Quantification of Detergents Complexed with Membrane Proteins - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Scientific Reports Année : 2017

Quantification of Detergents Complexed with Membrane Proteins

Frédéric Delolme
Luca Monticelli
Renaud Wagner
  • Fonction : Auteur
Anass Jawhari
  • Fonction : Auteur
H. Ronald Ronald Kaback
  • Fonction : Auteur

Résumé

Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies. Detergents play a major role in handling membrane proteins. They are indispensable tools for extracting membrane proteins from the membrane and maintaining them in a soluble and active state for further study. A typical problem that arises during extraction, purification and crystallization is difficulty in controlling detergent concentration and composition, especially when detergent mixtures are used. Indeed, little is known about the fundamentals of detergent behavior around membrane proteins. Pioneering and important studies were conducted with radioactive detergents, which allowed estimation of the ratio of detergent to hydrophobic area 1,2. Moreover, use of radiolabeled detergents led to crystallization of the bovine ADP/ATP carrier and sarcoplasmic reticulum Ca 2+-ATPase, which was found to be highly dependent on the detergent:membrane protein ratio 3,4. However, only a few types of radioactive detergents are available, making this approach generally untenable. Other methods developed to measure detergent concentrations include: (i) colorimetric assays to estimate the sugar moiety for specific detergents 5,6 ; (ii) Fourier transform Infrared spectroscopy 7 ; (iii) plain thin layer chromatography coupled with densitometric quantification, or more recently coupled with laser densitometry 8,9 ; drop-shape based quantification 10 ; (iv) liquid chromatography/ESI-MS 11 ; (v) size-exclusion chromatography coupled with multi-angle laser light scattering 12,13 and analytical ultracentrifugation 14. Although useful, these methods are laborious, difficult to implement routinely, limited to a given type of detergent or inapplicable to detergent mixtures. Moreover, hundreds of detergents are now commercially available, rendering these methods generally impractical.
Fichier principal
Vignette du fichier
Chaptal-Detergent-quantification-ScientifReports-2017.pdf (2.65 Mo) Télécharger le fichier
Origine : Fichiers éditeurs autorisés sur une archive ouverte
Loading...

Dates et versions

hal-02110228 , version 1 (25-04-2019)

Identifiants

Citer

Vincent Chaptal, Frédéric Delolme, Arnaud Kilburg, Sandrine Magnard, Cédric Montigny, et al.. Quantification of Detergents Complexed with Membrane Proteins. Scientific Reports, 2017, 7 (1), ⟨10.1038/srep41751⟩. ⟨hal-02110228⟩
101 Consultations
273 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More