Quantitative modeling of self-oligomerization of proteins in the nuclear envelope by fluorescence fluctuation analysis.
Anal Biochem
; 582: 113359, 2019 10 01.
Article
en En
| MEDLINE
| ID: mdl-31279795
ABSTRACT
Analysis of fluorescence fluctuation data through the time-shifted mean-segmented Q (tsMSQ) analysis method has recently been shown to successfully identify protein oligomerization and mobility in the nuclear envelope by properly accounting for local volume fluctuations of the nuclear envelope within living cells. However, by its nature, tsMSQ produces correlated data which poses unique challenges for applying goodness of fit tests and obtaining parameter uncertainties from individual measurements. In this paper, we overcome these challenges by introducing bootstrap tsMSQ which involves randomly resampling the fluorescence intensity data to eliminate the correlations in the tsMSQ data. This analysis technique was verified in both the cytoplasm and the lumen of the nuclear envelope with well-characterized proteins that served as model systems. Uncertainties and goodness of fit tests of individual measurements were compared to estimates obtained from sampling multiple experiments. We further applied bootstrapping to fluorescence fluctuation data of the luminal domain of the SUN domain-containing protein 2 in order to characterize its self-oligomerization within the nuclear envelope. Analysis of the concentration-dependent brightness suggests a monomer-trimer transition of the protein.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Espectrometría de Fluorescencia
/
Proteínas Nucleares
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Péptidos y Proteínas de Señalización Intracelular
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Multimerización de Proteína
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Proteínas de la Membrana
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Membrana Nuclear
Límite:
Humans
Idioma:
En
Revista:
Anal Biochem
Año:
2019
Tipo del documento:
Article
País de afiliación:
Estados Unidos