Name: how detergents solubilize membrane proteins
Uploaded: 2023-04-30T09:16:15.000Z
Duration: 1 min 18 s
Channel: JoVE

K12 Standards

Alabama Course of Study Science

9-12

Biology

from-molecules-to-organisms-structures-and-processes

From Molecules to Organisms: Structures and Processes

9-12.B.1.4.a

detergent-purification-of-membrane-proteins

Detergent Purification of Membrane Proteins

Les détergents contiennent des groupes de tête hydrophiles et des queues hydrophobes, qui leur permettent de former des micelles sphériques ; par conséquent, ils sont largement utilisés pour purifier les protéines membranaires.
Il existe trois types de détergents : ionique, non ionique et zwitterionique.
Les détergents ioniques tels que le dodécylsulfate de sodium ont des groupes de tête chargés, tandis que les détergents non ioniques tels que le Triton X-100 ne sont pas chargés.
Les détergents Zwitterionic comme CHAPS ont des groupes de tête avec des charges positives et négatives.
Les détergents non ioniques et zwitterioniques sont les plus couramment utilisés pour la purification des protéines car ils permettent à la protéine de conserver sa forme fonctionnelle. En revanche, les détergents ioniques peuvent dénaturer les protéines cibles.
Les protéines membranaires intégrales sont purifiées en trois étapes distinctes. Tout d’abord, la micelle du détergent interagit avec la bicouche externe de la membrane.
Ensuite, la membrane est solubilisée, avec la formation de complexes lipide-détergent et protéine-détergent.
Une fois les détergents éliminés, la protéine peut être purifiée à l’aide de techniques telles que la chromatographie d’affinité.

la purification détergente des protéines membranaires

Les détergents sont utilisés pour purifier les protéines intégrales de la membrane. La partie hydrophobe du détergent peut remplacer les phospholipides membranaires tout en solubilisant les protéines membranaires. Lorsque les monomères détergents atteignent une concentration spécifique dans une solution appelée concentration micellaire critique (CMC), ils forment des micelles. Au-dessus de la CMC, la concentration des monomères détergents reste en équilibre avec la micelle. Le nombre de monomères détergents présents dans la CMC varie pour chaque détergent et dépend de la taille et de la structure du monomère détergent. La formation de CMC est également affectée par la température et le pH. La structure et la concentration d'un détergent ainsi que sa structure micellaire déterminent sa capacité à solubiliser la bicouche lipidique.

Pour la purification des protéines, la concentration en protéines et en détergent doit être supérieure à la CMC. Il devrait y avoir au minimum une micelle pour chaque protéine membranaire. Le rapport détergent : lipide joue également un rôle essentiel dans la purification des protéines, et il est déterminé à l’aide de la méthode d’essais et d’erreurs pour chaque protéine cible. Certaines protéines peuvent être extraites avec un rapport détergent-lipide allant de 0,1:1 à 1:1, mais la bicouche lipidique reste intacte. Dans des rapports allant de 1:1 à 2:1, les molécules de détergent solubilisent la membrane, ce qui donne lieu à des micelles mixtes contenant des lipides, des protéines et du détergent. Si le rapport augmente à 10:1, toutes les molécules lipidiques du complexe lipide-protéine seront remplacées par la molécule détergente.

Le choix du détergent dépend du fait que la protéine purifiée doit être fonctionnellement active ou inactive. Les détergents avec une tête polaire plus grande ou une chaîne alkyle plus longue sont considérés comme plus doux que les détergents avec une tête polaire plus petite ou une chaîne alkyle plus courte. La composition de la membrane affecte également le choix des détergents. Par exemple, le Triton X-100 agit sur la membrane  bactérienne interne mais ne peut solubiliser la membrane externe. En revanche, la digitonine est connue pour agir spécifiquement sur la membrane plasmique eucaryote.

La purification détergente des protéines membranaires

Les détergents sont utilisés pour purifier les protéines intégrales de la membrane. La partie hydrophobe du détergent peut remplacer les phospholipides membranaires tandis que ...

Education

Enseignement

JoVE Core

Cell Biology

Biologie cellulaire

Unit 1

Unité 1

Membrane Structure and Components

Structure et composants de la membrane

KEY TERMS AND CONCEPTS

TERMES ET CONCEPTS CLÉS

how detergents solubilize membrane proteins

Detergents help solubilize membrane proteins so they can be purified from the lipid bilayer. Their hydrophobic region can replace membrane phospholipids and keep integral membrane proteins in solution.

A detergent works well only when its monomers reach the critical micelle concentration, or CMC. At that point, the molecules assemble into micelles, which are small clusters of detergent. Above the CMC, free detergent monomers stay in equilibrium with the micelles. The CMC varies with the detergent’s size and structure, and it is also affected by temperature and pH.

Both detergent structure and detergent amount affect how well a membrane is solubilized. For purification, the protein and detergent levels should be above the CMC, and there should be at least one micelle for each membrane protein. The detergent-to-lipid ratio is usually found by trial and error for each target protein.

Different ratios produce different results. At ratios from 0.1:1 to 1:1, some proteins can be extracted while the lipid bilayer stays intact. At ratios from 1:1 to 2:1, the membrane is solubilized and mixed micelles form with lipid, protein, and detergent. At a ratio of 10:1, the detergent can replace the lipid molecules in the lipid-protein complex.

The choice of detergent also depends on whether the purified protein should stay active or become inactive. Detergents with a larger polar head or a longer alkyl chain are considered milder than detergents with a smaller polar head or a shorter alkyl chain. Membrane type matters too. Triton X-100 acts on the bacterial inner membrane but does not solubilize the outer membrane. Digitonin, on the other hand, works specifically on the eukaryotic plasma membrane.

How Detergents Solubilize Membrane Proteins

how-detergents-solubilize-membrane-proteins

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membrane lipids

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detergent purification of membrane proteins

Detergents help solubilize membrane proteins so they can be purified from the lipid bilayer. Their hydrophobic region can replace membrane phospholipids and keep integral membrane proteins in solution. A detergent works well only when its monomers reach the critical micelle concentration, or CMC. At that point, the molecules assemble into micelles, which are small clusters of detergent. Above the CMC, free detergent monomers stay in equilibrium with the micelles. The CMC varies with the...

protein diffusion in the membrane

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Membrane proteins can move across the cell surface by lateral diffusion, and they can also rotate in place. Scientists confirmed lateral movement with a cell fusion experiment. Mouse and human cells were fused to make hybrid cells. The membrane proteins on the two cells were tagged with red and green fluorescent markers. At first, each color stayed on its own side of the fused cell. After 40 minutes, the colors mixed across the whole cell surface, showing that the proteins had moved laterally. <br...

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key terms and concepts

Learn how detergents solubilize membrane proteins, form micelles at the CMC, and help choose the right detergent for purification in high school biology.

How Detergents Solubilize Membrane Proteins | JoVE K12

cell biology

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How Detergents Solubilize Membrane Proteins

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