Nanoscale high-content analysis using compositional heterogeneities of single proteoliposomes

Signe Mathiasen; Sune M. Christensen; Juan José Fung; Søren G.F. Rasmussen; Jonathan F. Fay; Sune K. Jorgensen; Salome Veshaguri; David L. Farrens; Maria Kiskowski; Brian Kobilka; Dimitrios Stamou

doi:10.1038/nmeth.3062

Nanoscale high-content analysis using compositional heterogeneities of single proteoliposomes

Signe Mathiasen, Sune M. Christensen, Juan José Fung, Søren G.F. Rasmussen, Jonathan F. Fay, Sune K. Jorgensen, Salome Veshaguri, David L. Farrens, Maria Kiskowski, Brian Kobilka, Dimitrios Stamou

Chemical Physiology and Biochemistry

Research output: Contribution to journal › Article › peer-review

54 Scopus citations

Abstract

Proteoliposome reconstitution is a standard method to stabilize purified transmembrane proteins in membranes for structural and functional assays. Here we quantified intrareconstitution heterogeneities in single proteoliposomes using fluorescence microscopy. Our results suggest that compositional heterogeneities can severely skew ensemble-average proteoliposome measurements but also enable ultraminiaturized high-content screens. We took advantage of this screening capability to map the oligomerization energy of the b₂-adrenergic receptor using ~10⁹-fold less protein than conventional assays.

Original language	English (US)
Pages (from-to)	931-934
Number of pages	4
Journal	Nature Methods
Volume	11
Issue number	9
DOIs	https://doi.org/10.1038/nmeth.3062
State	Published - 2014

ASJC Scopus subject areas

Biotechnology
Biochemistry
Molecular Biology
Cell Biology

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10.1038/nmeth.3062

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title = "Nanoscale high-content analysis using compositional heterogeneities of single proteoliposomes",

abstract = "Proteoliposome reconstitution is a standard method to stabilize purified transmembrane proteins in membranes for structural and functional assays. Here we quantified intrareconstitution heterogeneities in single proteoliposomes using fluorescence microscopy. Our results suggest that compositional heterogeneities can severely skew ensemble-average proteoliposome measurements but also enable ultraminiaturized high-content screens. We took advantage of this screening capability to map the oligomerization energy of the b2-adrenergic receptor using ~109-fold less protein than conventional assays.",

author = "Signe Mathiasen and Christensen, {Sune M.} and Fung, {Juan Jos{\'e}} and Rasmussen, {S{\o}ren G.F.} and Fay, {Jonathan F.} and Jorgensen, {Sune K.} and Salome Veshaguri and Farrens, {David L.} and Maria Kiskowski and Brian Kobilka and Dimitrios Stamou",

note = "Funding Information: This work was supported by the Lundbeck Foundation, the Danish Councils for Independent and Strategic Research and the University of Copenhagen programs of excellence “Single molecule Nanoscience,” “BioScaRT” and “UNIK-Synthetic Biology.” We thank E.M. Danielsen and H. Flyvbjerg for helpful discussion.",

year = "2014",

doi = "10.1038/nmeth.3062",

language = "English (US)",

volume = "11",

pages = "931--934",

journal = "Nature Methods",

issn = "1548-7091",

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T1 - Nanoscale high-content analysis using compositional heterogeneities of single proteoliposomes

AU - Mathiasen, Signe

AU - Christensen, Sune M.

AU - Fung, Juan José

AU - Rasmussen, Søren G.F.

AU - Fay, Jonathan F.

AU - Jorgensen, Sune K.

AU - Veshaguri, Salome

AU - Farrens, David L.

AU - Kiskowski, Maria

AU - Kobilka, Brian

AU - Stamou, Dimitrios

N1 - Funding Information: This work was supported by the Lundbeck Foundation, the Danish Councils for Independent and Strategic Research and the University of Copenhagen programs of excellence “Single molecule Nanoscience,” “BioScaRT” and “UNIK-Synthetic Biology.” We thank E.M. Danielsen and H. Flyvbjerg for helpful discussion.

PY - 2014

Y1 - 2014

N2 - Proteoliposome reconstitution is a standard method to stabilize purified transmembrane proteins in membranes for structural and functional assays. Here we quantified intrareconstitution heterogeneities in single proteoliposomes using fluorescence microscopy. Our results suggest that compositional heterogeneities can severely skew ensemble-average proteoliposome measurements but also enable ultraminiaturized high-content screens. We took advantage of this screening capability to map the oligomerization energy of the b2-adrenergic receptor using ~109-fold less protein than conventional assays.

AB - Proteoliposome reconstitution is a standard method to stabilize purified transmembrane proteins in membranes for structural and functional assays. Here we quantified intrareconstitution heterogeneities in single proteoliposomes using fluorescence microscopy. Our results suggest that compositional heterogeneities can severely skew ensemble-average proteoliposome measurements but also enable ultraminiaturized high-content screens. We took advantage of this screening capability to map the oligomerization energy of the b2-adrenergic receptor using ~109-fold less protein than conventional assays.

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Nanoscale high-content analysis using compositional heterogeneities of single proteoliposomes

Abstract

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