Dwell Time Analysis of a Single-Molecule Mechanochemical Reaction†

Langmuir ◽  
2008 ◽  
Vol 24 (4) ◽  
pp. 1356-1364 ◽  
Author(s):  
Robert Szoszkiewicz ◽  
Sri Rama Koti Ainavarapu ◽  
Arun P. Wiita ◽  
Raul Perez-Jimenez ◽  
Jose M. Sanchez-Ruiz ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dwell Time ◽  
Mechanochemical Reaction ◽  
Time Analysis

Single-Molecule Force-Clamp Spectroscopy: Dwell Time Analysis and Practical Considerations†

Langmuir ◽  
2011 ◽  
Vol 27 (4) ◽  
pp. 1440-1447 ◽  
Author(s):  
Yi Cao ◽  
Hongbin Li
Keyword(s):  
Single Molecule ◽  
Dwell Time ◽  
Time Analysis ◽  
Force Clamp

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

10.3791/62112 ◽  
2021 ◽  
Author(s):  
Candice M. Etson ◽  
Petar Todorov ◽  
Nooshin Shatery Nejad ◽  
Nirmala Shrestha ◽  
David R. Walt
Keyword(s):  
Restriction Endonuclease ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Dwell Time ◽  
Time Analysis

scholarly journals Power-law behavior of transcription factor dynamics at the single-molecule level implies a continuum affinity model

2021 ◽  
Author(s):  
David A Garcia ◽  
Gregory Fettweis ◽  
Diego M Presman ◽  
Ville Paakinaho ◽  
Christopher Jarzynski ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Single Molecule ◽  
Power Law ◽  
Dwell Time ◽  
Specific Binding ◽  
Power Law Distribution ◽  
Single Molecule Level ◽  
Binding Behavior ◽  
Chromatin Template ◽  
Nuclear Domains

Abstract Single-molecule tracking (SMT) allows the study of transcription factor (TF) dynamics in the nucleus, giving important information regarding the diffusion and binding behavior of these proteins in the nuclear environment. Dwell time distributions obtained by SMT for most TFs appear to follow bi-exponential behavior. This has been ascribed to two discrete populations of TFs—one non-specifically bound to chromatin and another specifically bound to target sites, as implied by decades of biochemical studies. However, emerging studies suggest alternate models for dwell-time distributions, indicating the existence of more than two populations of TFs (multi-exponential distribution), or even the absence of discrete states altogether (power-law distribution). Here, we present an analytical pipeline to evaluate which model best explains SMT data. We find that a broad spectrum of TFs (including glucocorticoid receptor, oestrogen receptor, FOXA1, CTCF) follow a power-law distribution of dwell-times, blurring the temporal line between non-specific and specific binding, suggesting that productive binding may involve longer binding events than previously believed. From these observations, we propose a continuum of affinities model to explain TF dynamics, that is consistent with complex interactions of TFs with multiple nuclear domains as well as binding and searching on the chromatin template.

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.

How Powerful is the Dwell-Time Analysis of Multichannel Records?

1998 ◽  
Vol 165 (1) ◽  
pp. 19-35 ◽  
Author(s):  
R. Blunck ◽  
U. Kirst ◽  
T. Riessner ◽  
U.-P. Hansen
Keyword(s):  
Dwell Time ◽  
Time Analysis

Stability Analysis for a Class of Linear Switched Positive Systems

2012 ◽  
Vol 562-564 ◽  
pp. 2084-2087
Author(s):  
Hui Ding ◽  
Xu Yang Lou
Keyword(s):  
Lyapunov Functions ◽  
Dwell Time ◽  
Positive Systems ◽  
Time Analysis ◽  
Arbitrary Switching ◽  
The Family ◽  
The Common ◽  
The Stability ◽  
Switched Positive Systems ◽  
Explicit Relation

This paper addresses stability properties of linear switched positive systems composed of continuous-time subsystems and discrete-time subsystems. Based on the common linear copositive Lyapunov functions, stability of the positive systems is discussed under arbitrary switching. Moreover, a sufficient condition on the minimum dwell time that guarantees the stability of linear switched positive systems. The dwell time analysis interprets the stability of linear switched positive systems through the distance between the eigenvector sets. Thus, an explicit relation in view of stability is obtained between the family of the involved subsystems and the set of admissible switching signals.

scholarly journals Anomalous protein kinetics on low-fouling surfaces

2020 ◽  
Vol 22 (9) ◽  
pp. 5264-5271
Author(s):  
Mohammadhasan Hedayati ◽  
Matt J. Kipper ◽  
Diego Krapf
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Single Molecule ◽  
Bovine Serum ◽  
Time Distribution ◽  
Dwell Time ◽  
Protein Kinetics ◽  
Single Molecule Tracking ◽  
Heavy Tailed ◽  
Protein Bovine Serum Albumin

Single-molecule tracking reveals the protein bovine serum albumin exhibits anomalous kinetics with a heavy-tailed dwell time distribution on PEG surfaces. This effect is shown to be caused by the ability of the protein to oligomerize in solution.

