Name: membrane proteins in liposome models
Uploaded: 2023-04-30T06:31:34.000Z
Duration: 7 min 5 s
Channel: JoVE

K12 Standards

new-york-state-science-learning-standards

New York State Science Learning Standards

High School

Life Science

Structure and Function

HS-LS1-3.

Reconstitution of Membrane Proteins

reconstitución de proteínas de membrana

Reconstitución es el proceso de volver una biomolécula aislado a su forma o su función original. Esto es particularmente útil para el estudio de proteínas de la membrana, que permiten funciones celulares importantes y afectan el comportamiento de los lípidos cercano. Para estudiar la función purificada de proteínas de membrana in situ, debe ser reconstituidos por integrarlos en una membrana lipídica artificial.
Este video presenta la membrana proteína reconstitución conceptos y procedimientos relacionados, tales como aislamiento de proteínas con detergente, formación de vesículas artificiales con lípidos, incorporación de la proteína aislada en la vesícula artificial y separación del detergente de la solución. Por último, se cubren dos aplicaciones: reconstitución de proteínas de transporte de membrana y la reconstitución de proteínas recolección de luz.
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Reconstitución es el proceso de restauración de una biomolécula aislado a su forma original o su funcionalidad. A menudo se utiliza este enfoque al estudio de proteínas de la membrana, que permiten muchos procesos celulares importantes y afectan el comportamiento de los vecinos de lípidos. Sin embargo, la complejidad del entorno celular dificulta la membrana funciones de la proteína a estudiar en situ. Las proteínas pueden extraerse y purificarse, pero sus funciones reales no pueden ser evaluados sin una membrana. Por lo tanto, las proteínas de membrana aislado son reconstituidos por integración en una membrana lipídica artificial, como un liposoma. Este video presenta los principios de la reconstitución de proteínas de membrana, un procedimiento de reconstitución general y unas cuantas aplicaciones en bioquímica.
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Las membranas celulares consisten principalmente de fosfolípidos y proteínas de membrana. Los fosfolípidos forman una bicapa en la que las cabezas hidrofílicas de fosfato interactúan con el acuoso interior y exterior de la célula, mientras que las colas de ácidos grasos hidrofóbicos interactúan entre sí en la bicapa.
Algunas proteínas de la membrana sólo interactúan con la membrana por interacciones electrostáticas o no covalente. Otros, llamados 'proteínas integradas', se incrustan en la bicapa lipídica.
Como la bicapa proteínas integrales tienen extremos hidrofílicos y un centro hidrofóbico y se llevan a cabo en lugar por interacciones hidrofóbicas. Las proteínas integrales que abarcan la membrana entera se conocen como "proteínas transmembranales".
Las interacciones entre la membrana y estas proteínas son tan fuertes que incluso lisis de las células a no separarlos. Un surfactante especial llamado un detergente se utiliza para extraer las proteínas. Similar a los fosfolípidos, detergentes tienen cabezas hidrofílicas y colas lipofílicas y pueden entrar libremente en la membrana.
Dentro de la membrana, las colas lipofílicas del detergente interactúan con el núcleo hidrofóbico de la proteína. Esto rodea la proteína con una concha de las cabezas hidrofílicas de detergente, que interrumpen las interacciones proteína-lípido.
El complejo proteína-detergente ahora se separa fácilmente de la membrana. El detergente hace el complejo soluble en soluciones acuosas y listos para su reconstitución en una membrana artificial.
