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

Los detergentes contienen grupos de cabeza hidrofílicos y colas hidrofóbicas, que les permiten formar micelas esféricas; por lo tanto, son ampliamente utilizados para purificar proteínas de membrana.
Hay tres tipos de detergentes: iónicos, no iónicos y zwitteriónicos.
Los detergentes iónicos, como el dodecil sulfato de sodio, tienen grupos principales cargados, mientras que los detergentes no iónicos, como el Triton X-100, no están cargados.
Los detergentes zwitteriónicos como CHAPS tienen grupos de cabeza con cargas positivas y negativas.
Los detergentes no iónicos y zwitteriónicos son los más utilizados para la purificación de proteínas, ya que permiten que la proteína conserve su forma funcional. Por el contrario, los detergentes iónicos pueden desnaturalizar las proteínas objetivo.
Las proteínas integrales de membrana se purifican en tres etapas distintas. En primer lugar, la micela detergente interactúa con la bicapa exterior de la membrana.
A continuación, la membrana se solubiliza, con la formación de complejos lípidos-detergente y proteínas-detergente.
Una vez retirados los detergentes, la proteína puede purificarse mediante técnicas como la cromatografía de afinidad.

purificación de detergentes de proteínas de membrana

Se utilizan detergentes para purificar las proteínas integrales de la membrana. La porción hidrofóbica del detergente puede reemplazar los fosfolípidos de la membrana mientras solubiliza las proteínas de la membrana. Cuando los monómeros de detergente alcanzan una concentración específica en una solución llamada concentración micelar crítica (CMC), forman micelas. Por encima de CMC, la concentración de los monómeros detergentes permanece en equilibrio con la micela. El número de monómeros detergentes presentes en la CMC varía para cada detergente y depende del tamaño y la estructura del monómero detergente. La formación de CMC también se ve afectada por la temperatura y el pH. La estructura y concentración de un detergente y su estructura micelar determinan su capacidad para solubilizar la bicapa lipídica.

Para la purificación de proteínas, la concentración de proteínas y detergente debe ser mayor que la CMC. Debe haber un mínimo de una micela por cada proteína de membrana. La relación detergente:lípidos también juega un papel esencial en la purificación de proteínas y se determina mediante el método de prueba y error para cada proteína objetivo. Algunas proteínas se pueden extraer con una proporción detergente-lípido que oscila entre 0,1:1 y 1:1, pero la bicapa lipídica permanece intacta. Mientras que en proporciones que varían de 1:1 a 2:1, las moléculas de detergente solubilizan la membrana, dando como resultado micelas mixtas con lípidos, proteínas y detergente. Si la proporción aumenta a 10:1, todas las moléculas de lípidos en el complejo lípido-proteína serán reemplazadas por la molécula de detergente.

La elección del detergente depende de si la proteína purificada debe ser funcionalmente activa o inactiva. Los detergentes con una cabeza polar más grande o una cadena alquílica más larga se consideran más suaves que los detergentes con una cabeza polar más pequeña o una cadena alquílica más corta. La composición de la membrana también influye en la elección de los detergentes. Por ejemplo, Triton X-100 actúa sobre la membrana interna bacteriana pero no puede solubilizar la membrana externa. Por el contrario, se sabe que la digitonina actúa específicamente en la membrana plasmática de las eucariotas.

Purificación de detergentes de proteínas de membrana

Los detergentes se utilizan para purificar las proteínas integrales de la membrana. La parte hidrofóbica del detergente puede sustituir a los fosfolípidos de membrana, mientras que ...

Education

Educación

JoVE Core

Cell Biology

Biología Celular

Unit 1

Unidad 1

Membrane Structure and Components

Estructura y componentes de la membrana

KEY TERMS AND CONCEPTS

TÉRMINOS Y CONCEPTOS CLAVE

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

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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...

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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 proteins

Facilitated Transport Across Cell Membranes

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lipids as anchors

Lipid Anchors in Membrane Proteins

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How Membrane Proteins Span Cell Membranes

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multi-pass transmembrane proteins and &#946;-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 domain formation

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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

JoVE Core Biología Celular ilustra procesos celulares complejos mediante completos vídeos animados fáciles de entender. Esta colección se centra en los componentes esenciales de las células y su funcionamiento, incluyendo los orgánulos y las membranas, el transporte celular y el metabolismo energético celular. Todo este material se complementa con los vídeos de científicos en acción, que muestran la aplicación de la biología celular en experimentos reales de investigación.

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9-12.B.1.2.a

How Detergents Solubilize Membrane Proteins

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