PMID- 25945577
OWN - NLM
STAT- MEDLINE
DCOM- 20160204
LR  - 20171116
IS  - 1399-0047 (Electronic)
IS  - 0907-4449 (Linking)
VI  - 71
IP  - Pt 5
DP  - 2015 May
TI  - Development of a Thermofluor assay for stability determination of membrane 
      proteins using the Na(+)/H(+) antiporter NhaA and cytochrome c oxidase.
PG  - 1112-22
LID - 10.1107/S1399004715004058 [doi]
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.
FAU - Kohlstaedt, Martin
AU  - Kohlstaedt M
AD  - Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, 
      Max-von-Laue-Strasse 3, 60438 Frankfurt, Germany.
FAU - von der Hocht, Iris
AU  - von der Hocht I
AD  - Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, 
      Max-von-Laue-Strasse 3, 60438 Frankfurt, Germany.
FAU - Hilbers, Florian
AU  - Hilbers F
AD  - Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, 
      Max-von-Laue-Strasse 3, 60438 Frankfurt, Germany.
FAU - Thielmann, Yvonne
AU  - Thielmann Y
AD  - Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, 
      Max-von-Laue-Strasse 3, 60438 Frankfurt, Germany.
FAU - Michel, Hartmut
AU  - Michel H
AD  - Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, 
      Max-von-Laue-Strasse 3, 60438 Frankfurt, Germany.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20150424
PL  - United States
TA  - Acta Crystallogr D Biol Crystallogr
JT  - Acta crystallographica. Section D, Biological crystallography
JID - 9305878
RN  - 0 (Anilino Naphthalenesulfonates)
RN  - 0 (Detergents)
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Sodium-Hydrogen Exchangers)
RN  - 630I4V6051 (1-anilino-8-naphthalenesulfonate)
RN  - EC 1.9.3.1 (Electron Transport Complex IV)
SB  - IM
MH  - Anilino Naphthalenesulfonates/*chemistry
MH  - Crystallography, X-Ray
MH  - Detergents/*chemistry
MH  - Electron Transport Complex IV/*chemistry/metabolism
MH  - Escherichia coli/metabolism
MH  - Fluorescent Dyes/chemistry
MH  - Fluorometry/*methods
MH  - Hydrogen-Ion Concentration
MH  - Paracoccus denitrificans/enzymology
MH  - Protein Conformation
MH  - Protein Denaturation
MH  - Sodium-Hydrogen Exchangers/*chemistry/metabolism
MH  - Temperature
OTO - NOTNLM
OT  - Thermofluor assay
OT  - differential scanning calorimetry
OT  - membrane proteins
EDAT- 2015/05/07 06:00
MHDA- 2016/02/05 06:00
CRDT- 2015/05/07 06:00
PHST- 2014/08/05 00:00 [received]
PHST- 2015/02/26 00:00 [accepted]
PHST- 2015/05/07 06:00 [entrez]
PHST- 2015/05/07 06:00 [pubmed]
PHST- 2016/02/05 06:00 [medline]
AID - S1399004715004058 [pii]
AID - 10.1107/S1399004715004058 [doi]
PST - ppublish
SO  - Acta Crystallogr D Biol Crystallogr. 2015 May;71(Pt 5):1112-22. doi: 
      10.1107/S1399004715004058. Epub 2015 Apr 24.