Detection of Intensity Change Points in Time-Resolved Single-Molecule Measurements

Watkins, Lucas P.; Yang, Haw

doi:10.1021/jp0467548

Cited by 321 publications

(498 citation statements)

References 53 publications

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“…This report represents the application of several recent developments in fluorescence single-molecule techniques based on information theory that use the information carried by each detected photon in an unbiased way (31,32,45). Our data-driven approach allows us to quantitatively account for photon-detection statistics, follow the time-dependent FRET distance changes photon-by-photon with the best possible time and distance resolution, and to unambiguously recover the entire FRET distance distribution.…”

Section: Discussionmentioning

confidence: 99%

“…To minimize error and bias in calculating time-dependent donor-acceptor distances from single-molecule trajectories (see SI Materials and Methods), a change-point analysis (45) was applied to the data to determine the precise moment at which the dyes bleach. Distance vs. time data were extracted from trajectories using the maximumlikelihood analysis method (31).…”

Section: Methodsmentioning

confidence: 99%

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Illuminating the mechanistic roles of enzyme conformational dynamics

Hanson

Duderstadt

Watkins

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Many enzymes mold their structures to enclose substrates in their active sites such that conformational remodeling may be required during each catalytic cycle. In adenylate kinase (AK), this involves a large-amplitude rearrangement of the enzyme's lid domain. Using our method of high-resolution single-molecule FRET, we directly followed AK's domain movements on its catalytic time scale. To quantitatively measure the enzyme's entire conformational distribution, we have applied maximum entropy-based methods to remove photon-counting noise from single-molecule data. This analysis shows unambiguously that AK is capable of dynamically sampling two distinct states, which correlate well with those observed by x-ray crystallography. Unexpectedly, the equilibrium favors the closed, active-site-forming configurations even in the absence of substrates. Our experiments further showed that interaction with substrates, rather than locking the enzyme into a compact state, restricts the spatial extent of conformational fluctuations and shifts the enzyme's conformational equilibrium toward the closed form by increasing the closing rate of the lid. Integrating these microscopic dynamics into macroscopic kinetics allows us to model lid opening-coupled product release as the enzyme's rate-limiting step.conformational equilibrium ͉ rate-limiting step ͉ single-molecule FRET ͉ adenylate kinase P roteins such as enzymes are flexible with a range of motions spanning from picoseconds for localized vibrations to seconds for concerted global conformational rearrangements (1). Despite their randomly fluctuating environment, in which stochastic collisions with solvent molecules drive changes in tertiary structure, enzymes have evolved to catalyze reactions efficiently and specifically. Indeed, conformational transitions have been postulated to play a central role in enzyme functions in a wide variety of ways, including direct contribution to catalysis (2), allosteric regulation (3), and large-scale conformational changes in response to ligand binding (4). Most of our current understanding of structural motions in solution comes from NMR experiments (5) as well as from molecular dynamics simulations (6), approaches that are best suited to study dynamics in the picoto millisecond time scales. Because catalysis in enzymes frequently occurs in the submillisecond to minute time regime, our current understanding of the relationship between enzyme function and conformational dynamics comes from NMR experiments involving relatively localized motions of active site forming loops on the submillisecond time scale (7-10). However, many enzymes contain active sites located in between domains in which large-amplitude, low-frequency domain motions are required to complete their Michaelis-Menten enzyme-substrate complexes. Even simple questions regarding these transitions remain generally unanswered: What is the number and range of conformational states accessible to enzymes during their catalytic cycle? How does the enzyme's conformation respond to interac...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Illuminating the mechanistic roles of enzyme conformational dynamics

Hanson

Duderstadt

Watkins

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

276

430

View full text Add to dashboard Cite

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“…S2b. Below the main concepts and procedures of the change point detection developed by Watkins et al 1 and employed in this work are summarized.…”

Section: Change Point Analysis Of the Simulated Photon Arrival Time Smentioning

confidence: 99%

“…Following Watkins et al, 1 the detection of an intensity change point for a given photon arrival time trace is treated as a hypothesis test problem. We compare statistically the following two hypotheses for each photon in the trace: a) the photon under consideration is not a change point (called the null hypothesis and denoted by H 0 ); b) the photon under consideration is a change point (called the alternative hypothesis and denoted by H A ).…”

Section: Change Point Detection As a Hypothesis Testmentioning

confidence: 99%

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Dynamic Disorder in Single-Enzyme Experiments: Facts and Artifacts

et al. 2011

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Using a single molecule fluorescence approach, the time series of catalytic events of an enzymatic reaction can be monitored yielding a sequence of fluorescent "on"- and "off"-states. An accurate on/off-assignment is complicated by the intrinsic and extrinsic noise in every single molecule fluorescence experiment. Using simulated data, the performance of the most widely employed binning and thresholding approach was systematically compared to change point analysis. It is shown that the underlying on- and off-histograms as well as the off-autocorrelation are not necessarily extracted from the "signal" buried in noise. The shapes of the on- and off-histograms are affected by artifacts introduced by the analysis procedure and depend on the signal-to-noise ratio and the overall fluorescence intensity. For experimental data where the background Intensity is not constant over time we consider change point analysis to be more accurate. When using change point analysis for data of the enzyme α-chymotrypsin, no characteristics of dynamic disorder were found. In light of these results, dynamic disorder might not be a general characteristic of enzymatic reactions

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Single‐Molecule FRET of Protein‐Folding Dynamics

Nettels

Schuler

2011

Single‐Molecule Biophysics

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Detection of Intensity Change Points in Time-Resolved Single-Molecule Measurements

Cited by 321 publications

References 53 publications

Illuminating the mechanistic roles of enzyme conformational dynamics

Illuminating the mechanistic roles of enzyme conformational dynamics

Dynamic Disorder in Single-Enzyme Experiments: Facts and Artifacts

Single‐Molecule FRET of Protein‐Folding Dynamics

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