
Atp Synthases

AbstractATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed theBacillusPS3 ATP synthase inEschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunitεshows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.

scholarly journals Targeting Mycobacterial F-ATP Synthase C-Terminal α Subunit Interaction Motif on Rotary Subunit γ

Antibiotics ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1456
Author(s):  
Amaravadhi Harikishore ◽  
Chui-Fann Wong ◽  
Priya Ragunathan ◽  
Dennis Litty ◽  
Volker Müller ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Membrane Vesicles ◽  
Α Subunit ◽  
Rotational States ◽  
C Terminus ◽  
Inverted Membrane Vesicles ◽  
Hydrolysis Of ◽  
Micromolar Range

Mycobacteria regulate their energy (ATP) levels to sustain their survival even in stringent living conditions. Recent studies have shown that mycobacteria not only slow down their respiratory rate but also block ATP hydrolysis of the F-ATP synthase (α3:β3:γ:δ:ε:a:b:b’:c9) to maintain ATP homeostasis in situations not amenable for growth. The mycobacteria-specific α C-terminus (α533-545) has unraveled to be the major regulative of latent ATP hydrolysis. Its deletion stimulates ATPase activity while reducing ATP synthesis. In one of the six rotational states of F-ATP synthase, α533-545 has been visualized to dock deep into subunit γ, thereby blocking rotation of γ within the engine. The functional role(s) of this C-terminus in the other rotational states are not clarified yet and are being still pursued in structural studies. Based on the interaction pattern of the docked α533-545 region with subunit γ, we attempted to study the druggability of the α533-545 motif. In this direction, our computational work has led to the development of an eight-featured α533-545 peptide pharmacophore, followed by database screening, molecular docking, and pose selection, resulting in eleven hit molecules. ATP synthesis inhibition assays using recombinant ATP synthase as well as mycobacterial inverted membrane vesicles show that one of the hits, AlMF1, inhibited the mycobacterial F-ATP synthase in a micromolar range. The successful targeting of the α533-545-γ interaction motif demonstrates the potential to develop inhibitors targeting the α site to interrupt rotary coupling with ATP synthesis.

scholarly journals Effect of Tentoxin on the Activation and on the Catalytic Reaction of Reconstituted H+-ATPase from Chloroplasts

1992 ◽  
Vol 47 (3-4) ◽  
pp. 239-244 ◽  
Author(s):  
Petra Fromme Max-Volmer ◽  
Ingo Dahse Friedrich-Schiller ◽  
Peter Gräber
Keyword(s):  
Energy Transfer ◽  
Catalytic Reaction ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Nucleotide Release ◽  
Transfer Inhibitor ◽  
Proton Translocating Atpase

Abstract The proton-translocating ATPase from chloroplasts, CF0F1, was isolated, purified and reconstituted into asolectin liposomes. The effect of the energy transfer inhibitor, tentoxin, on different functions of the enzyme was investigated. Tentoxin does not inhibit the nucleotide release during energization by a pH /ΔΨ jump, i.e. the activation of the enzyme is not influenced. ATP synthesis driven by a pH /ΔΨ jump and multi-site ATP hydrolysis are completely inhibited by tentoxin, whereas uni-site ATP hydrolysis is not influenced.

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