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

I detergenti contengono gruppi di testa idrofili e code idrofobiche, che consentono loro di formare micelle sferiche; pertanto, sono ampiamente utilizzati per purificare le proteine di membrana.
Esistono tre tipi di detergenti: ionici, non ionici e zwitterionici.
I detergenti ionici come il sodio dodecil solfato hanno gruppi di testa caricati, mentre i detergenti non ionici come Triton X-100 sono scarichi.
I detersivi zwitterionici come CHAPS hanno gruppi di testa con cariche sia positive che negative.
I detergenti non ionici e zwitterionici sono più comunemente usati per la purificazione delle proteine in quanto consentono alla proteina di mantenere la sua forma funzionale. Al contrario, i detergenti ionici possono denaturare le proteine bersaglio.
Le proteine integrali di membrana vengono purificate in tre fasi distinte. In primo luogo, la micella detergente interagisce con il doppio strato esterno della membrana.
Successivamente, la membrana viene solubilizzata, con la formazione di complessi lipidi-detergente e proteina-detergente.
Una volta rimossi i detergenti, la proteina può essere purificata utilizzando tecniche come la cromatografia di affinità.

purificazione detergente delle proteine di membrana

I detergenti vengono utilizzati per purificare le proteine integrali della membrana. La porzione idrofobica del detergente può sostituire i fosfolipidi di membrana solubilizzando le proteine di membrana. Quando i monomeri detergenti raggiungono una concentrazione specifica in una soluzione chiamata concentrazione micellare critica (CMC), si formano le micelle. Dopo la CMC, la concentrazione dei monomeri detergenti rimane in equilibrio con la micella. Il numero di monomeri detergenti presenti nella CMC varia per ciascun detergente e dipende dalle dimensioni e dalla struttura del monomero detergente. La formazione della CMC è influenzata anche dalla temperatura e dal pH. La struttura e la concentrazione di un detergente e la sua struttura micellare determinano la sua capacità di solubilizzare il doppio strato lipidico.

Per la purificazione delle proteine, la concentrazione di proteine e detergente dovrebbe essere superiore alla CMC. Dovrebbe esserci almeno una micella per ciascuna proteina di membrana. Anche il rapporto detergente:lipidi svolge un ruolo essenziale nella purificazione delle proteine e viene determinato utilizzando il metodo per tentativi ed errori per ciascuna proteina bersaglio. Alcune proteine possono essere estratte con un rapporto detergente-lipide compreso tra 0,1:1 e 1:1, ma il doppio strato lipidico rimane indisturbato. Mentre in rapporti che vanno da 1:1 a 2:1, le molecole del detergente solubilizzano la membrana, risultando in micelle miste con lipidi, proteine e detergente. Se il rapporto viene aumentato a 10:1, tutte le molecole lipidiche del complesso lipide-proteico verranno sostituite dalla molecola detergente.

La scelta del detergente dipende dal fatto che la proteina purificata debba essere funzionalmente attiva o inattiva. I detergenti con una testa polare più grande o una catena alchilica più lunga sono considerati più delicati rispetto ai detersivi con una testa polare più piccola o una catena alchilica più corta. La composizione della membrana influisce anche sulla scelta dei detergenti. Ad esempio, il Triton X-100 agisce sulla membrana interna dei batteri ma non può solubilizzare la membrana esterna. Al contrario, è noto che la digitonina agisce specificamente sulla membrana plasmatica eucariotica.

Purificazione detergente delle proteine di membrana

I detergenti vengono utilizzati per purificare le proteine integrali della membrana. La parte idrofoba del detergente può sostituire i fosfolipidi di membrana mentre...

Education

Didattica

JoVE Core

Cell Biology

Biologia cellulare

Unit 1

Unità 1

Membrane Structure and Components

Struttura e componenti della membrana

KEY TERMS AND CONCEPTS

TERMINI E CONCETTI CHIAVE

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

what are membranes?

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membrane fluidity

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fluid mosaic model

Fluid Mosaic Model of the Membrane

The fluid mosaic model explains the structure and function of the plasma membrane. Scientists first identified the plasma membrane in the 1890s, and by 1915 they had identified its main chemical parts: lipids and proteins. Early electron micrographs showed a railroad track pattern, which helped lead to the first widely accepted membrane model in 1935.

membrane lipids

Membrane Lipids in Cells and Tissues

Membrane lipids give biological membranes their structure and help set their properties. In mammalian cells, lipids make up about 50% of the membrane on average. The amount can be much lower in the inner mitochondrial membrane or much higher in the myelin sheath around nerve cells. Several phospholipids are common in mammalian membranes. These include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. At physiological pH, phosphatidylserine carries a negative...

asymmetric lipid bilayer

Membrane Lipid Unevenness

Biological membranes have an uneven mix of lipids between the inner and outer layers. This membrane asymmetry gives each side a different chemical makeup and supports different cell functions. Evidence for this uneven layout came from treating an erythrocyte membrane with phospholipase. Phospholipase is an enzyme that breaks lipids into fatty acids and hydrophilic groups. It acted only on the outer layer, while the inner layer stayed intact. The treatment degraded about 80% of total membrane...

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membrane-asymmetry-regulating-transporters

Membrane Lipid Flipping and Transport

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Membrane carbohydrates help give cells their surface identity and support plasma membrane function. The plasma membrane is a dynamic barrier made of lipids, proteins, and carbohydrates. These sugar chains have unique structural and chemical traits that help the membrane work properly for cell growth and survival. Membrane carbohydrates do not have a hydrophobic region, so they are found only on the outer surface of the cell. Sugar addition, or glycosylation, of proteins occurs in the lumens of...

membrane proteins

Facilitated Transport Across Cell Membranes

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lipids as anchors

Lipid Anchors in Membrane Proteins

Lipid anchors help attach proteins to the plasma membrane. In eukaryotic cells, the main lipid anchors are prenyl groups, fatty acyl groups, and glycosylphosphatidylinositol, or GPI, groups. Prenyl and fatty acyl groups anchor proteins on the cytosolic side of the membrane. GPI anchors proteins on the extracellular side. Prenylated proteins often have a CaaX motif at the carboxy terminus. In this sequence, C means cysteine, a means any aliphatic amino acid, and X means any other amino acid...

single-pass transmembrane proteins

How Membrane Proteins Span Cell Membranes

Membrane proteins help cells communicate, send signals, stick to other cells, and move molecules across the membrane. Integral membrane proteins are tightly linked to the cell membrane and carry out these jobs. Some integral membrane proteins sit only in one membrane layer. Fatty acid amide hydrolase is one example, and it is found on the cytoplasmic side of the membrane monolayer. Other integral membrane proteins are transmembrane proteins, which span across the membrane. These proteins have...

multi-pass transmembrane proteins and &#946;-barrels

multi-pass-transmembrane-proteins-and-barrels

Membrane-Spanning Helices and β-Barrels

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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

How Membrane Proteins Move Side to Side

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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mechanisms of membrane-bending

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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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9-12.B.1.2.a

How Detergents Solubilize Membrane Proteins

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