scholarly journals Single-molecule tracking of tau reveals fast kiss-and-hop interaction with microtubules in living neurons

2014 ◽  
Vol 25 (22) ◽  
pp. 3541-3551 ◽  
Author(s):  
Dennis Janning ◽  
Maxim Igaev ◽  
Frederik Sündermann ◽  
Jörg Brühmann ◽  
Oliver Beutel ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dwell Time ◽  
Quantitative Imaging ◽  
Fluorescence Decay ◽  
Microtubule Dynamics ◽  
Microtubule Assembly ◽  
Apparent Paradox ◽  
Single Molecule Tracking ◽  
Dependent Transport ◽  
Living Neurons

The microtubule-associated phosphoprotein tau regulates microtubule dynamics and is involved in neurodegenerative diseases collectively called tauopathies. It is generally believed that the vast majority of tau molecules decorate axonal microtubules, thereby stabilizing them. However, it is an open question how tau can regulate microtubule dynamics without impeding microtubule-dependent transport and how tau is also available for interactions other than those with microtubules. Here we address this apparent paradox by fast single-molecule tracking of tau in living neurons and Monte Carlo simulations of tau dynamics. We find that tau dwells on a single microtubule for an unexpectedly short time of ∼40 ms before it hops to the next. This dwell time is 100-fold shorter than previously reported by ensemble measurements. Furthermore, we observed by quantitative imaging using fluorescence decay after photoactivation recordings of photoactivatable GFP–tagged tubulin that, despite this rapid dynamics, tau is capable of regulating the tubulin–microtubule balance. This indicates that tau's dwell time on microtubules is sufficiently long to influence the lifetime of a tubulin subunit in a GTP cap. Our data imply a novel kiss-and-hop mechanism by which tau promotes neuronal microtubule assembly. The rapid kiss-and-hop interaction explains why tau, although binding to microtubules, does not interfere with axonal transport.

scholarly journals Introducing Point-of-Interest as an alternative to Area-of-Interest for fixation duration analysis

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0250170
Author(s):  
Nak Won Rim ◽  
Kyoung Whan Choe ◽  
Coltan Scrivner ◽  
Marc G. Berman
Keyword(s):  
Eye Tracking ◽  
Social Interactions ◽  
Dwell Time ◽  
Fixation Duration ◽  
Weighted Sum ◽  
Time Analysis ◽  
Point Of Interest ◽  
Area Of Interest ◽  
Points Of Interest ◽  
Alternative Approach

Many eye-tracking data analyses rely on the Area-of-Interest (AOI) methodology, which utilizes AOIs to analyze metrics such as fixations. However, AOI-based methods have some inherent limitations including variability and subjectivity in shape, size, and location of AOIs. In this article, we propose an alternative approach to the traditional AOI dwell time analysis: Weighted Sum Durations (WSD). This approach decreases the subjectivity of AOI definitions by using Points-of-Interest (POI) while maintaining interpretability. In WSD, the durations of fixations toward each POI is weighted by the distance from the POI and summed together to generate a metric comparable to AOI dwell time. To validate WSD, we reanalyzed data from a previously published eye-tracking study (n = 90). The re-analysis replicated the original findings that people gaze less towards faces and more toward points of contact when viewing violent social interactions.

scholarly journals Mapping the stochastic sequence of individual ligand-receptor binding events to cellular activation: T cells act on the rare events

2019 ◽  
Vol 12 (564) ◽  
pp. eaat8715 ◽  
Author(s):  
Jenny J. Y. Lin ◽  
Shalini T. Low-Nam ◽  
Katherine N. Alfieri ◽  
Darren B. McAffee ◽  
Nicole C. Fay ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Single Molecule ◽  
Cellular Response ◽  
Dwell Time ◽  
Cell Activation ◽  
Cell Receptor ◽  
Cellular Activation ◽  
Spatially Correlated ◽  
Order Of Magnitude

T cell receptor (TCR) binding to agonist peptide major histocompatibility complex (pMHC) triggers signaling events that initiate T cell responses. This system is remarkably sensitive, requiring only a few binding events to successfully activate a cellular response. On average, activating pMHC ligands exhibit mean dwell times of at least a few seconds when bound to the TCR. However, a T cell accumulates pMHC-TCR interactions as a stochastic series of discrete, single-molecule binding events whose individual dwell times are broadly distributed. With activation occurring in response to only a handful of such binding events, individual cells are unlikely to experience the average binding time. Here, we mapped the ensemble of pMHC-TCR binding events in space and time while simultaneously monitoring cellular activation. Our findings revealed that T cell activation hinges on rare, long–dwell time binding events that are an order of magnitude longer than the average agonist pMHC-TCR dwell time. Furthermore, we observed that short pMHC-TCR binding events that were spatially correlated and temporally sequential led to cellular activation. These observations indicate that T cell antigen discrimination likely occurs by sensing the tail end of the pMHC-TCR binding dwell time distribution rather than its average properties.

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