scholarly journals Rotary catalysis of bovine mitochondrial F1-ATPase studied by single-molecule experiments

2020 ◽  
Vol 117 (3) ◽  
pp. 1447-1456 ◽  
Author(s):  
Ryohei Kobayashi ◽  
Hiroshi Ueno ◽  
Chun-Biu Li ◽  
Hiroyuki Noji
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Atp Hydrolysis ◽  
Angular Position ◽  
Magnetic Tweezers ◽  
Reaction Scheme ◽  
Atp Binding ◽  
High Speed Imaging ◽  
Hydrolysis Process ◽  
Rotary Catalysis

The reaction scheme of rotary catalysis and the torque generation mechanism of bovine mitochondrial F1 (bMF1) were studied in single-molecule experiments. Under ATP-saturated concentrations, high-speed imaging of a single 40-nm gold bead attached to the γ subunit of bMF1 showed 2 types of intervening pauses during the rotation that were discriminated by short dwell and long dwell. Using ATPγS as a slowly hydrolyzing ATP derivative as well as using a functional mutant βE188D with slowed ATP hydrolysis, the 2 pausing events were distinctively identified. Buffer-exchange experiments with a nonhydrolyzable analog (AMP-PNP) revealed that the long dwell corresponds to the catalytic dwell, that is, the waiting state for hydrolysis, while it remains elusive which catalytic state short pause represents. The angular position of catalytic dwell was determined to be at +80° from the ATP-binding angle, mostly consistent with other F1s. The position of short dwell was found at 50 to 60° from catalytic dwell, that is, +10 to 20° from the ATP-binding angle. This is a distinct difference from human mitochondrial F1, which also shows intervening dwell that probably corresponds to the short dwell of bMF1, at +65° from the binding pause. Furthermore, we conducted “stall-and-release” experiments with magnetic tweezers to reveal how the binding affinity and hydrolysis equilibrium are modulated by the γ rotation. Similar to thermophilic F1, bMF1 showed a strong exponential increase in ATP affinity, while the hydrolysis equilibrium did not change significantly. This indicates that the ATP binding process generates larger torque than the hydrolysis process.

scholarly journals Extreme parsimony in ATP consumption by 20S complexes in the global disassembly of single SNARE complexes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Changwon Kim ◽  
Min Ju Shon ◽  
Sung Hyun Kim ◽  
Gee Sung Eun ◽  
Je-Kyung Ryu ◽  
...  
Keyword(s):  
Energy Efficiency ◽  
Single Molecule ◽  
Molecular Motors ◽  
Atp Hydrolysis ◽  
Snare Complex ◽  
Atp Binding ◽  
Attachment Protein ◽  
Receptor Complexes ◽  
Protein Receptor ◽  
Sensitive Factor

AbstractFueled by ATP hydrolysis in N-ethylmaleimide sensitive factor (NSF), the 20S complex disassembles rigid SNARE (soluble NSF attachment protein receptor) complexes in single unraveling step. This global disassembly distinguishes NSF from other molecular motors that make incremental and processive motions, but the molecular underpinnings of its remarkable energy efficiency remain largely unknown. Using multiple single-molecule methods, we found remarkable cooperativity in mechanical connection between NSF and the SNARE complex, which prevents dysfunctional 20S complexes that consume ATP without productive disassembly. We also constructed ATP hydrolysis cycle of the 20S complex, in which NSF largely shows randomness in ATP binding but switches to perfect ATP hydrolysis synchronization to induce global SNARE disassembly, minimizing ATP hydrolysis by non-20S complex-forming NSF molecules. These two mechanisms work in concert to concentrate ATP consumption into functional 20S complexes, suggesting evolutionary adaptations by the 20S complex to the energetically expensive mechanical task of SNARE complex disassembly.

scholarly journals Real-time detection of condensin-driven DNA compaction reveals a multistep binding mechanism

2017 ◽  
Author(s):  
Jorine M. Eeftens ◽  
Shveta Bisht ◽  
Jacob Kerssemakers ◽  
Christian H. Haering ◽  
Cees Dekker
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Electrostatic Interactions ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Magnetic Tweezers ◽  
Condensed State ◽  
Binding Modes ◽  
Dna Molecules ◽  
Dna Compaction

