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Thermostability Assays: a Generic and Versatile Tool for Studying the Functional and Structural Properties of Membrane Proteins in Detergents

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Biophysics of Membrane Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2168))

Abstract

There are very few generic methods to assess the stability and functional properties of membrane proteins solubilized in detergent. For this purpose, a thiol-reactive fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) can be used. An unfolding profile is obtained when the fluorochrome becomes fluorescent on reaction with cysteine residues that have been exposed during thermal denaturation of the protein population. The method was initially developed to optimize the stability of membrane proteins for crystallization studies, but in the course of our work we found many other applications. First, the assay can be used to study the binding of inhibitors, substrates, lipids, and other effectors to membrane proteins. Second, the assay can be used to understand the dynamics of proteins, allowing states to be defined by changes in accessibility of cysteine residues or by changes in specific amino acid interactions. Finally, the assay can be used to study state-dependent domain interactions, for example, as part of regulatory mechanisms. The CPM thermostability assay represents a broadly applicable and versatile tool for a wide range of applications in the functional and structural analysis of membrane proteins.

This research was supported by the Medical Research Council (grant MC_UU_00015/1).

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Abbreviations

AAC:

Mitochondrial ADP/ATP carrier

APC:

Mitochondrial ATP-Mg/Pi carrier

ATR:

Atractyloside

BKA:

Bongkrekic acid

CATR:

Carboxyatractyloside

CPM:

N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]

DDM:

Dodecyl maltoside

DM:

Decyl maltoside

DMNG:

Decyl maltose neopentyl glycol

LMNG:

Lauryl maltose neopentyl glycol

PC:

Phosphocholine

T m :

Apparent melting temperature

TOCL/CL:

Tetraoleoyl cardiolipin (18:1)

UDM:

Undecyl maltoside

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Author information

Authors and Affiliations

  1. School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK

    Steven P. D. Harborne

  2. Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge Biomedical Campus, Hills road, Cambridge, UK

    Martin S. King & Edmund R. S. Kunji

Authors
  1. Steven P. D. Harborne
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  2. Martin S. King
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  3. Edmund R. S. Kunji
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Corresponding author

Correspondence to Edmund R. S. Kunji .

Editor information

Editors and Affiliations

  1. School of Clinical and Applied Sciences, Leeds Beckett University, Leeds, UK

    Vincent L. G. Postis

  2. Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences; Molecular and Integrative Biosciences, Faculty of Biological and Environmental Sciences, University of Leeds, Leeds, UK; University of Helsinki, Helsinki, Finland

    Adrian Goldman

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Harborne, S.P.D., King, M.S., Kunji, E.R.S. (2020). Thermostability Assays: a Generic and Versatile Tool for Studying the Functional and Structural Properties of Membrane Proteins in Detergents. In: Postis, V.L.G., Goldman, A. (eds) Biophysics of Membrane Proteins. Methods in Molecular Biology, vol 2168. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0724-4_5

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  • DOI: https://doi.org/10.1007/978-1-0716-0724-4_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0723-7

  • Online ISBN: 978-1-0716-0724-4

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