A menudo se reconstituyen las proteínas en las membranas de liposomas, que son vesículas artificiales. Para preparar liposomas, lípidos secados hidratados y agitados para inducir la formación de la vesícula. Cuando un detergente se agrega, se incorpora a las membranas de liposomas.
Para reconstituir la proteína, se combinan las proteínas solubilizadas y liposomas, y luego se elimina el detergente de la solución por diálisis o adsorción química. Las proteínas y liposomas rápidamente montan en proteoliposomes, tan sólo los grupos hidrofílicos están expuestos. Las proteínas funcionan entonces como en una membrana de la célula y puede ser investigados de manera aislada.
Ahora que hemos cubierto los fundamentos de la reconstitución de proteínas, vamos un protocolo para la reconstitución de proteínas de la membrana en los liposomas.
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Para comenzar a aislar las proteínas de membrana, las células son sometidas a lisis. Las células intactas se quitan con centrifugación.
El sobrenadante es centrifugado a una velocidad superior a las membranas de la pelotilla. El sedimento se suspende de nuevo y se añade un detergente para extraer las proteínas.
Los restos de células restantes se elimina por centrifugación adicional. La proteína es purificada del sobrenadante con cromatografía en columna y luego concentrada o purificada aún más según sea necesario.
Para comenzar a preparar los liposomas, una suspensión de fosfolípidos en solvente orgánico se seca bajo nitrógeno o argón.
Los fosfolípidos son hidratados con tampón de hidratación, y la mezcla se sonicó para terminar la creación de los liposomas.
Detergente se agrega para solubilizar los liposomas, que se combina con las proteínas.
El detergente se elimina luego por adsorción sobre granos del poliestireno, diálisis o de una columna de enlace de detergente. El proteoliposomes resultantes está listos para ser purificada y utilizada en experimentos posteriores.
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Ahora que usted está familiarizado con los fundamentos de un procedimiento de reconstitución de proteínas de membrana, echemos un vistazo a algunas aplicaciones de la reconstitución de proteínas en bioquímica.
Una proteína transportadora de membrana se reconstituyó para obtener una comprensión más clara de su mecanismo de transporte. Su reconstitución después de la función se verificó con un eflujo de iones yoduro. Luego, se estudió la actividad de transporte en presencia de diferentes inhibidores de molécula pequeña ion channel y potenciadotes. De esta manera, podrían estudiarse las interacciones directas de estas pequeñas moléculas con la proteína de transporte.
Clorofila y carotenoides-proteínas de membrana en plantas cosechan luz, promoción la separación del cargo y mitigación daños ligeros. Por reconstituir estas proteínas de recolección de luz, se pueden estudiar su dinámica y la interacción con pigmentos plegable. Las proteínas de recolección de luz reconstituidas con esta técnica tenían propiedades ópticas muy similares a las proteínas nativas. Espectroscopia de emisión de fluorescencia puede utilizarse entonces para estudiar la transferencia de energía de los pigmentos a las proteínas reconstituidas de recolección de luz.
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Sólo has visto video de Zeus durante la reconstitución de proteínas de membrana. Reconstitución es una manera de transferir proteínas importantes para un imitador de celda para análisis adicionales. Este video cubre los principios de la reconstitución de proteínas, un protocolo de reconstitución y unas cuantas aplicaciones en bioquímica. ¡Gracias por ver!
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Reconstitución de proteínas de membrana

