scholarly journals CHD4 slides nucleosomes by decoupling entry- and exit-side DNA translocation

2019 ◽  
Author(s):  
Yichen Zhong ◽  
Bishnu Prasad Paudel ◽  
Daniel P. Ryan ◽  
Jason K. K. Low ◽  
Charlotte Franck ◽  
...  
Keyword(s):  
Binding Energy ◽  
Complex Formation ◽  
Single Molecule ◽  
Conformational Changes ◽  
Published Data ◽  
Entry And Exit ◽  
Dna Translocation ◽  
Exit Side ◽  
Nucleosomal Dna ◽  
Histone Octamers

SummaryChromatin remodellers hydrolyse ATP to move nucleosomal DNA against histone octamers. The mechanism, however, is only partially resolved, and unclear if it is conserved among the four remodeller families. Here we use single-molecule assays to examine the mechanism of action of CHD4, which is part of the least well understood family of remodellers. We demonstrate that the binding energy for CHD4-nucleosome complex formation – even in the absence of nucleotide – triggers significant conformational changes in DNA at the entry side, effectively priming the system for remodelling. During remodelling, flanking DNA enters the nucleosome in a continuous, gradual manner but exits in concerted 4–6 base-pair steps. This decoupling of entry- and exit-side translocation suggests that ATP-driven movement of entry-side DNA builds up strain inside the nucleosome that is subsequently released at the exit side by DNA expulsion. We propose a mechanism for nucleosome sliding based on these and published data.

scholarly journals Conformational Change of Syntaxin-3b in Regulating SNARE Complex Assembly in the Ribbon Synapses

2021 ◽  
Author(s):  
Claire Gething ◽  
Joshua Ferrar ◽  
Bishal Misra ◽  
Giovanni Howells ◽  
Ucheor B. Choi
Keyword(s):  
Plasma Membrane ◽  
Complex Formation ◽  
Synaptic Vesicle ◽  
Conformational Change ◽  
Single Molecule ◽  
Conformational Changes ◽  
Snare Complex ◽  
Complex Assembly ◽  
Ribbon Synapses ◽  
Snare Complex Formation

AbstractNeurotransmitter release of synaptic vesicles relies on the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of syntaxin and SNAP-25 on the plasma membrane and synaptobrevin on the synaptic vesicle. The formation of the SNARE complex progressively zippers towards the membranes, which drives membrane fusion between the plasma membrane and the synaptic vesicle. However, the underlying molecular mechanism of SNARE complex regulation is unclear. In this study, we investigate the syntaxin-3b isoform found in the retinal ribbon synapses using single-molecule fluorescence resonance energy transfer (smFRET) to monitor the conformational changes of syntaxin-3b that modulate the SNARE complex formation. We found that syntaxin-3b is predominantly in a self-inhibiting closed conformation, inefficiently forming the ternary SNARE complex. Conversely, a phosphomimetic mutation (T14E) at the N-terminal region of syntaxin-3b promoted the open conformation, similar to the constitutively open form of syntaxin LE mutant. When syntaxin-3b is bound to Munc18-1, SNARE complex formation is almost completely blocked. Surprisingly, the T14E mutation of syntaxin-3b partially abolishes Munc18-1 regulation, acting as a conformational switch to trigger SNARE complex assembly. Thus, we suggest a model where the conformational change of syntaxin-3b induced by phosphorylation initiates the release of neurotransmitters in the ribbon synapses.

Single Molecule Manipulation and Sensing with the Atomic Force Microscope

2000 ◽  
Vol 6 (S2) ◽  
pp. 972-973
Author(s):  
S.M. Lindsay ◽  
S. H. Leuba ◽  
J. Zlatanova ◽  
M. A. Karymov ◽  
R. Bash ◽  
...  
Keyword(s):  
Atomic Force Microscope ◽  
Single Molecule ◽  
Optical Tweezer ◽  
Linker Histones ◽  
Atomic Force ◽  
Nucleosomal Dna ◽  
Order Of Magnitude ◽  
Force Curves ◽  
Histone Octamers ◽  
Glass Support

