Single-molecule mechanics of actomyosin measured by an optical tweezers ; Role of the neck region.

The structure of promoter chromatin determines the ability of transcription factors (TFs) to bind to DNA and therefore has a profound effect on the expression levels of genes. However, the role of spontaneous nucleosome movements in this process is not fully understood. Here, we developed a single-molecule optical tweezers assay capable of simultaneously characterizing the base pair-scale diffusion of a nucleosome on DNA and the binding of a TF, using the luteinizing hormone β subunit gene (Lhb) promoter and Egr-1 as a model system. Our results demonstrate that nucleosomes undergo confined diffusion, and that the incorporation of the histone variant H2A.Z serves to partially relieve this confinement, inducing a different type of nucleosome repositioning. The increase in diffusion leads to exposure of a TF’s binding site and facilitates its association with the DNA, which, in turn, biases the subsequent movement of the nucleosome. Our findings suggest the use of mobile nucleosomes as a general transcriptional regulatory mechanism.

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The regulatory function of the AAA4 ATPase domain of cytoplasmic dynein

10.1101/2020.09.20.305243 ◽

2020 ◽

Author(s):

Xinglei Liu ◽

Lu Rao ◽

Arne Gennerich

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Essential Role ◽

Cytoplasmic Dynein ◽

Regulatory Function ◽

Atpase Domain ◽

Single Molecule Fluorescence ◽

Mechanochemical Cycle ◽

Aaa Atpases

AbstractCytoplasmic dynein is the primary motor for microtubule minus-end-directed transport and is indispensable to eukaryotic cells. Although each motor domain of dynein contains three active AAA+ ATPases (AAA1, 3, and 4), only the functions of AAA1 and 3 are known. Here, we use single-molecule fluorescence and optical tweezers studies to elucidate the role of AAA4 in dynein’s mechanochemical cycle. We demonstrate that AAA4 controls the priming stroke of the motion-generating linker, which connects the dimerizing tail of the motor to the AAA+ ring. Before ATP binds to AAA4, dynein remains incapable of generating motion. However, when AAA4 is bound to ATP, the gating of AAA1 by AAA3 prevails and dynein motion can occur. Thus, AAA1, 3, and 4 work together to regulate dynein function. Our work elucidates an essential role for AAA4 in dynein’s stepping cycle and underscores the complexity and crosstalk among the motor’s multiple AAA+ domains.

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Role of the Kinesin Neck Region in Processive Microtubule-based Motility

The Journal of Cell Biology ◽

10.1083/jcb.140.6.1407 ◽

1998 ◽

Vol 140 (6) ◽

pp. 1407-1416 ◽

Cited By ~ 106

Author(s):

Laura Romberg ◽

Daniel W. Pierce ◽

Ronald D. Vale

Keyword(s):

Single Molecule ◽

Fluorescent Protein ◽

Catalytic Domain ◽

Coiled Coil ◽

Neck Region ◽

Structural Features ◽

Peptide Sequence ◽

Mechanochemical Cycle ◽

Green Fluorescent

Kinesin is a dimeric motor protein that can move along a microtubule for several microns without releasing (termed processive movement). The two motor domains of the dimer are thought to move in a coordinated, hand-over-hand manner. A region adjacent to kinesin's motor catalytic domain (the neck) contains a coiled coil that is sufficient for motor dimerization and has been proposed to play an essential role in processive movement. Recent models have suggested that the neck enables head-to-head communication by creating a stiff connection between the two motor domains, but also may unwind during the mechanochemical cycle to allow movement to new tubulin binding sites. To test these ideas, we mutated the neck coiled coil in a 560-amino acid (aa) dimeric kinesin construct fused to green fluorescent protein (GFP), and then assayed processivity using a fluorescence microscope that can visualize single kinesin–GFP molecules moving along a microtubule. Our results show that replacing the kinesin neck coiled coil with a 28-aa residue peptide sequence that forms a highly stable coiled coil does not greatly reduce the processivity of the motor. This result argues against models in which extensive unwinding of the coiled coil is essential for movement. Furthermore, we show that deleting the neck coiled coil decreases processivity 10-fold, but surprisingly does not abolish it. We also demonstrate that processivity is increased by threefold when the neck helix is elongated by seven residues. These results indicate that structural features of the neck coiled coil, although not essential for processivity, can tune the efficiency of single molecule motility.