Reconstitution is the process of returning an isolated biomolecule to its original form or function. This is particularly useful for studying membrane ...

La reconstitución es el proceso de restaurar una biomolécula aislada a su forma o funcionalidad original. Este enfoque se utiliza a menudo cuando se estudian las proteínas de membrana, que permiten muchos procesos celulares importantes y afectan el comportamiento de los lípidos vecinos. Sin embargo, la complejidad del entorno celular hace que las funciones de las proteínas de membrana sean difíciles de estudiar in situ. Las proteínas pueden extraerse y purificarse, pero sus funciones reales no pueden evaluarse sin una membrana. Por lo tanto, las proteínas de membrana aisladas se reconstituyen mediante la integración en una membrana lipídica artificial, como un liposoma. Este video presentará los principios de la reconstitución de proteínas de membrana, un procedimiento general de reconstitución y algunas aplicaciones en bioquímica. Las membranas celulares están formadas principalmente por fosfolípidos y proteínas de membrana. Los fosfolípidos forman una bicapa en la que las cabezas de fosfato hidrofílico interactúan con el interior y el exterior acuosos de la célula, mientras que las colas de ácidos grasos hidrofóbicos interactúan entre sí en la bicapa. Algunas proteínas de membrana solo interactúan con la membrana mediante interacciones electrostáticas o no covalentes. Otras, llamadas "proteínas integrales", están incrustadas en la bicapa lipídica. Al igual que la bicapa, las proteínas integrales tienen extremos hidrofílicos y un centro hidrofóbico, y se mantienen en su lugar mediante interacciones hidrofóbicas. Las proteínas integrales que abarcan toda la membrana se conocen como "proteínas transmembrana". Las interacciones entre estas proteínas y la membrana son tan fuertes que incluso la lisis de las células no las separará. Se utiliza un tensioactivo especial llamado detergente para extraer las proteínas. Al igual que los fosfolípidos, los detergentes tienen cabezas hidrofílicas y colas lipofílicas, y pueden entrar libremente en la membrana. Dentro de la membrana, las colas lipofílicas del detergente interactúan con el núcleo de la proteína hidrofóbica. Esto rodea la proteína con una capa de cabezas de detergente hidrófilo, lo que interrumpe las interacciones proteína-lípido. El complejo proteína-detergente ahora se separa fácilmente de la membrana. El detergente hace que el complejo sea soluble en soluciones acuosas y esté listo para su reconstitución en una membrana artificial. Las proteínas a menudo se reconstituyen en las membranas de los liposomas, que son vesículas artificiales. Para preparar liposomas, los lípidos secos se hidratan y agitan para inducir la formación de vesículas. Cuando se agrega un detergente, se incorpora a las membranas de los liposomas. Para reconstituir la proteína, se combinan las proteínas solubilizadas y los liposomas, y luego el detergente se elimina de la solución mediante diálisis o adsorción química. Las proteínas y los liposomas se ensamblan rápidamente en proteolisomas, por lo que solo quedan expuestos los grupos hidrofílicos. Las proteínas funcionan como lo harían en una membrana celular y pueden investigarse de forma aislada. Ahora que hemos cubierto los conceptos básicos de la reconstitución de proteínas, repasemos un protocolo para reconstituir proteínas de membrana en liposomas. Para comenzar a aislar las proteínas de membrana, se lisan las células. Las células intactas se eliminan con centrifugación. El sobrenadante se centrifuga a una velocidad más alta para granular las membranas. El pellet se vuelve a suspender y se añade un detergente para extraer las proteínas. Los restos de celda restantes se eliminan mediante centrifugación adicional. La proteína se purifica a partir del sobrenadante con cromatografía en columna y luego se concentra o purifica aún más según sea necesario. Para comenzar a preparar los liposomas, se seca una suspensión de fosfolípidos en solvente orgánico bajo nitrógeno o argón. Los fosfolípidos se hidratan con tampón de hidratación, y la mezcla se sonica para terminar de crear los liposomas. Se añade detergente para solubilizar los liposomas, que luego se combina con las proteínas. A continuación, el detergente se elimina por adsorción sobre perlas de poliestireno, diálisis o una columna de unión de detergente. Los proteoliposomas resultantes están listos para ser purificados y utilizados en experimentos posteriores. Ahora que está familiarizado con los conceptos básicos de un procedimiento de reconstitución de proteínas de membrana, veamos algunas aplicaciones de la reconstitución de proteínas en bioquímica. Se reconstituyó una proteína transportadora de membrana para obtener una comprensión más clara de su mecanismo de transporte. Su función post-reconstitución se verificó con un eflujo de iones yoduro. A continuación, se estudió la actividad de transporte en presencia de varios inhibidores y potenciadores de canales iónicos de moléculas pequeñas. De esta forma, se podrían estudiar las interacciones directas de estas pequeñas moléculas con la proteína transportadora. La clorofila y las proteínas de membrana de unión a carotenoides en las plantas recolectan luz, promueven la separación de carga y mitigan el daño de la luz. Al reconstituir estas proteínas recolectoras de luz, se puede estudiar su dinámica de plegamiento e interacción con los pigmentos. Las proteínas recolectoras de luz reconstituidas con esta técnica tenían propiedades ópticas muy similares a las proteínas nativas. La espectroscopia de emisión de fluorescencia se puede utilizar para estudiar la transferencia de energía de los pigmentos a las proteínas reconstituidas que captan la luz. Acabas de ver el vídeo de JoVE sobre la reconstitución de las proteínas de membrana. La reconstitución es una forma de transferir proteínas importantes a una célula para su posterior investigación. Este video cubrió los principios de la reconstitución de proteínas, un protocolo de reconstitución y algunas aplicaciones en bioquímica. ¡Gracias por mirar!

