Introducing a fluorescence-based standard to quantify protein partitioning into membranes

Franziska A. Thomas, Ilaria Visco, Zdeněk Petrášek, Fabian Heinemann, Petra Schwille

Research output: Contribution to journalArticleResearchpeer-review

Abstract

The affinity of peripheral membrane proteins for a lipid bilayer can be described using the partition coefficient (KP). Although several methods to determine KP are known, all possess limitations. To address some of these issues, we developed both: a versatile method based on single molecule detection and fluorescence imaging for determining KP, and a simple measurement standard employing hexahistidine-tagged enhanced green fluorescent protein (eGFP-His6) and free standing membranes of giant unilamellar vesicles (GUVs) functionalized with NTA(Ni) lipids as binding sites. To ensure intrinsic control, our method features two measurement modes. In the single molecule mode, fluorescence correlation spectroscopy (FCS) is applied to quantify free and membrane associated protein concentrations at equilibrium and calculate KP. In the imaging mode, confocal fluorescence images of GUVs are recorded and analyzed with semi-automated software to extract protein mean concentrations used to derive KP. Both modes were compared by determining the affinity of our standard, resulting in equivalent KP values. As observed in other systems, eGFP-His6 affinity for membranes containing increasing amounts of NTA(Ni) lipids rises in a stronger-than-linear fashion. We compared our dual approach with a FCS-based assay that uses large unilamellar vesicles (LUVs), which however fails to capture the stronger-than-linear trend for our NTA(Ni)-His6 standard. Hence, we determined the KP of the MARCKS effector domain with our FCS approach on GUVs, whose results are consistent with previously published data using LUVs. We finally provide a practical manual on how to measure KP and understand it in terms of molecules per lipid surface.

Original languageEnglish
Pages (from-to)2932-2941
Number of pages10
JournalBiochimica et Biophysica Acta / Biomembranes
Volume1848
Issue number11
DOIs
Publication statusPublished - 1 Nov 2015

Fingerprint

Unilamellar Liposomes
Fluorescence
Fluorescence Spectrometry
Membranes
His-His-His-His-His-His
Spectroscopy
Proteins
Lipids
Molecules
Membrane Proteins
Imaging techniques
Lipid bilayers
Optical Imaging
Lipid Bilayers
Assays
Software
Binding Sites

Keywords

  • Confocal imaging
  • Fluorescence correlation spectroscopy
  • Giant unilamellar vesicles
  • Partition coefficient

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Biophysics
  • Medicine(all)

Cite this

Introducing a fluorescence-based standard to quantify protein partitioning into membranes. / Thomas, Franziska A.; Visco, Ilaria; Petrášek, Zdeněk; Heinemann, Fabian; Schwille, Petra.

In: Biochimica et Biophysica Acta / Biomembranes, Vol. 1848, No. 11, 01.11.2015, p. 2932-2941.

Research output: Contribution to journalArticleResearchpeer-review

Thomas, Franziska A. ; Visco, Ilaria ; Petrášek, Zdeněk ; Heinemann, Fabian ; Schwille, Petra. / Introducing a fluorescence-based standard to quantify protein partitioning into membranes. In: Biochimica et Biophysica Acta / Biomembranes. 2015 ; Vol. 1848, No. 11. pp. 2932-2941.
@article{b058915f71884f5b830974d9878029c2,
title = "Introducing a fluorescence-based standard to quantify protein partitioning into membranes",
abstract = "The affinity of peripheral membrane proteins for a lipid bilayer can be described using the partition coefficient (KP). Although several methods to determine KP are known, all possess limitations. To address some of these issues, we developed both: a versatile method based on single molecule detection and fluorescence imaging for determining KP, and a simple measurement standard employing hexahistidine-tagged enhanced green fluorescent protein (eGFP-His6) and free standing membranes of giant unilamellar vesicles (GUVs) functionalized with NTA(Ni) lipids as binding sites. To ensure intrinsic control, our method features two measurement modes. In the single molecule mode, fluorescence correlation spectroscopy (FCS) is applied to quantify free and membrane associated protein concentrations at equilibrium and calculate KP. In the imaging mode, confocal fluorescence images of GUVs are recorded and analyzed with semi-automated software to extract protein mean concentrations used to derive KP. Both modes were compared by determining the affinity of our standard, resulting in equivalent KP values. As observed in other systems, eGFP-His6 affinity for membranes containing increasing amounts of NTA(Ni) lipids rises in a stronger-than-linear fashion. We compared our dual approach with a FCS-based assay that uses large unilamellar vesicles (LUVs), which however fails to capture the stronger-than-linear trend for our NTA(Ni)-His6 standard. Hence, we determined the KP of the MARCKS effector domain with our FCS approach on GUVs, whose results are consistent with previously published data using LUVs. We finally provide a practical manual on how to measure KP and understand it in terms of molecules per lipid surface.",
keywords = "Confocal imaging, Fluorescence correlation spectroscopy, Giant unilamellar vesicles, Partition coefficient",
author = "Thomas, {Franziska A.} and Ilaria Visco and Zdeněk Petr{\'a}šek and Fabian Heinemann and Petra Schwille",
year = "2015",
month = "11",
day = "1",
doi = "10.1016/j.bbamem.2015.09.001",
language = "English",
volume = "1848",
pages = "2932--2941",
journal = "Biochimica et Biophysica Acta / Biomembranes",
issn = "0005-2736",
publisher = "Elsevier B.V.",
number = "11",

