scholarly journals Examining kinesin processivity within a general gating framework

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Johan OL Andreasson ◽  
Bojan Milic ◽  
Geng-Yuan Chen ◽  
Nicholas R Guydosh ◽  
William O Hancock ◽  
...  
Keyword(s):  
Single Molecule ◽  
General Framework ◽  
Optical Trapping ◽  
Atp Hydrolysis ◽  
Wild Type ◽  
Structural Element ◽  
Gating Mechanisms ◽  
Motor Velocity

Kinesin-1 is a dimeric motor that transports cargo along microtubules, taking 8.2-nm steps in a hand-over-hand fashion. The ATP hydrolysis cycles of its two heads are maintained out of phase by a series of gating mechanisms, which lead to processive runs averaging ∼1 μm. A key structural element for inter-head coordination is the neck linker (NL), which connects the heads to the stalk. To examine the role of the NL in regulating stepping, we investigated NL mutants of various lengths using single-molecule optical trapping and bulk fluorescence approaches in the context of a general framework for gating. Our results show that, although inter-head tension enhances motor velocity, it is crucial neither for inter-head coordination nor for rapid rear-head release. Furthermore, cysteine-light mutants do not produce wild-type motility under load. We conclude that kinesin-1 is primarily front-head gated, and that NL length is tuned to enhance unidirectional processivity and velocity.

scholarly journals A Generalized Kinetic Model for Coupling between Stepping and ATP Hydrolysis of Kinesin Molecular Motors

2019 ◽  
Vol 20 (19) ◽  
pp. 4911 ◽  
Author(s):  
Xie ◽  
Guo ◽  
Chen
Keyword(s):  
Kinetic Model ◽  
Atpase Activity ◽  
Single Molecule ◽  
Rate Constants ◽  
Molecular Motors ◽  
Atp Hydrolysis ◽  
Power Production ◽  
Wild Type ◽  
Chemomechanical Coupling ◽  
Generalized Kinetic Model

A general kinetic model is presented for the chemomechanical coupling of dimeric kinesin molecular motors with and without extension of their neck linkers (NLs). A peculiar feature of the model is that the rate constants of ATPase activity of a kinesin head are independent of the strain on its NL, implying that the heads of the wild-type kinesin dimer and the mutant with extension of its NLs have the same force-independent rate constants of the ATPase activity. Based on the model, an analytical theory is presented on the force dependence of the dynamics of kinesin dimers with and without extension of their NLs at saturating ATP. With only a few adjustable parameters, diverse available single molecule data on the dynamics of various kinesin dimers, such as wild-type kinesin-1, kinesin-1 with mutated residues in the NLs, kinesin-1 with extension of the NLs and wild-type kinesin-2, under varying force and ATP concentration, can be reproduced very well. Additionally, we compare the power production among different kinesin dimers, showing that the mutation in the NLs reduces the power production and the extension of the NLs further reduces the power production.

scholarly journals 150-kDa oxygen-regulated protein (ORP150) functions as a novel molecular chaperone in MDCK cells

2000 ◽  
Vol 278 (6) ◽  
pp. C1172-C1182 ◽  
Author(s):  
Yoshio Bando ◽  
Satoshi Ogawa ◽  
Atsushi Yamauchi ◽  
Keisuke Kuwabara ◽  
Kentaro Ozawa ◽  
...  
Keyword(s):  
Molecular Chaperone ◽  
Protein Transport ◽  
Atp Hydrolysis ◽  
Mdck Cells ◽  
Secretory Protein ◽  
Metabolic Labeling ◽  
Wild Type ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney

To assess the participation of the 150-kDa oxygen-regulated protein (ORP150) in protein transport, its function in Madin-Darby canine kidney (MDCK) cells was studied. Exposure of MDCK cells to hypoxia resulted in an increase of ORP150 antigen and increased binding of ORP150 to GP80/clusterin (80-kDa glycoprotein), a natural secretory protein in this cell line. In ORP150 antisense transformant MDCK cells, GP80 was retained within the endoplasmic reticulum after exposure to hypoxia. Metabolic labeling showed the delay of GP80 maturation in antisense transformants in hypoxia, whereas its matured form was detected in wild-type cells, indicating a role of ORP150 in protein transport, especially in hypoxia. The affinity chromatographic analysis of ORP150 suggested its ability to bind to ATP-agarose. Furthermore, the ATP hydrolysis analysis showed that ORP150 can release GP80 at a lower ATP concentration. These data indicate that ORP150 may function as a unique molecular chaperone in renal epithelial cells by facilitating protein transport/maturation in an environment where less ATP is accessible.