ABSTRACTCondensin, a conserved member of the SMC protein family of ring-shaped multi-subunit protein complexes, is essential for structuring and compacting chromosomes. Despite its key role, its molecular mechanism has remained largely unknown. Here, we employ single-molecule magnetic tweezers to measure, in real-time, the compaction of individual DNA molecules by the budding yeast condensin complex. We show that compaction proceeds in large (~200nm) steps, driving DNA molecules into a fully condensed state against forces of up to 2pN. Compaction can be reversed by applying high forces or adding buffer of high ionic strength. While condensin can stably bind DNA in the absence of ATP, ATP hydrolysis by the SMC subunits is required for rendering the association salt-insensitive and for subsequent compaction. Our results indicate that the condensin reaction cycle involves two distinct steps, where condensin first binds DNA through electrostatic interactions before using ATP hydrolysis to encircle the DNA topologically within its ring structure, which initiates DNA compaction. The finding that both binding modes are essential for its DNA compaction activity has important implications for understanding the mechanism of chromosome compaction.

Solid-State Nanopores: From Fabrication to Application

2012 ◽  
Vol 20 (5) ◽  
pp. 24-29 ◽  
Author(s):  
Adam R. Hall
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
High Speed ◽  
Super Resolution ◽  
Magnetic Tweezers ◽  
Fluorescent Imaging ◽  
Force Microscopy ◽  
Atomic Force ◽  
Labeling Requirements ◽  
Molecular Manipulation

There are relatively few technologies for measurement at the single-molecule scale. Fluorescent imaging, for example, can be used to directly visualize molecules and their interactions, but diffraction limitations and labeling requirements may push the system from its native state. Although recent advances in super-resolution imaging have been able to break this resolution barrier, important challenges remain. Atomic force microscopy (AFM) is capable of imaging molecules at high resolution and at high speed. However, AFM imaging is a surface technique, requiring sample preparation and some immobilization. Other technologies such as optical tweezers and magnetic tweezers are capable of molecular manipulation and spectroscopy to great effect but require a significant apparatus and have limited inherent analytical capabilities.

scholarly journals The 3 × 120° rotary mechanism ofParacoccus denitrificansF1-ATPase is different from that of the bacterial and mitochondrial F1-ATPases

2020 ◽  
Vol 117 (47) ◽  
pp. 29647-29657 ◽  
Author(s):  
Mariel Zarco-Zavala ◽  
Ryo Watanabe ◽  
Duncan G. G. McMillan ◽  
Toshiharu Suzuki ◽  
Hiroshi Ueno ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Single Molecule ◽  
Paracoccus Denitrificans ◽  
Thermus Thermophilus ◽  
Angular Position ◽  
Adenosine Diphosphate ◽  
Atp Binding ◽  
Clear Trend ◽  
Inhibition Experiment ◽  
Atp Binding And Hydrolysis

The rotation ofParacoccus denitrificansF1-ATPase (PdF1) was studied using single-molecule microscopy. At all concentrations of adenosine triphosphate (ATP) or a slowly hydrolyzable ATP analog (ATPγS), above or belowKm, PdF1showed three dwells per turn, each separated by 120°. Analysis of dwell time between steps showed that PdF1executes binding, hydrolysis, and probably product release at the same dwell. The comparison of ATP binding and catalytic pauses in single PdF1molecules suggested that PdF1executes both elementary events at the same rotary position. This point was confirmed in an inhibition experiment with a nonhydrolyzable ATP analog (AMP-PNP). Rotation assays in the presence of adenosine diphosphate (ADP) or inorganic phosphate at physiological concentrations did not reveal any obvious substeps. Although the possibility of the existence of substeps remains, all of the datasets show that PdF1is principally a three-stepping motor similar to bacterial vacuolar (V1)-ATPase fromThermus thermophilus. This contrasts with all other known F1-ATPases that show six or nine dwells per turn, conducting ATP binding and hydrolysis at different dwells. Pauses by persistent Mg-ADP inhibition or the inhibitory ζ-subunit were also found at the same angular position of the rotation dwell, supporting the simplified chemomechanical scheme of PdF1. Comprehensive analysis of rotary catalysis of F1from different species, including PdF1, suggests a clear trend in the correlation between the numbers of rotary steps of F1and Fodomains of F-ATP synthase. F1motors with more distinctive steps are coupled with proton-conducting Forings with fewer proteolipid subunits, giving insight into the design principle the F1Foof ATP synthase.