Education

Educación

JoVE Science Education

Chemistry

Química

Biochemistry

Bioquímica

membrane proteins in liposome models

Membrane proteins can be moved into liposome models so their function can be studied outside a cell. Reconstitution means restoring an isolated biomolecule to a working form. For membrane proteins, that usually means placing a purified protein into an artificial lipid membrane.

Cell membranes are made mainly of phospholipids and membrane proteins. The phospholipids form a bilayer with hydrophilic phosphate heads facing water and hydrophobic fatty acid tails facing inward. Some membrane proteins attach by noncovalent or electrostatic interactions, while integral proteins are embedded in the bilayer. Transmembrane proteins are integral proteins that span the full membrane, and their strong interactions with the membrane are not broken just by lysing cells.

Detergent is used to extract these proteins. Detergents have hydrophilic heads and lipophilic tails, so they can enter the membrane. Their tails interact with the hydrophobic protein core and surround it with detergent heads, which breaks protein-lipid interactions. The protein-detergent complex becomes soluble in water and is ready for reconstitution.

Liposomes are artificial vesicles often used for this step. They are made by hydrating dried lipids and agitating them to form vesicles. When detergent is added, it becomes part of the liposome membrane. The purified protein and liposomes are then mixed, and the detergent is removed by dialysis or chemical adsorption. As the detergent leaves, the protein and lipids assemble into proteoliposomes, with hydrophilic groups exposed to water. The reconstituted proteins can then work in a membrane-like setting and be studied in isolation.

A general liposome protocol starts with cell lysis. Unbroken cells are removed by centrifugation, and the membranes are pelleted at higher speed. The pellet is resuspended, detergent is added to extract the proteins, and more centrifugation removes leftover debris. The protein is then purified by column chromatography and concentrated if needed.

Liposome preparation follows a separate path. Phospholipids in organic solvent are dried under nitrogen or argon, then hydrated with buffer. Sonication finishes the liposome formation. After detergent is added to solubilize the liposomes, the proteins are combined with them. The detergent is removed with polystyrene beads, dialysis, or a detergent-binding column, leaving proteoliposomes that can be purified and used in later experiments.

Reconstituted membrane proteins have several uses in biochemistry. One transport protein was reconstituted to better understand how it moves molecules. Its activity was checked by the efflux of iodide ions. Researchers also tested small-molecule ion channel inhibitors and potentiators to study how they interact with the transport protein directly.

Plant light-harvesting proteins are another example. These chlorophyll- and carotenoid-binding membrane proteins help harvest light, support charge separation, and reduce light damage. After reconstitution, scientists can study folding dynamics and pigment interactions. The reconstituted proteins showed optical properties very similar to native proteins, and fluorescence emission spectroscopy can be used to follow energy transfer from pigments to the proteins.

Membrane Proteins in Liposome Models

dialysis: diffusion based separation

Dialysis for Buffer Exchange in Biochemistry

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enzyme assays and kinetics

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chromatography-based biomolecule purification methods

chromatography-based-biomolecule-purification-methods

Protein Purification with SEC and IMAC

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two-dimensional gel electrophoresis

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electrophoretic mobility shift assay (emsa)

electrophoretic-mobility-shift-assay-emsa

Detecting Protein-DNA Binding by EMSA

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photometric protein determination

Measuring Protein Concentration with Absorbance

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density gradient ultracentrifugation

Sucrose Gradient Separation in an Ultracentrifuge

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co-immunoprecipitation and pull-down assays

co-immunoprecipitation-and-pull-down-assays

Protein Interaction Capture Methods in Cells

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reconstitution of membrane proteins

Membrane proteins can be moved into liposome models so their function can be studied outside a cell. Reconstitution means restoring an isolated biomolecule to a working form. For membrane proteins, that usually means placing a purified protein into an artificial lipid membrane. Cell membranes are made mainly of phospholipids and membrane proteins. The phospholipids form a bilayer with hydrophilic phosphate heads facing water and hydrophobic fatty acid tails facing inward. Some membrane...