scholarly journals RecA polymerization on double-stranded DNA by using single-molecule manipulation: The role of ATP hydrolysis

1999 ◽  
Vol 96 (14) ◽  
pp. 7916-7921 ◽  
Author(s):  
G. V. Shivashankar ◽  
M. Feingold ◽  
O. Krichevsky ◽  
A. Libchaber
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Double Stranded Dna ◽  
Single Molecule Manipulation

scholarly journals Single mutations in the ε subunit from thermophilic Bacillus PS3 generate a high binding affinity site for ATP

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5505 ◽  
Author(s):  
Alexander Krah ◽  
Peter J. Bond
Keyword(s):  
Binding Affinity ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Evolutionary Analysis ◽  
Binding Affinities ◽  
Wild Type ◽  
Dynamics Simulations ◽  
High Binding Affinity ◽  
Atp Synthases

The ε subunit from ATP synthases acts as an ATP sensor in the bacterial cell to prevent ATP hydrolysis and thus the waste of ATP under conditions of low ATP concentration. However, the ATP binding affinities from various bacterial organisms differ markedly, over several orders of magnitude. For example, the ATP synthases from thermophilic Bacillus PS3 and Escherichia coli exhibit affinities of 4 µM and 22 mM, respectively. The recently reported R103A/R115A double mutant of Bacillus PS3 ATP synthase demonstrated an increased binding affinity by two orders of magnitude with respect to the wild type. Here, we used atomic-resolution molecular dynamics simulations to determine the role of the R103A and R115A single mutations. These lead us to predict that both single mutations also cause an increased ATP binding affinity. Evolutionary analysis reveals R103 and R115 substitutions in the ε subunit from other bacillic organisms, leading us to predict they likely have a higher ATP binding affinity than previously expected.

scholarly journals Arp2/3 complex ATP hydrolysis promotes lamellipodial actin network disassembly but is dispensable for assembly

2013 ◽  
Vol 200 (5) ◽  
pp. 619-633 ◽  
Author(s):  
Elena Ingerman ◽  
Jennifer Ying Hsiao ◽  
R. Dyche Mullins
Keyword(s):  
Atp Hydrolysis ◽  
Leading Edge ◽  
S2 Cells ◽  
Actin Network ◽  
Wild Type ◽  
Actin Networks ◽  
Dendritic Networks ◽  
A Cell

We examined the role of ATP hydrolysis by the Arp2/3 complex in building the leading edge of a cell by studying the effects of hydrolysis defects on the behavior of the complex in the lamellipodial actin network of Drosophila S2 cells and in a reconstituted, in vitro, actin-based motility system. In S2 cells, nonhydrolyzing Arp2 and Arp3 subunits expanded and delayed disassembly of lamellipodial actin networks and the effect of mutant subunits was additive. Arp2 and Arp3 ATP hydrolysis mutants remained in lamellipodial networks longer and traveled greater distances from the plasma membrane, even in networks still containing wild-type Arp2/3 complex. In vitro, wild-type and ATP hydrolysis mutant Arp2/3 complexes each nucleated actin and built similar dendritic networks. However, networks constructed with Arp2/3 hydrolysis-defective mutants were more resistant to disassembly by cofilin. Our results indicate that ATP hydrolysis on both Arp2 and Arp3 contributes to dissociation of the complex from the actin network but is not strictly necessary for lamellipodial network disassembly.

scholarly journals Cortical stiffness of keratinocytes measured by lateral indentation with optical tweezers

2020 ◽  
Author(s):  
Špela Zemljič Jokhadar ◽  
Biljana Stojković ◽  
Marko Vidak ◽  
Mirjana Liovic ◽  
Marcos Gouveia ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Small Difference ◽  
Cell Cortex ◽  
Wild Type ◽  
Structural Element ◽  
Mechanical Elements ◽  
Mutant Cells ◽  
Keratin Intermediate Filaments ◽  
Bulk Elasticity

