Development of a Thermofluor assay for stability determination of membrane proteins using the Na+/H+ antiporter NhaA and cytochrome c oxidase

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Development of a Thermofluor assay for stability determination of membrane proteins using the Na+/H+ antiporter NhaA and cytochrome c oxidase. / Kohlstaedt, Martin; Von Der Hocht, Iris; Hilbers, Florian; Thielmann, Yvonne; Michel, Hartmut.

I: Acta crystallographica. Section D, Structural biology, Bind 71, 2015, s. 1112-1122.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

Harvard

Kohlstaedt, M, Von Der Hocht, I, Hilbers, F, Thielmann, Y & Michel, H 2015, 'Development of a Thermofluor assay for stability determination of membrane proteins using the Na+/H+ antiporter NhaA and cytochrome c oxidase', Acta crystallographica. Section D, Structural biology, bind 71, s. 1112-1122. https://doi.org/10.1107/S1399004715004058

APA

Kohlstaedt, M., Von Der Hocht, I., Hilbers, F., Thielmann, Y., & Michel, H. (2015). Development of a Thermofluor assay for stability determination of membrane proteins using the Na+/H+ antiporter NhaA and cytochrome c oxidase. Acta crystallographica. Section D, Structural biology, 71, 1112-1122. https://doi.org/10.1107/S1399004715004058

CBE

Kohlstaedt M, Von Der Hocht I, Hilbers F, Thielmann Y, Michel H. 2015. Development of a Thermofluor assay for stability determination of membrane proteins using the Na+/H+ antiporter NhaA and cytochrome c oxidase. Acta crystallographica. Section D, Structural biology. 71:1112-1122. https://doi.org/10.1107/S1399004715004058

MLA

Vancouver

Kohlstaedt M, Von Der Hocht I, Hilbers F, Thielmann Y, Michel H. Development of a Thermofluor assay for stability determination of membrane proteins using the Na+/H+ antiporter NhaA and cytochrome c oxidase. Acta crystallographica. Section D, Structural biology. 2015;71:1112-1122. https://doi.org/10.1107/S1399004715004058

Author

Kohlstaedt, Martin ; Von Der Hocht, Iris ; Hilbers, Florian ; Thielmann, Yvonne ; Michel, Hartmut. / Development of a Thermofluor assay for stability determination of membrane proteins using the Na+/H+ antiporter NhaA and cytochrome c oxidase. I: Acta crystallographica. Section D, Structural biology. 2015 ; Bind 71. s. 1112-1122.

Bibtex

@article{9fd722a3d83543fa96798dacceccf3db,
title = "Development of a Thermofluor assay for stability determination of membrane proteins using the Na+/H+ antiporter NhaA and cytochrome c oxidase",
abstract = "Crystallization of membrane proteins is very laborious and time-consuming, yielding well diffracting crystals in only a minority of projects. Therefore, a rapid and easy method is required to optimize the conditions for initial crystallization trials. The Thermofluor assay has been developed as such a tool. However, its applicability to membrane proteins is still limited because either large hydrophilic extramembranous regions or cysteine residues are required for the available dyes to bind and therefore act as reporters in this assay. No probe has been characterized to discriminate between the hydrophobic surfaces of detergent micelles, folded and detergent-covered membrane proteins and denatured membrane proteins. Of the four dyes tested, the two dyes 1-anilinonaphthalene-8-sulfonic acid (ANS) and SYPRO Orange were systematically screened for compatibility with five detergents commonly used in the crystallization of membrane proteins. ANS showed the weakest interactions with all of the detergents screened. It was possible to determine the melting temperature of the sodium ion/proton antiporter NhaA, a small membrane protein without large hydrophilic domains, over a broad pH range using ANS. Furthermore, cytochrome c oxidase (CcO) was used to apply the method to a four-subunit membrane protein complex. It was possible to obtain preliminary information on the temperature-dependent denaturation of this complex using the dye ANS. Application of the dye 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM) to CcO in the Thermofluor assay enabled the determination of the melting temperatures of distinct subunits of the complex.",
keywords = "differential scanning calorimetry, membrane proteins, Thermofluor assay",
author = "Martin Kohlstaedt and {Von Der Hocht}, Iris and Florian Hilbers and Yvonne Thielmann and Hartmut Michel",
year = "2015",
doi = "10.1107/S1399004715004058",
language = "English",
volume = "71",
pages = "1112--1122",
journal = "Acta crystallographica. Section D, Structural biology",
issn = "2059-7983",