f&#246;rster resonance energy transfer (fret)

FRET as a Molecular Ruler in Cells

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surface plasmon resonance (spr)

SPR Biosensors for Tracking Binding Kinetics

Surface plasmon resonance, or SPR, is a way to track biomolecular binding on a label-free biosensor. It is used to measure molecular affinity, kinetics, specificity, and concentration. The method works with a thin metal layer on a prism, where changes in reflected light reveal interactions in real time. SPR sensors use a thin layer of a noble metal on the face of a prism. Light is reflected from the prism-metal interface into a photodetector. At one specific angle, called the surface plasmon...

biochemistry

Esta colección presenta varios métodos de purificación comúnmente usados, como la cromatografía de afinidad y métodos analíticos, como MALDI-TOF. Además, los videos muestran métodos para evaluar la interacción y la función de diversas biomoléculas como en la coprecipitación y el etiquetado metabólico.

See how membrane proteins are extracted with detergent and reinserted into liposomes so students can study transport and light-harvesting function.

Membrane Proteins in Liposome Models | JoVE K12

matter-and-energy-in-organisms-and-ecosystems

Matter and Energy in Organisms and Ecosystems

HS-LS1-5.

HS-LS1-6.

Overview

Principles of Membrane Protein Reconstitution

Protocol for Reconstitution of Membrane Proteins in Liposomes

Applications

Summary

Characteristics of Life

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Cell Structure

Background
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What are Cells?

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Cytoplasm

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The Nucleus

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The Central Dogma

The DNA Helix

Nucleic Acids

DNA as a Genetic Template

Structure of a Gene

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Chromosome Structure

Base-pairing and DNA Repair

What is Gene Expression?

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RNA Structure

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Types of RNA

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Transcription

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Translation

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Ribosomes

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Protein and Protein Structures

Proteins: From Genes to Degradation

From DNA to Protein

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Protein Folding

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Chromatin Immunoprecipitation

Histones are proteins that help organize DNA in eukaryotic nuclei by serving as &#8220;scaffolds&#8221; around which DNA can be wrapped, forming a complex ...

An Overview of Epigenetics

Since the early days of genetics research, scientists have noted certain heritable phenotypic differences that are not due to differences in the ...

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Community DNA Extraction from Bacterial Colonies

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Luisa Ikner
Traditional methods of analysis ...

Cytogenetics

Cytogenetics is the field of study devoted to chromosomes, and involves the direct observation of a cell&#8217;s chromosomal number and structure, ...

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Embryonic Stem Cell Culture and Differentiation

Culturing embryonic stem (ES) cells requires conditions that maintain these cells in an undifferentiated state to preserve their capacity for self-renewal ...

DNA Gel Electrophoresis

DNA gel electrophoresis is a technique used for the detection and separation of DNA molecules. An electric field is applied to a gel matrix comprised of ...

Enzyme Assays and Kinetics

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DNA Ligation Reactions

In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond.  An enzyme known as a ligase ...

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Explant Culture for Developmental Studies

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DNA Methylation Analysis

Methylation at CpG dinucleotides is a chemical modification of DNA hypothesized to play important roles in regulating gene expression. In particular, the ...

Expression Profiling with Microarrays

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Density Gradient Ultracentrifugation

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F&#246;rster Resonance Energy Transfer (FRET)

F&#246;rster resonance energy transfer (FRET) is a phenomenon used to investigate close-range biochemical interactions. In FRET, a donor photoluminescent ...

detecting-environmental-microorganisms-with-polymerase-chain-reaction

Detecting Environmental Microorganisms with the Polymerase Chain Reaction and Gel Electrophoresis

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Bradley Schmitz
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Gel Purification

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Detecting Reactive Oxygen Species

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Gene Silencing with Morpholinos

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Development and Reproduction of the Laboratory Mouse

Successful breeding of the laboratory mouse (Mus musculus) is critical to the establishment and maintenance of a productive animal colony. Additionally, ...

Construct an explanation based on evidence for how the structure of DNA determines the structure of proteins which carry out the essential functions of life through systems of specialized cells.

HS-LS1-1.

Membrane Proteins in Liposome Models

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