The atomic force microscope (AFM) can be used both to manipulate molecules and to make measurements on individual molecules. Here, we describe manipulation of chromatin constructs as an example of the first type of measurement, and electronic measurements on single molecules as an example of the second type.An AFM was used to both image and stretch single synthetic chromatin fibers consisting of twelve core nucleosomes with no linker histones. Peaks in the force-curves are attributed to sequential detachment of nucleosomes from the glass support (Figure 1). The short distances between peaks and reversibility of the pulling process show that the nucleosomes remain intact even at tensions on the order of 350 picoNewtons (pN). This is more than an order of magnitude larger than the force required to de-spool histone octamers from the nucleosomal DNA in laser optical tweezer measurements made with longer molecules, suggesting that loading rates and sample size are important factors in determining the force required to break inter-molecular bonds.

scholarly journals Mapping protein binding stability on nucleosomal DNA by single-molecule approach

2014 ◽  
Vol 70 (a1) ◽  
pp. C111-C111
Author(s):  
Jianshi Jin ◽  
Teng-fei Lian ◽  
Xiaoliang Xie ◽  
Xiao-Dong Su
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interaction ◽  
Binding Sites ◽  
Binding Domain ◽  
Shielding Effect ◽  
Dna Binding Domain ◽  
Nucleosomal Dna ◽  
Dna Protein Interaction

The conformation of nucleosomal DNA is significantly different from that of a canonical B-form double stranded DNA (dsDNA), and is generally regarded to be less flexible and less accessible than free dsDNA due to the tight association of histone cores. Previous studies have demonstrated that the key mechanism involved in nucleosomal DNA-protein interaction is the protein accessibility to the DNA binding site. In this work, we used single molecule assays to measure the stability of two transcriptional factors (glucocorticoid receptor DNA binding domain (GRDBD) and estrogen receptor DNA-binding domain (ERDBD)) bound to their binding sites on different positions of the nucleosomal DNA. Interestingly, the results demonstrated that the nucleosomal DNA-GRDBD binding is not always consistent with the histone shielding effect, but adjusted by additional structural changes. Furthermore, the changes of these DNA-GRDBD interaction profiles were confirmed using molecular modeling and docking approaches based on their crystal structures. Very differently, ERDBD essentially is unable to bind to the nucleosomal DNA anywhere including the unblocked positions. We thus have concluded that the nucleosomal DNA-protein interaction is regulated not only by the histone shielding of the DNA binding sites, but also by the conformational changes of the nucleosomal DNA.

scholarly journals Structure and mechanism of the ATPase that powers viral genome packaging

2015 ◽  
Vol 112 (29) ◽  
pp. E3792-E3799 ◽  
Author(s):  
Brendan J. Hilbert ◽  
Janelle A. Hayes ◽  
Nicholas P. Stone ◽  
Caroline M. Duffy ◽  
Banumathi Sankaran ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Model ◽  
Atp Hydrolysis ◽  
Mechanistic Model ◽  
Published Data ◽  
Dna Translocation ◽  
Genome Packaging ◽  
Large Rotation ◽  
Arginine Finger ◽  
Motor Structure

Many viruses package their genomes into procapsids using an ATPase machine that is among the most powerful known biological motors. However, how this motor couples ATP hydrolysis to DNA translocation is still unknown. Here, we introduce a model system with unique properties for studying motor structure and mechanism. We describe crystal structures of the packaging motor ATPase domain that exhibit nucleotide-dependent conformational changes involving a large rotation of an entire subdomain. We also identify the arginine finger residue that catalyzes ATP hydrolysis in a neighboring motor subunit, illustrating that previous models for motor structure need revision. Our findings allow us to derive a structural model for the motor ring, which we validate using small-angle X-ray scattering and comparisons with previously published data. We illustrate the model’s predictive power by identifying the motor’s DNA-binding and assembly motifs. Finally, we integrate our results to propose a mechanistic model for DNA translocation by this molecular machine.