}

RIS

TY - JOUR

T1 - Development of a Thermofluor assay for stability determination of membrane proteins using the Na+/H+ antiporter NhaA and cytochrome c oxidase

AU - Kohlstaedt, Martin

AU - Von Der Hocht, Iris

AU - Hilbers, Florian

AU - Thielmann, Yvonne

AU - Michel, Hartmut

PY - 2015

Y1 - 2015

N2 - Crystallization of membrane proteins is very laborious and time-consuming, yielding well diffracting crystals in only a minority of projects. Therefore, a rapid and easy method is required to optimize the conditions for initial crystallization trials. The Thermofluor assay has been developed as such a tool. However, its applicability to membrane proteins is still limited because either large hydrophilic extramembranous regions or cysteine residues are required for the available dyes to bind and therefore act as reporters in this assay. No probe has been characterized to discriminate between the hydrophobic surfaces of detergent micelles, folded and detergent-covered membrane proteins and denatured membrane proteins. Of the four dyes tested, the two dyes 1-anilinonaphthalene-8-sulfonic acid (ANS) and SYPRO Orange were systematically screened for compatibility with five detergents commonly used in the crystallization of membrane proteins. ANS showed the weakest interactions with all of the detergents screened. It was possible to determine the melting temperature of the sodium ion/proton antiporter NhaA, a small membrane protein without large hydrophilic domains, over a broad pH range using ANS. Furthermore, cytochrome c oxidase (CcO) was used to apply the method to a four-subunit membrane protein complex. It was possible to obtain preliminary information on the temperature-dependent denaturation of this complex using the dye ANS. Application of the dye 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM) to CcO in the Thermofluor assay enabled the determination of the melting temperatures of distinct subunits of the complex.

AB - Crystallization of membrane proteins is very laborious and time-consuming, yielding well diffracting crystals in only a minority of projects. Therefore, a rapid and easy method is required to optimize the conditions for initial crystallization trials. The Thermofluor assay has been developed as such a tool. However, its applicability to membrane proteins is still limited because either large hydrophilic extramembranous regions or cysteine residues are required for the available dyes to bind and therefore act as reporters in this assay. No probe has been characterized to discriminate between the hydrophobic surfaces of detergent micelles, folded and detergent-covered membrane proteins and denatured membrane proteins. Of the four dyes tested, the two dyes 1-anilinonaphthalene-8-sulfonic acid (ANS) and SYPRO Orange were systematically screened for compatibility with five detergents commonly used in the crystallization of membrane proteins. ANS showed the weakest interactions with all of the detergents screened. It was possible to determine the melting temperature of the sodium ion/proton antiporter NhaA, a small membrane protein without large hydrophilic domains, over a broad pH range using ANS. Furthermore, cytochrome c oxidase (CcO) was used to apply the method to a four-subunit membrane protein complex. It was possible to obtain preliminary information on the temperature-dependent denaturation of this complex using the dye ANS. Application of the dye 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM) to CcO in the Thermofluor assay enabled the determination of the melting temperatures of distinct subunits of the complex.

KW - differential scanning calorimetry

KW - membrane proteins

KW - Thermofluor assay

UR - http://www.scopus.com/inward/record.url?scp=84929242226&partnerID=8YFLogxK

U2 - 10.1107/S1399004715004058

DO - 10.1107/S1399004715004058

M3 - Journal article

C2 - 25945577

AN - SCOPUS:84929242226

VL - 71

SP - 1112

EP - 1122

JO - Acta crystallographica. Section D, Structural biology

JF - Acta crystallographica. Section D, Structural biology

SN - 2059-7983

ER -