}

TY - JOUR

T1 - Introducing a fluorescence-based standard to quantify protein partitioning into membranes

AU - Thomas, Franziska A.

AU - Visco, Ilaria

AU - Petrášek, Zdeněk

AU - Heinemann, Fabian

AU - Schwille, Petra

PY - 2015/11/1

Y1 - 2015/11/1

N2 - The affinity of peripheral membrane proteins for a lipid bilayer can be described using the partition coefficient (KP). Although several methods to determine KP are known, all possess limitations. To address some of these issues, we developed both: a versatile method based on single molecule detection and fluorescence imaging for determining KP, and a simple measurement standard employing hexahistidine-tagged enhanced green fluorescent protein (eGFP-His6) and free standing membranes of giant unilamellar vesicles (GUVs) functionalized with NTA(Ni) lipids as binding sites. To ensure intrinsic control, our method features two measurement modes. In the single molecule mode, fluorescence correlation spectroscopy (FCS) is applied to quantify free and membrane associated protein concentrations at equilibrium and calculate KP. In the imaging mode, confocal fluorescence images of GUVs are recorded and analyzed with semi-automated software to extract protein mean concentrations used to derive KP. Both modes were compared by determining the affinity of our standard, resulting in equivalent KP values. As observed in other systems, eGFP-His6 affinity for membranes containing increasing amounts of NTA(Ni) lipids rises in a stronger-than-linear fashion. We compared our dual approach with a FCS-based assay that uses large unilamellar vesicles (LUVs), which however fails to capture the stronger-than-linear trend for our NTA(Ni)-His6 standard. Hence, we determined the KP of the MARCKS effector domain with our FCS approach on GUVs, whose results are consistent with previously published data using LUVs. We finally provide a practical manual on how to measure KP and understand it in terms of molecules per lipid surface.

AB - The affinity of peripheral membrane proteins for a lipid bilayer can be described using the partition coefficient (KP). Although several methods to determine KP are known, all possess limitations. To address some of these issues, we developed both: a versatile method based on single molecule detection and fluorescence imaging for determining KP, and a simple measurement standard employing hexahistidine-tagged enhanced green fluorescent protein (eGFP-His6) and free standing membranes of giant unilamellar vesicles (GUVs) functionalized with NTA(Ni) lipids as binding sites. To ensure intrinsic control, our method features two measurement modes. In the single molecule mode, fluorescence correlation spectroscopy (FCS) is applied to quantify free and membrane associated protein concentrations at equilibrium and calculate KP. In the imaging mode, confocal fluorescence images of GUVs are recorded and analyzed with semi-automated software to extract protein mean concentrations used to derive KP. Both modes were compared by determining the affinity of our standard, resulting in equivalent KP values. As observed in other systems, eGFP-His6 affinity for membranes containing increasing amounts of NTA(Ni) lipids rises in a stronger-than-linear fashion. We compared our dual approach with a FCS-based assay that uses large unilamellar vesicles (LUVs), which however fails to capture the stronger-than-linear trend for our NTA(Ni)-His6 standard. Hence, we determined the KP of the MARCKS effector domain with our FCS approach on GUVs, whose results are consistent with previously published data using LUVs. We finally provide a practical manual on how to measure KP and understand it in terms of molecules per lipid surface.

KW - Confocal imaging

KW - Fluorescence correlation spectroscopy

KW - Giant unilamellar vesicles

KW - Partition coefficient

UR - http://www.scopus.com/inward/record.url?scp=84941662973&partnerID=8YFLogxK

U2 - 10.1016/j.bbamem.2015.09.001

DO - 10.1016/j.bbamem.2015.09.001

M3 - Article

VL - 1848

SP - 2932

EP - 2941

JO - Biochimica et Biophysica Acta / Biomembranes

JF - Biochimica et Biophysica Acta / Biomembranes

SN - 0005-2736

IS - 11

ER -