scholarly journals CHD4 slides nucleosomes by decoupling entry- and exit-side DNA translocation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Yichen Zhong ◽  
Bishnu P. Paudel ◽  
Daniel P. Ryan ◽  
Jason K. K. Low ◽  
Charlotte Franck ◽  
...  
Keyword(s):  
Entry And Exit ◽  
Dna Translocation ◽  
Exit Side

scholarly journals Studies of Conformational Changes of Tubulin Induced by Interaction with Kinesin Using Atomistic Molecular Dynamics Simulations

2021 ◽  
Vol 22 (13) ◽  
pp. 6709
Author(s):  
Xiao-Xuan Shi ◽  
Peng-Ye Wang ◽  
Hong Chen ◽  
Ping Xie
Keyword(s):  
Molecular Dynamics ◽  
High Resolution ◽  
Binding Energy ◽  
Molecular Dynamics Simulations ◽  
Conformational Changes ◽  
Weak Interactions ◽  
Molecular Motor ◽  
Structural Data ◽  
Calculated Binding Energy ◽  
Dynamics Simulations

The transition between strong and weak interactions of the kinesin head with the microtubule, which is regulated by the change of the nucleotide state of the head, is indispensable for the processive motion of the kinesin molecular motor on the microtubule. Here, using all-atom molecular dynamics simulations, the interactions between the kinesin head and tubulin are studied on the basis of the available high-resolution structural data. We found that the strong interaction can induce rapid large conformational changes of the tubulin, whereas the weak interaction cannot. Furthermore, we found that the large conformational changes of the tubulin have a significant effect on the interaction of the tubulin with the head in the weak-microtubule-binding ADP state. The calculated binding energy of the ADP-bound head to the tubulin with the large conformational changes is only about half that of the tubulin without the conformational changes.

scholarly journals Real-time analysis of RAG complex activity in V(D)J recombination

2016 ◽  
Vol 113 (42) ◽  
pp. 11853-11858 ◽  
Author(s):  
Jennifer Zagelbaum ◽  
Noriko Shimazaki ◽  
Zitadel Anne Esguerra ◽  
Go Watanabe ◽  
Michael R. Lieber ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Conformational Changes ◽  
Signal Sequence ◽  
High Mobility ◽  
Time Analysis ◽  
Complex Activity ◽  
Synaptic Complex ◽  
Single Molecule Fret ◽  
Real Time Analysis

Single-molecule FRET (smFRET) and single-molecule colocalization (smCL) assays have allowed us to observe the recombination-activating gene (RAG) complex reaction mechanism in real time. Our smFRET data have revealed distinct bending modes at recombination signal sequence (RSS)-conserved regions before nicking and synapsis. We show that high mobility group box 1 (HMGB1) acts as a cofactor in stabilizing conformational changes at the 12RSS heptamer and increasing RAG1/2 binding affinity for 23RSS. Using smCL analysis, we have quantitatively measured RAG1/2 dwell time on 12RSS, 23RSS, and non-RSS DNA, confirming a strict RSS molecular specificity that was enhanced in the presence of a partner RSS in solution. Our studies also provide single-molecule determination of rate constants that were previously only possible by indirect methods, allowing us to conclude that RAG binding, bending, and synapsis precede catalysis. Our real-time analysis offers insight into the requirements for RSS–RSS pairing, architecture of the synaptic complex, and dynamics of the paired RSS substrates. We show that the synaptic complex is extremely stable and that heptamer regions of the 12RSS and 23RSS substrates in the synaptic complex are closely associated in a stable conformational state, whereas nonamer regions are perpendicular. Our data provide an enhanced and comprehensive mechanistic description of the structural dynamics and associated enzyme kinetics of variable, diversity, and joining [V(D)J] recombination.

scholarly journals Analysis of Protein Complex Formation on Membrane Surfaces by Single Molecule Diffusion

2012 ◽  
Vol 102 (3) ◽  
pp. 183a
Author(s):  
Brian P. Ziemba ◽  
Jefferson D. Knight ◽  
Joseph J. Falke
Keyword(s):  
Complex Formation ◽  
Single Molecule ◽  
Protein Complex ◽  
Membrane Surfaces ◽  
Protein Complex Formation
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