Single-molecule spectroscopic methods

Haustein, Elke; Schwille, Petra

doi:10.1016/j.sbi.2004.09.004

Cited by 184 publications

(158 citation statements)

References 53 publications

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“…In FCS, the autocorrelation function, G(), measures the self-similarity of the fluorescent signal after a lag time () (40,55,56). Initially, attempts were made to fit the data using the autocorrelation function, G() (Equation 1), and fitting software provided by Zeiss.…”

Section: Methodsmentioning

confidence: 99%

Dynamic Imaging by Fluorescence Correlation Spectroscopy Identifies Diverse Populations of Polyglutamine Oligomers Formed in Vivo

Beam

Silva

Morimoto

2012

Journal of Biological Chemistry

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Background: Protein aggregation is implicated in numerous diseases. Results: Polyglutamine oligomers maintain a heterogeneous distribution that reaches an equilibrium during aging and can be altered by genetic modifiers that suppress aggregation. Conclusion: Shifts in populations of oligomers do not correlate with toxicity. Significance: The dynamics of oligomerization and aggregation to inert species is essential for protein misfolding and toxicity.

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Section: Methodsmentioning

confidence: 99%

Dynamic Imaging by Fluorescence Correlation Spectroscopy Identifies Diverse Populations of Polyglutamine Oligomers Formed in Vivo

Beam

Silva

Morimoto

2012

Journal of Biological Chemistry

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“…To study fast binding kinetics (on the timescale of a millisecond and less), solution-based approaches have been developed that are based on measuring the fluorescence intensity fluctuations of small numbers of labeled molecules traversing a focused laser beam [36,37]. Colocalization [38] and FRET-based [39] strategies can be used in combination with this fluorescence correlation spectroscopy (FCS) method to obtain accurate information on the kinetic and thermodynamic properties of supramolecular complex formation or ligand binding [40 ].…”

Section: Strategies To Visualize Binding Kineticsmentioning

confidence: 99%

Single-molecule approaches to characterizing kinetics of biomolecular interactions

Oijen

2011

Current Opinion in Biotechnology

113

111

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Link to publication in University of Groningen/UMCG research database Citation for published version (APA): van Oijen, A. M. (2011). Single-molecule approaches to characterizing kinetics of biomolecular interactions. Current Opinion in Biotechnology, 22(1), 75-80. DOI: 10.101675-80. DOI: 10. /j.copbio.2010 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Available online at www.sciencedirect.com Single-molecule approaches to characterizing kinetics of biomolecular interactions Antoine M van OijenSingle-molecule fluorescence techniques have emerged as powerful tools to study biological processes at the molecular level. This review describes the application of these methods to the characterization of the kinetics of interaction between biomolecules. A large number of single-molecule assays have been developed that visualize association and dissociation kinetics in vitro by fluorescently labeling binding partners and observing their interactions over time. Even though recent progress has been significant, there are certain limitations to this approach. To allow the observation of individual, fluorescently labeled molecules requires low, nanomolar concentrations. I will discuss how such concentration requirements in single-molecule experiments limit their applicability to investigate intermolecular interactions and how recent technical advances deal with this issue.

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“…The important issue is whether BAX activation requires protein oligomerization or whether active BAX conformation can be generated from a single protein monomer. To solve this issue we used two single-molecule sensitivity techniques: fluorescence correlation spectroscopy (FCS) (17) and fluorescence-intensity distribution analysis (FIDA) (18). Combined use of FCS and FIDA allows simultaneous determination of the apparent molecular weight and the number of fluorescently labeled BAX monomers per protein-detergent micelle.…”

Section: Baxmentioning

confidence: 99%

Detergent-activated BAX Protein Is a Monomer

Ivashyna

García‐Sáez

Ries

et al. 2009

Journal of Biological Chemistry

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BAX is a pro-apoptotic member of the BCL-2 protein family. At the onset of apoptosis, monomeric, cytoplasmic BAX is activated and translocates to the outer mitochondrial membrane, where it forms an oligomeric pore. The chemical mechanism of BAX activation is controversial, and several in vitro and in vivo methods of its activation are known. One of the most commonly used in vitro methods is activation withdetergents,suchasn-octylglucoside.DuringBAXactivationwith n-octyl glucoside, it has been shown that BAX forms high molecular weight complexes that are larger than the combined molecular weight of BAX monomer and one detergent micelle. These large complexes have been ascribed to the oligomerization of BAX prior to its membrane insertion and pore formation. This is in contrast to the in vivo studies that suggest that active BAX inserts into the outer mitochondrial membrane as a monomer and then undergoes oligomerization. Here, to simultaneously determine the molecular weight and the number of BAX proteins per BAX-detergent micelle during detergent activation, we have used an approach that combines two single-molecule sensitivity technique, fluorescence correlation spectroscopy, and fluorescence-intensity distribution analysis. We have tested a range of detergents as follows: n-octyl glucoside, dodecyl maltoside, Triton X-100, Tween 20, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and cholic acid. With these detergents we observe that BAX is a monomer before, during, and after interaction with micelles. We conclude that detergent activation of BAX is not congruent with oligomerization and that in physiologic buffer conditions BAX can assume two stable monomeric conformations, one inactive and one active.

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Single-molecule spectroscopic methods

Cited by 184 publications

References 53 publications

Dynamic Imaging by Fluorescence Correlation Spectroscopy Identifies Diverse Populations of Polyglutamine Oligomers Formed in Vivo

Dynamic Imaging by Fluorescence Correlation Spectroscopy Identifies Diverse Populations of Polyglutamine Oligomers Formed in Vivo

Single-molecule approaches to characterizing kinetics of biomolecular interactions

Detergent-activated BAX Protein Is a Monomer

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