Single-Molecule Investigation of Rad-51 Presynaptic Filament Assembly and the Role of Mediator Proteins

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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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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Role of the ribosomal protein L27 revealed by single-molecule FRET study

Protein Science ◽

10.1002/pro.2149 ◽

2012 ◽

Vol 21 (11) ◽

pp. 1696-1704 ◽

Cited By ~ 7

Author(s):

Yuhong Wang ◽

Ming Xiao

Keyword(s):

Ribosomal Protein ◽

Single Molecule ◽

Single Molecule Fret

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Receptor compaction and GTPase rearrangement drive SRP-mediated cotranslational protein translocation into the ER

Science Advances ◽

10.1126/sciadv.abg0942 ◽

2021 ◽

Vol 7 (21) ◽

pp. eabg0942

Author(s):

Jae Ho Lee ◽

Ahmad Jomaa ◽

SangYoon Chung ◽

Yu-Hsien Hwang Fu ◽

Ruilin Qian ◽

...

Keyword(s):

Single Molecule ◽

Protein Translocation ◽

Molecular Model ◽

Genetic Diseases ◽

Signal Recognition Particle ◽

Biological Regulation ◽

Single Molecule Studies ◽

Guanosine Triphosphatase ◽

Gtpase Cycle

The conserved signal recognition particle (SRP) cotranslationally delivers ~30% of the proteome to the eukaryotic endoplasmic reticulum (ER). The molecular mechanism by which eukaryotic SRP transitions from cargo recognition in the cytosol to protein translocation at the ER is not understood. Here, structural, biochemical, and single-molecule studies show that this transition requires multiple sequential conformational rearrangements in the targeting complex initiated by guanosine triphosphatase (GTPase)–driven compaction of the SRP receptor (SR). Disruption of these rearrangements, particularly in mutant SRP54G226E linked to severe congenital neutropenia, uncouples the SRP/SR GTPase cycle from protein translocation. Structures of targeting intermediates reveal the molecular basis of early SRP-SR recognition and emphasize the role of eukaryote-specific elements in regulating targeting. Our results provide a molecular model for the structural and functional transitions of SRP throughout the targeting cycle and show that these transitions provide important points for biological regulation that can be perturbed in genetic diseases.

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The role of surface corrugation and tip oscillation in single-molecule manipulation with a non-contact atomic force microscope

Beilstein Journal of Nanotechnology ◽

10.3762/bjnano.5.22 ◽

2014 ◽

Vol 5 ◽

pp. 202-209 ◽

Cited By ~ 9

Author(s):

Christian Wagner ◽

Norman Fournier ◽

F Stefan Tautz ◽

Ruslan Temirov

Keyword(s):

Frequency Shift ◽

Single Molecule ◽

Oscillation Amplitude ◽

Surface Corrugation ◽

Atomic Force ◽

Data Points ◽

Atomic Surface ◽

Tetracarboxylic Dianhydride ◽

Single Molecule Manipulation

Scanning probe microscopy (SPM) plays an important role in the investigation of molecular adsorption. The possibility to probe the molecule–surface interaction while tuning its strength through SPM tip-induced single-molecule manipulation has particularly promising potential to yield new insights. We recently reported experiments, in which 3,4,9,10-perylene-tetracarboxylic-dianhydride (PTCDA) molecules were lifted with a qPlus-sensor and analyzed these experiments by using force-field simulations. Irrespective of the good agreement between the experiment and those simulations, systematic inconsistencies remained that we attribute to effects omitted from the initial model. Here we develop a more realistic simulation of single-molecule manipulation by non-contact AFM that includes the atomic surface corrugation, the tip elasticity, and the tip oscillation amplitude. In short, we simulate a full tip oscillation cycle at each step of the manipulation process and calculate the frequency shift by solving the equation of motion of the tip. The new model correctly reproduces previously unexplained key features of the experiment, and facilitates a better understanding of the mechanics of single-molecular junctions. Our simulations reveal that the surface corrugation adds a positive frequency shift to the measurement that generates an apparent repulsive force. Furthermore, we demonstrate that the scatter observed in the experimental data points is related to the sliding of the molecule across the surface.

