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Detection and Analysis of Protein Aggregation with Confocal Single Molecule Fluorescence Spectroscopy


Hillger, Frank; Nettels, Daniel; Dorsch, Simone; Schuler, Benjamin (2007). Detection and Analysis of Protein Aggregation with Confocal Single Molecule Fluorescence Spectroscopy. Journal of Fluorescence, 17(6):759-765.

Abstract

The misfolding and aggregation of proteins is a common phenomenon both in the cell, in in vitro protein refolding, and the corresponding biotechnological applications. Most importantly, it is involved in a wide range of diseases, including some of the most prevalent neurodegenerative disorders. However, the range of methods available to analyze this highly heterogeneous process and the resulting aggregate structures has been very limited. Here we present an approach that uses confocal single molecule detection of FRET-labeled samples employing four detection channels to obtain information about diffusivity, anisotropy, fluorescence lifetimes and Förster transfer efficiencies from a single measurement. By combining these observables, this method allows the separation of subpopulations of folded and misfolded proteins in solution with high sensitivity and a differentiation of aggregates generated under different conditions. We demonstrate the versatility of the method with experiments on rhodanese, an aggregation-prone two-domain protein

Abstract

The misfolding and aggregation of proteins is a common phenomenon both in the cell, in in vitro protein refolding, and the corresponding biotechnological applications. Most importantly, it is involved in a wide range of diseases, including some of the most prevalent neurodegenerative disorders. However, the range of methods available to analyze this highly heterogeneous process and the resulting aggregate structures has been very limited. Here we present an approach that uses confocal single molecule detection of FRET-labeled samples employing four detection channels to obtain information about diffusivity, anisotropy, fluorescence lifetimes and Förster transfer efficiencies from a single measurement. By combining these observables, this method allows the separation of subpopulations of folded and misfolded proteins in solution with high sensitivity and a differentiation of aggregates generated under different conditions. We demonstrate the versatility of the method with experiments on rhodanese, an aggregation-prone two-domain protein

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:National licences > 142-005
Dewey Decimal Classification:Unspecified
Scopus Subject Areas:Life Sciences > Biochemistry
Social Sciences & Humanities > Clinical Psychology
Social Sciences & Humanities > Social Sciences (miscellaneous)
Social Sciences & Humanities > Sociology and Political Science
Physical Sciences > Spectroscopy
Life Sciences > Clinical Biochemistry
Social Sciences & Humanities > Law
Language:English
Date:7 November 2007
Deposited On:03 Jul 2019 12:50
Last Modified:31 Jul 2020 02:49
Publisher:Springer
ISSN:1053-0509
OA Status:Green
Publisher DOI:https://doi.org/10.1007/s10895-007-0187-z
Related URLs:https://www.swissbib.ch/Search/Results?lookfor=nationallicencespringer101007s108950070187z (Library Catalogue)
PubMed ID:17447125

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