AbstractKeratin intermediate filaments are the principal structural element of epithelial cells. Their importance in providing bulk cellular stiffness is well recognized, but their role in the mechanics of cell cortex is less understood. In this study, we therefore compared the cortical stiffness of three keratinocyte lines: primary wild type cells (NHEK2), immortalized wild type cells (NEB1) and immortalized mutant cells (KEB7). The cortical stiffness was measured by lateral indentation of cells with AOD-steered optical tweezers without employing any moving mechanical elements. The method was validated on fixed cells and Cytochalasin-D treated cells to ensure that the observed variations in stiffness within a single cell line were not a consequence of low measurement precision. The measurements of the cortical stiffness showed that primary wild type cells were significantly stiffer than immortalized wild type cells, which was also detected in previous studies of bulk elasticity. In addition, a small difference between the mutant and the wild type cells was detected, showing that mutation of keratin impacts also the cell cortex. Thus, our results indicate that the role of keratins in cortical stiffness is not negligible and call for further investigation of the mechanical interactions between keratins and elements of the cell cortex.

scholarly journals Cortical stiffness of keratinocytes measured by lateral indentation with optical tweezers

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0231606
Author(s):  
Špela Zemljič Jokhadar ◽  
Biljana Stojković ◽  
Marko Vidak ◽  
Tjaša Sorčan ◽  
Mirjana Liovic ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Small Difference ◽  
Cell Cortex ◽  
Wild Type ◽  
Structural Element ◽  
Mechanical Elements ◽  
Mutant Cells ◽  
Keratin Intermediate Filaments ◽  
Bulk Elasticity

Keratin intermediate filaments are the principal structural element of epithelial cells. Their importance in providing bulk cellular stiffness is well recognized, but their role in the mechanics of cell cortex is less understood. In this study, we therefore compared the cortical stiffness of three keratinocyte lines: primary wild type cells (NHEK2), immortalized wild type cells (NEB1) and immortalized mutant cells (KEB7). The cortical stiffness was measured by lateral indentation of cells with AOD-steered optical tweezers without employing any moving mechanical elements. The method was validated on fixed cells and Cytochalasin-D treated cells to ensure that the observed variations in stiffness within a single cell line were not a consequence of low measurement precision. The measurements of the cortical stiffness showed that primary wild type cells were significantly stiffer than immortalized wild type cells, which was also detected in previous studies of bulk elasticity. In addition, a small difference between the mutant and the wild type cells was detected, showing that mutation of keratin impacts also the cell cortex. Thus, our results indicate that the role of keratins in cortical stiffness is not negligible and call for further investigation of the mechanical interactions between keratins and elements of the cell cortex.

scholarly journals Decoupling the bridge helix of Cas12a results in a reduced trimming activity and impaired conformational transitions

2021 ◽  
Author(s):  
Elisabeth Wörle ◽  
Leonhard Jakob ◽  
Andreas Schmidbauer ◽  
Gabriel Zinner ◽  
Dina Grohmann
Keyword(s):  
Single Molecule ◽  
Conformational Transitions ◽  
Structural Element ◽  
Francisella Novicida ◽  
Closed State ◽  
Single Molecule Fret ◽  
Type V ◽  
Single Effector ◽  
Cas Proteins

AbstractThe widespread and versatile prokaryotic CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats and associated Cas proteins) constitute powerful weapons against foreign nucleic acids. Recently, the single-effector nuclease Cas12a that belongs to the type V CRISPR-Cas system was added to the Cas enzymes repertoire employed for gene editing purposes. Cas12a is a bilobal enzyme composed of the REC and Nuc lobe connected by a central structural element, the so-called bridge helix (BH). We generated BH mutants and integrated biochemical and single-molecule FRET (smFRET) studies to elucidate the role of the BH for the enzymatic activity and conformational flexibility of Francisella novicida Cas12a. We demonstrate that the BH impacts the trimming activity of Cas12a resulting in Cas12a variants with improved cleavage accuracy. Single-molecule FRET measurements reveal the hitherto unknown open and closed state of apo Cas12a. BH mutants preferentially adopt the open state. Transition to the closed state of the Cas12a-crRNA complex is inefficient in BH mutants but the semi-closed state of the ternary complex can be adopted even if the BH is deleted in its entirety. Taken together, these insights reveal that the BH is a structural element that influences the catalytic activity and impacts conformational transitions of FnCas12a.