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Multiple Roles of Flagellar Export Chaperones for Efficient and Robust Flagellar Filament Formation in Salmonella

Frontiers in Microbiology ◽

10.3389/fmicb.2021.756044 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tohru Minamino ◽

Yusuke V. Morimoto ◽

Miki Kinoshita ◽

Keiichi Namba

Keyword(s):

Type Iii Secretion ◽

Culture Media ◽

Multiple Roles ◽

Filament Formation ◽

Dynamic Interactions ◽

Filament Assembly ◽

Suppressor Mutations ◽

Folded Structure ◽

Flagellar Filament

FlgN, FliS, and FliT are flagellar export chaperones specific for FlgK/FlgL, FliC, and FliD, respectively, which are essential component proteins for filament formation. These chaperones facilitate the docking of their cognate substrates to a transmembrane export gate protein, FlhA, to facilitate their subsequent unfolding and export by the flagellar type III secretion system (fT3SS). Dynamic interactions of the chaperones with FlhA are thought to determine the substrate export order. To clarify the role of flagellar chaperones in filament assembly, we constructed cells lacking FlgN, FliS, and/or FliT. Removal of either FlgN, FliS, or FliT resulted in leakage of a large amount of unassembled FliC monomers into the culture media, indicating that these chaperones contribute to robust and efficient filament formation. The ∆flgN ∆fliS ∆fliT (∆NST) cells produced short filaments similarly to the ∆fliS mutant. Suppressor mutations of the ∆NST cells, which lengthened the filament, were all found in FliC and destabilized the folded structure of FliC monomer. Deletion of FliS inhibited FliC export and filament elongation only after FliC synthesis was complete. We propose that FliS is not involved in the transport of FliC upon onset of filament formation, but FliS-assisted unfolding of FliC by the fT3SS becomes essential for its rapid and efficient export to form a long filament when FliC becomes fully expressed in the cytoplasm.

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Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases

eLife ◽

10.7554/elife.18574 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 38

Author(s):

Cosimo Pinto ◽

Kristina Kasaciunaite ◽

Ralf Seidel ◽

Petr Cejka

Keyword(s):

Single Molecule ◽

Nuclease Activity ◽

Molecular Machine ◽

Dna Unwinding ◽

Metabolic Processes ◽

Nuclease Domain

Human DNA2 (hDNA2) contains both a helicase and a nuclease domain within the same polypeptide. The nuclease of hDNA2 is involved in a variety of DNA metabolic processes. Little is known about the role of the hDNA2 helicase. Using bulk and single-molecule approaches, we show that hDNA2 is a processive helicase capable of unwinding kilobases of dsDNA in length. The nuclease activity prevents the engagement of the helicase by competing for the same substrate, hence prominent DNA unwinding by hDNA2 alone can only be observed using the nuclease-deficient variant. We show that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine. This collectively suggests that the hDNA2 motor promotes the enzyme's capacity to degrade dsDNA in conjunction with BLM or WRN and thus promote the repair of broken DNA.

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TRPV channel OCR-2 is distributed along C. elegans chemosensory cilia by diffusion in a local interplay with intraflagellar transport

10.1101/2020.11.19.390005 ◽

2020 ◽

Author(s):

Jaap van Krugten ◽

Noémie Danné ◽

Erwin J.G. Peterman

Keyword(s):

Single Molecule ◽

Transmembrane Proteins ◽

Intraflagellar Transport ◽

Single Molecule Tracking ◽

C Elegans ◽

Normal Diffusion ◽

Cellular Signal ◽

Underlying Mechanisms ◽

And Function

AbstractSensing and reacting to the environment is essential for survival and procreation of most organisms. Caenorhabditis elegans senses soluble chemicals with transmembrane proteins (TPs) in the cilia of its chemosensory neurons. Development, maintenance and function of these cilia relies on intraflagellar transport (IFT), in which motor proteins transport cargo, including sensory TPs, back and forth along the ciliary axoneme. Here we use live fluorescence imaging to show that IFT machinery and the sensory TP OCR-2 reversibly redistribute along the cilium after exposure to repellant chemicals. To elucidate the underlying mechanisms, we performed single-molecule tracking experiments and found that OCR-2 distribution depends on an intricate interplay between IFT-driven transport, normal diffusion and subdiffusion that depends on the specific location in the cilium. These insights in the role of IFT on the dynamics of cellular signal transduction contribute to a deeper understanding of the regulation of sensory TPs and chemosensing.

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Dynamics of Tpm1.8 domains on actin filaments with single molecule resolution

10.1101/2020.06.15.152033 ◽

2020 ◽

Cited By ~ 1

Author(s):

Ilina Bareja ◽

Hugo Wioland ◽

Miro Janco ◽

Philip R. Nicovich ◽

Antoine Jégou ◽

...

Keyword(s):

Actin Filament ◽

Single Molecule ◽

Actin Filaments ◽

High Probability ◽

Actin Binding ◽

Single Molecule Level ◽

Filament Dynamics ◽

Kinetics Of ◽

Insight Into

ABSTRACTTropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

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