scholarly journals Thermodynamic analysis of F1 -ATPase rotary catalysis using high-speed imaging

2014 ◽  
Vol 23 (12) ◽  
pp. 1773-1779 ◽  
Author(s):  
Rikiya Watanabe ◽  
Yoshihiro Minagawa ◽  
Hiroyuki Noji
Keyword(s):  
Thermodynamic Analysis ◽  
High Speed ◽  
High Speed Imaging ◽  
F1 Atpase ◽  
Rotary Catalysis

scholarly journals Single-molecule live cell imaging of Rep reveals the dynamic interplay between an accessory replicative helicase and the replisome

2019 ◽  
Vol 47 (12) ◽  
pp. 6287-6298 ◽  
Author(s):  
Aisha H Syeda ◽  
Adam J M Wollman ◽  
Alex L Hargreaves ◽  
Jamieson A L Howard ◽  
Jan-Gert Brüning ◽  
...  
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Atp Hydrolysis ◽  
Average Dwell Time ◽  
Genetic Studies ◽  
Replication Restart ◽  
Barrier Removal ◽  
Dynamic Interplay ◽  
Rapid Binding

Abstract DNA replication must cope with nucleoprotein barriers that impair efficient replisome translocation. Biochemical and genetic studies indicate accessory helicases play essential roles in replication in the presence of nucleoprotein barriers, but how they operate inside the cell is unclear. With high-speed single-molecule microscopy we observed genomically-encoded fluorescent constructs of the accessory helicase Rep and core replisome protein DnaQ in live Escherichia coli cells. We demonstrate that Rep colocalizes with 70% of replication forks, with a hexameric stoichiometry, indicating maximal occupancy of the single DnaB hexamer. Rep associates dynamically with the replisome with an average dwell time of 6.5 ms dependent on ATP hydrolysis, indicating rapid binding then translocation away from the fork. We also imaged PriC replication restart factor and observe Rep-replisome association is also dependent on PriC. Our findings suggest two Rep-replisome populations in vivo: one continually associating with DnaB then translocating away to aid nucleoprotein barrier removal ahead of the fork, another assisting PriC-dependent reloading of DnaB if replisome progression fails. These findings reveal how a single helicase at the replisome provides two independent ways of underpinning replication of protein-bound DNA, a problem all organisms face as they replicate their genomes.

scholarly journals Resolving the step size in condensin-driven DNA loop extrusion identifies ATP binding as the step-generating process

2020 ◽  
Author(s):  
Je-Kyung Ryu ◽  
Sang-Hyun Rah ◽  
Richard Janissen ◽  
Jacob W. J. Kerssemakers ◽  
Cees Dekker
Keyword(s):  
Conformational Changes ◽  
Protein Complex ◽  
Atp Hydrolysis ◽  
Magnetic Tweezers ◽  
Atp Binding ◽  
Globular Domain ◽  
Step Size ◽  
Chromosomal Structure ◽  
Dna Loop

SUMMARYThe condensin SMC protein complex organizes chromosomal structure by extruding loops of DNA. Its ATP-dependent motor mechanism remains unclear but likely involves steps associated with large conformational changes within the ~50 nm protein complex. Here, using magnetic tweezers, we resolve single steps in loop extrusion by individual yeast condensins. Step sizes range between 20-45 nm at forces of 1.0-0.2 pN, respectively, comparable to the complex size. The large steps show that condensin reels in DNA in very sizeable amounts, up to ~600 bp per extrusion step, consistent with the non-stretched DNA polymer at these low forces. Using ATP-binding-impaired and ATP-hydrolysis-deficient mutants, we find that ATP binding is the primary step-generating stage underlying DNA loop extrusion. We discuss the findings in terms of a scrunching model where a stepwise DNA loop extrusion is generated by an ATP-binding-induced engagement of the hinge and the globular domain of the SMC complex.

scholarly journals High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F 1 -ATPase

2013 ◽  
Vol 368 (1611) ◽  
pp. 20120023 ◽  
Author(s):  
Thomas Bilyard ◽  
Mayumi Nakanishi-Matsui ◽  
Bradley C. Steel ◽  
Teuta Pilizota ◽  
Ashley L. Nord ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Single Molecule ◽  
Room Temperature ◽  
Atp Hydrolysis ◽  
Chemical Processes ◽  
Atp Binding ◽  
Γ Subunit ◽  
Rotary Motor ◽  
Resolution Single