scholarly journals Effect of directional pulling on mechanical protein degradation by ATP-dependent proteolytic machines

2017 ◽  
Vol 114 (31) ◽  
pp. E6306-E6313 ◽  
Author(s):  
Adrian O. Olivares ◽  
Hema Chandra Kotamarthi ◽  
Benjamin J. Stein ◽  
Robert T. Sauer ◽  
Tania A. Baker
Keyword(s):  
Escherichia Coli ◽  
Protein Degradation ◽  
Single Molecule ◽  
Wild Type ◽  
N Terminus ◽  
Multiple Copies ◽  
Rate Limiting ◽  
Hydrolysis Of ◽  
Translocation Speed

AAA+ proteases and remodeling machines couple hydrolysis of ATP to mechanical unfolding and translocation of proteins following recognition of sequence tags called degrons. Here, we use single-molecule optical trapping to determine the mechanochemistry of two AAA+ proteases, Escherichia coli ClpXP and ClpAP, as they unfold and translocate substrates containing multiple copies of the titinI27 domain during degradation initiated from the N terminus. Previous studies characterized degradation of related substrates with C-terminal degrons. We find that ClpXP and ClpAP unfold the wild-type titinI27 domain and a destabilized variant far more rapidly when pulling from the N terminus, whereas translocation speed is reduced only modestly in the N-to-C direction. These measurements establish the role of directionality in mechanical protein degradation, show that degron placement can change whether unfolding or translocation is rate limiting, and establish that one or a few power strokes are sufficient to unfold some protein domains.

scholarly journals Role of Vma21p in Assembly and Transport of the Yeast Vacuolar ATPase

2004 ◽  
Vol 15 (11) ◽  
pp. 5075-5091 ◽  
Author(s):  
Per Malkus ◽  
Laurie A. Graham ◽  
Tom H. Stevens ◽  
Randy Schekman
Keyword(s):  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Wild Type ◽  
Vitro Assay ◽  
Accessory Factor ◽  
Transport Vesicles ◽  
Er Export

The Saccharomyces cerevisiae vacuolar H+-ATPase (V-ATPase) is a multisubunit complex composed of a peripheral membrane sector (V1) responsible for ATP hydrolysis and an integral membrane sector (V0) required for proton translocation. Biogenesis of V0 requires an endoplasmic reticulum (ER)-localized accessory factor, Vma21p. We found that in vma21Δ cells, the major proteolipid subunit of V0 failed to interact with the 100-kDa V0 subunit, Vph1p, indicating that Vma21p is necessary for V0 assembly. Immunoprecipitation of Vma21p from wild-type membranes resulted in coimmunoprecipitation of all five V0 subunits. Analysis of vmaΔ strains showed that binding of V0 subunits to Vma21p was mediated by the proteolipid subunit Vma11p. Although Vma21p/proteolipid interactions were independent of Vph1p, Vma21p/Vph1p association was dependent on all other V0 subunits, indicating that assembly of V0 occurs in a defined sequence, with Vph1p recruitment into a Vma21p/proteolipid/Vma6p complex representing the final step. An in vitro assay for ER export was used to demonstrate preferential packaging of the fully assembled Vma21p/proteolipid/Vma6p/Vph1p complex into COPII-coated transport vesicles. Pulse-chase experiments showed that the interaction between Vma21p and V0 was transient and that Vma21p/V0 dissociation was concomitant with V0/V1 assembly. Blocking ER export in vivo stabilized the interaction between Vma21p and V0 and abrogated assembly of V0/V1. Although a Vma21p mutant lacking an ER-retrieval signal remained associated with V0 in the vacuole, this interaction did not affect the assembly of vacuolar V0/V1 complexes. We conclude that Vma21p is not involved in regulating the interaction between V0 and V1 sectors, but that it has a crucial role in coordinating the assembly of V0 subunits and in escorting the assembled V0 complex into ER-derived transport vesicles.

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