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The regulatory function of the AAA4 ATPase domain of cytoplasmic dynein

Nature Communications ◽

10.1038/s41467-020-19477-3 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Xinglei Liu ◽

Lu Rao ◽

Arne Gennerich

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Essential Role ◽

Cytoplasmic Dynein ◽

Regulatory Function ◽

Atpase Domain ◽

Single Molecule Fluorescence ◽

Mechanochemical Cycle ◽

Aaa Atpases

AbstractCytoplasmic dynein is the primary motor for microtubule minus-end-directed transport and is indispensable to eukaryotic cells. Although each motor domain of dynein contains three active AAA+ ATPases (AAA1, 3, and 4), only the functions of AAA1 and 3 are known. Here, we use single-molecule fluorescence and optical tweezers studies to elucidate the role of AAA4 in dynein’s mechanochemical cycle. We demonstrate that AAA4 controls the priming stroke of the motion-generating linker, which connects the dimerizing tail of the motor to the AAA+ ring. Before ATP binds to AAA4, dynein remains incapable of generating motion. However, when AAA4 is bound to ATP, the gating of AAA1 by AAA3 prevails and dynein motion can occur. Thus, AAA1, 3, and 4 work together to regulate dynein function. Our work elucidates an essential role for AAA4 in dynein’s stepping cycle and underscores the complexity and crosstalk among the motor’s multiple AAA+ domains.

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Cytidine triphosphate promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional spreading from parS

10.1101/2021.02.11.430778 ◽

2021 ◽

Author(s):

Francisco de Asis Balaguer ◽

Clara Aicart-Ramos ◽

Gemma LM Fisher ◽

Sara de Bragança ◽

Cesar L. Pastrana ◽

...

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Dna Sequences ◽

Specific Interaction ◽

Magnetic Tweezers ◽

One Dimensional ◽

Sliding Clamp ◽

Absolute Requirement ◽

Bacterial Chromosomes

SUMMARYFaithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-like parS DNA sequences and the ParB CTPase. Using a combined dual optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced 4-fold by the presence of CTP or the non-hydrolysable analogue CTPγS. However, ParB proteins are also detected at a lower density in distal non-specific regions of DNA. This requires the presence of a parS loading site and is prevented by roadblocks on DNA, consistent with one dimensional diffusion by a sliding clamp. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation of parS-containing DNA molecules at low nanomolar protein concentrations. We propose a model in which ParB-CTP-Mg2+ complexes move along DNA following loading at parS sites and protein:protein interactions result in the localised condensation of DNA within ParB networks.

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The localized electron: magnetic properties

10.1093/oso/9780198814597.003.0002 ◽

2018 ◽

Author(s):

Jean-Pierre Launay ◽

Michel Verdaguer

Keyword(s):

Single Molecule ◽

Model Systems ◽

Ferromagnetic Coupling ◽

Single Chain ◽

Single Molecule Magnets ◽

Magnetic Systems ◽

Orbital Interactions ◽

Prussian Blue Analogues ◽

Mononuclear Complexes

After preliminaries about electron properties, and definitions in magnetism, one treats the magnetism of mononuclear complexes, in particular spin cross-over, showing the role of cooperativity and the sensitivity to external perturbations. Orbital interactions and exchange interaction are explained in binuclear model systems, using orbital overlap and orthogonality concepts to explain antiferromagnetic or ferromagnetic coupling. The phenomenologically useful Spin Hamiltonian is defined. The concepts are then applied to extended molecular magnetic systems, leading to molecular magnetic materials of various dimensionalities exhibiting bulk ferro- or ferrimagnetism. An illustration is provided by Prussian Blue analogues. Magnetic anisotropy is introduced. It is shown that in some cases, a slow relaxation of magnetization arises and gives rise to appealing single-ion magnets, single-molecule magnets or single-chain magnets, a route to store information at the molecular level.

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Optical tweezers in single-molecule biophysics

Nature Reviews Methods Primers ◽

10.1038/s43586-021-00021-6 ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Carlos J. Bustamante ◽

Yann R. Chemla ◽

Shixin Liu ◽

Michelle D. Wang

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Single Molecule Biophysics

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Optical tweezers in single-molecule biophysics

Nature Reviews Methods Primers ◽

10.1038/s43586-021-00027-0 ◽

2021 ◽

Vol 1 (1) ◽

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Single Molecule Biophysics

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Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

International Journal of Molecular Sciences ◽

10.3390/ijms22031018 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1018

Author(s):

Hiroaki Yokota

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Acid ◽

Single Molecule ◽

Underlying Mechanism ◽

Amino Acid Sequences ◽

E Coli ◽

X Ray Crystallography ◽

Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

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