The rotary motor F 1 -ATPase from the thermophilic Bacillus PS3 (TF 1 ) is one of the best-studied of all molecular machines. F 1 -ATPase is the part of the enzyme F 1 F O -ATP synthase that is responsible for generating most of the ATP in living cells. Single-molecule experiments have provided a detailed understanding of how ATP hydrolysis and synthesis are coupled to internal rotation within the motor. In this work, we present evidence that mesophilic F 1 -ATPase from Escherichia coli (EF 1 ) is governed by the same mechanism as TF 1 under laboratory conditions. Using optical microscopy to measure rotation of a variety of marker particles attached to the γ-subunit of single surface-bound EF 1 molecules, we characterized the ATP-binding, catalytic and inhibited states of EF 1 . We also show that the ATP-binding and catalytic states are separated by 35±3°. At room temperature, chemical processes occur faster in EF 1 than in TF 1 , and we present a methodology to compensate for artefacts that occur when the enzymatic rates are comparable to the experimental temporal resolution. Furthermore, we show that the molecule-to-molecule variation observed at high ATP concentration in our single-molecule assays can be accounted for by variation in the orientation of the rotating markers.

scholarly journals Thermodynamics of CFTR Channel Gating: A Spreading Conformational Change Initiates an Irreversible Gating Cycle

2006 ◽  
Vol 128 (5) ◽  
pp. 523-533 ◽  
Author(s):  
László Csanády ◽  
Angus C. Nairn ◽  
David C. Gadsby
Keyword(s):  
Transition State ◽  
Single Molecule ◽  
Large Scale ◽  
Atp Hydrolysis ◽  
Normal Closure ◽  
Atp Binding ◽  
Free Energies ◽  
Binding Domains ◽  
Channel Opening ◽  
Opening And Closing

CFTR is the only ABC (ATP-binding cassette) ATPase known to be an ion channel. Studies of CFTR channel function, feasible with single-molecule resolution, therefore provide a unique glimpse of ABC transporter mechanism. CFTR channel opening and closing (after regulatory-domain phosphorylation) follows an irreversible cycle, driven by ATP binding/hydrolysis at the nucleotide-binding domains (NBD1, NBD2). Recent work suggests that formation of an NBD1/NBD2 dimer drives channel opening, and disruption of the dimer after ATP hydrolysis drives closure, but how NBD events are translated into gate movements is unclear. To elucidate conformational properties of channels on their way to opening or closing, we performed non-equilibrium thermodynamic analysis. Human CFTR channel currents were recorded at temperatures from 15 to 35°C in inside-out patches excised from Xenopus oocytes. Activation enthalpies(ΔH‡) were determined from Eyring plots. ΔH‡ was 117 ± 6 and 69 ± 4 kJ/mol, respectively, for opening and closure of partially phosphorylated, and 96 ± 6 and 73 ± 5 kJ/mol for opening and closure of highly phosphorylated wild-type (WT) channels. ΔH‡ for reversal of the channel opening step, estimated from closure of ATP hydrolysis–deficient NBD2 mutant K1250R and K1250A channels, and from unlocking of WT channels locked open with ATP+AMPPNP, was 43 ± 2, 39 ± 4, and 37 ± 6 kJ/mol, respectively. Calculated upper estimates of activation free energies yielded minimum estimates of activation entropies (ΔS‡), allowing reconstruction of the thermodynamic profile of gating, which was qualitatively similar for partially and highly phosphorylated CFTR. ΔS‡ appears large for opening but small for normal closure. The large ΔH‡ and ΔS‡ (TΔS‡ ≥ 41 kJ/mol) for opening suggest that the transition state is a strained channel molecule in which the NBDs have already dimerized, while the pore is still closed. The small ΔS‡ for normal closure is appropriate for cleavage of a single bond (ATP's beta-gamma phosphate bond), and suggests that this transition state does not require large-scale protein motion and hence precedes rehydration (disruption) of the dimer interface.

scholarly journals Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
William John Allen ◽  
Robin Adam Corey ◽  
Peter Oatley ◽  
Richard Barry Sessions ◽  
Steve A Baldwin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Protein Translocation ◽  
Md Simulations ◽  
Atp Binding ◽  
Nucleotide Exchange ◽  
Brownian Ratchet ◽  
Ratchet Mechanism ◽  
Single Molecule Fret ◽  
Directional Movement

The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids.

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