Single molecule kinetics. II. Numerical Bayesian approach

Witkoskie, James B.; Cao, Jianshu

doi:10.1063/1.1785784

Cited by 37 publications

(32 citation statements)

References 18 publications

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“…At the same time, there has been a welcome increase in contributions from the theoretical community to the analysis and interpretation of single-molecule data (17,(119)(120)(121)(122)(123). With many new investigators continuing to enter the field, the applications of single-molecule fluorescence imaging methods should continue to expand.…”

Section: Discussionmentioning

confidence: 99%

New directions in single-molecule imaging and analysis

Moerner

2007

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

418

331

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Optical imaging and analysis of single molecules continue to unfold as powerful ways to study the individual behavior of biological systems, unobscured by ensemble averaging. Current expansion of interest in this field is great, as evidenced by new meetings, journal special issues, and the large number of new investigators. Selected recent advances in biomolecular analysis are described, and two new research directions are summarized: superresolution imaging using single-molecule fluorescence and trapping of single molecules in solution by direct suppression of Brownian motion.

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Section: Discussionmentioning

confidence: 99%

New directions in single-molecule imaging and analysis

Moerner

2007

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

418

331

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“…Alternating and pulsed lasers have been used to investigate dye bleaching, fluorescence lifetimes, quantum yield inhomogeneities, anisotropies, and dye-biomolecule stoichiometry [9–13]. Bayesian approaches [14–22] and complementary maximum entropy methods [23–25] have been implemented for single-molecule data analysis.…”

Section: Introductionmentioning

confidence: 99%

Reconstruction of calmodulin single-molecule FRET states, dye interactions, and CaMKII peptide binding by MultiNest and classic maximum entropy

DeVore¹,

Gull²,

Johnson³

2013

Chemical Physics

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We analyze single molecule FRET burst measurements using Bayesian nested sampling. The MultiNest algorithm produces accurate FRET efficiency distributions from single-molecule data. FRET efficiency distributions recovered by MultiNest and classic maximum entropy are compared for simulated data and for calmodulin labeled at residues 44 and 117. MultiNest compares favorably with maximum entropy analysis for simulated data, judged by the Bayesian evidence. FRET efficiency distributions recovered for calmodulin labeled with two different FRET dye pairs depended on the dye pair and changed upon Ca2+ binding. We also looked at the FRET efficiency distributions of calmodulin bound to the calcium/calmodulin dependent protein kinase II (CaMKII) binding domain. For both dye pairs, the FRET efficiency distribution collapsed to a single peak in the case of calmodulin bound to the CaMKII peptide. These measurements strongly suggest that consideration of dye-protein interactions is crucial in forming an accurate picture of protein conformations from FRET data.

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“…New methods are needed to bring out the underlying information on kinetics, dynamics, and conformations. Work is starting to appear along these lines based on maximum likelihood estimators and statistical analysis (65)(66)(67)(68)(69)(70). Other recent analytical approaches are aimed at improved precision of measurement in analysis of FRET efficiency distributions (71).…”

Section: Fret In Single Moleculesmentioning

confidence: 99%

Calmodulin, Conformational States, and Calcium Signaling. A Single-Molecule Perspective

Johnson

2006

Biochemistry

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Single-molecule fluorescence measurements can provide a new perspective on the conformations, dynamics, and interactions of proteins. Recent examples are described illustrating the application of single-molecule fluorescence spectroscopy to calcium signaling proteins with an emphasis on the new information available in single-molecule fluorescence burst measurements, resonance energy transfer, and polarization modulation methods. Calcium signaling pathways are crucial in many cellular processes. The calcium binding protein calmodulin (CaM) serves as a molecular switch to regulate a network of calcium signaling pathways. Single-molecule spectroscopic methods can yield insights into conformations and dynamics of CaM and CaM-regulated proteins. Examples include studies of the conformations and dynamics of CaM, binding of target peptides, and interaction with the plasma-membrane Ca 2+ pump. Single-molecule resonance energy transfer measurements revealed conformational substates of CaM, and single-molecule polarization modulation spectroscopy was used to probe interactions between CaM and the plasma-membrane Ca 2+ -ATPase.Single-molecule fluorescence methods have the potential to resolve details about protein conformations, interactions, and dynamics that were previously hidden by ensemble averaging. As the practice of single-molecule methods expands, it is useful to examine what new information can be obtained about biomolecules and what the limitations of the methods are. This paper will examine these questions in the context of recent applications of single-molecule fluorescence techniques in the author's laboratory to the conformations, dynamics, and interactions of the calcium signaling protein calmodulin (CaM) (see Fig. 1).Proteins are dynamic molecules. Multiple conformational states are required for them to function. CaM is no exception, and appears to sample an unusually wide range of conformations (1). How can the distribution of such conformations be characterized? Conventional bulk measurements detect the average properties of many (typically > 10 10 ) molecules and therefore do not resolve the range of conformations that are actually present. One way to attack this problem is to detect and characterize molecules one at a time.Introduced in the early 1990s (2-6), single-molecule fluorescence methods can now address real problems in biology (see recent reviews (7-10)). Conformational states and conformational motions can be detected that may be missed by other methods. Dynamic processes can be probed under either equilibrium or non-equilibrium conditions by following the fluctuations of an appropriate single-molecule spectroscopic signal. In kinetic *To whom correspondence should be addressed. E-mail: ckjohnson@ku.edu. † We acknowledge support for this research from NIH R01 GM58715, the American Heart Association (99513262 and 0455487Z) and a Research Corporation Research Opportunity Award. B.D.S. and J.R.U. acknowledge support from training grant NIH GM08545, and E.S.P. acknowledges support from t...

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Single molecule kinetics. II. Numerical Bayesian approach

Cited by 37 publications

References 18 publications

New directions in single-molecule imaging and analysis

New directions in single-molecule imaging and analysis

Reconstruction of calmodulin single-molecule FRET states, dye interactions, and CaMKII peptide binding by MultiNest and classic maximum entropy

Calmodulin, Conformational States, and Calcium Signaling. A Single-Molecule Perspective

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