Cell biology/Membrane Structure: Dynamics

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Here is the link to the ITunes U Lecture from Berkeley. Membrane Structure: Proteins


Membrane Structure Dynamics[edit]

Membrane proteins are asymmetrically placed and fixed in the membrane so that one side is always externally directed and one side internally directed. Experiments to prove this are explained below.

Sugar Where?[edit]

Gold molecule-membrane.jpg

You can determine the placement of sugars connected to glycoproteins using the technique below. Which has empirically proven that glycolipids and proteins contain sugars only on the external side of the cell membrane.

  1. Plants produce protein, called lectin, that binds sugars when released during cell lysis. The enzyme binds to the sugars that are found on the outside of bacteria acting like a simple antibiotic.
  2. ConA is a type of lectin that binds manose and [[w:Glucose|glucose].
  3. Gold particles can be attached to ConA that can later be bonded to a red blood cell (RBC) which has a great deal of manose sugars attached to the outside of the cell membrane.
  4. After the gold and ConA are allowed to attach to the manose, the cell can then be prepared for electron microscopy and the gold can be seen (because gold scatters electrons).
  5. An additional preparation can be created where the cell is gently lysed introducing cracks where the gold ConA can come within the cell. This can then be visualized with electron microscopy and compared to the control (intact cell)
  6. This study demonstrates that the manose sugars around only on the outside of the cell.

SDS PAGE Technique[edit]

SDS-PAGE with Taq DNA Polymerase.JPG

Proteins are treated with a material that gives them a charge in proportion to their mass. They are then placed at the starting line of a gel that has a current running through it. The molecules travel down the gel like electrons travel through an power line, this is called electrophoresis. Since the proteins have the same charge/mass ratio, they travel down the gel according to their size. The smallest proteins go the furthest. These results can then be compared in many different ways. For more information see Details on Electrophoresis or Details on SDS-PAGE.

Breaking Stuff is Always Fun[edit]

1. Peripheral protein on cytoplasmic side of membrane, 2. Peripheral protein on extracellular side of membrane, 3. Integral protein spanning both sides of membrane, 4. Integral protein on cytoplasmic side of membrane, 5. Peripheral protein attached to integral protein-not embedded in membrane.

If you expose an intact cell to a protease enzyme, you can shave off the protein found on the outside of the cell. If the cell is broken, you can remove proteins from the inside of the membrane as well, leaving only the parts of proteins within the cell wall intact.

  • Experiment
  1. Expose intact cell to protease.
  2. Expose intact cell with detergent (to release proteins from the cell membrane) and protease.
  3. Create a control where nothing is done with the intact cell.
  4. Centrifuge to break cells.
  5. Perform an SDS page of the pellet (pieces of the cell that are centrifuged to the bottom of the cell) of each group.
  6. Then compare results.
  • Findings
  1. Compared to the control the group treated with only the protease looses part of proteins 2 and 3 and consequently they travel further down the SDS gel.
  2. When the group treated with the protease and detergent is examined, Proteins 5 and 1 are absent. Protein 4 is reduced in size and protein 3 is reduced in size further than it was in the previous trial. The size reduction is know because the proteins travel further when their size is removed during electrophoresis.

This experiment shows where the proteins are embedded or attached to the membrane. Real cells have more than 5 membrane proteins and therefore much more complicated. This method has to be modified to work on a real cell.

Immunoblotting or the Western Blot Technique[edit]

SDS-PAGE Coomasie stained.jpg

This is a technique that uses an SDS-PAGE. After the electrophoresis has run, the gel is turned upside down onto a piece of nitrocellulose filter paper. Electrophoresis is ran again at a perpendicular angle to to the gel, which causes the proteins to transfer into the paper. The paper is then placed into a buffer that has antibodies with fluorescence ability or radioactive particles. The paper is then exposed to UV light so the antibodies with fluoresce or exposed to x-ray sensitive paper. Additionally, the proteins can be tagged through many other markers. For more information see Western Blot.


Antibodies-Doesn't Your Doctor Prescribe Those???[edit]

An antibody can be used to select a desired protein so the previous experiment can be used in real cells.

  • Glycophorin is a protein found within red blood cells (RBC) and it spans the membrane one time. The following study was designed to identify it's directionality.
  1. To produce the necessary antibodies to glycophorin, human glycophorin is injected into a rabbit. The antibodies is then extracted from the rabbit's blood serum. Multiple antibodies are produced that attached to different parts of the protein.
  2. The next parts of the experiment are the same as above. Cells are reacted with proteases with or without detergent. An SDS is then produced, and that SDS is then treated with the Western Blot method described above identifying pieces of only the glycophorin protein.
  3. This technique can also be used to find the location of the N-terminus (amino acid end) and C-terminus (carboxyl end of the protein). For this, antibodies specific to to N-terminus or the C-terminus are used. These antibodies can be produced by a rabbit or created in the laboratory (small amino acid chains can be manufactured through laboratory methods-large ones are still not feasible). To analyze the results, simply determine which side is present when the cell is treated with proteases without detergent. The terminus present on the inside of the cell will not be destroyed.

Really, After All That Work Some Proteins Are Still Illusive!!![edit]

  • Unfortunately, not all proteins are as simple as this one and they snake their way back and forth through the membrane multiple times. With the previous method it is difficult to analyze these proteins.
  • To get a good three dimensional analysis through laboratory methods, the protein needs to be crystallized and pure samples can then be analyzed through x-ray crystallography. This can be done with proteins in general but membrane bound proteins are difficult. Membrane proteins without detergents or lipids denature. They can only maintain their structure with detergents; these same detergents prevent the closeness required for crystallization. There are approximately a dozen membrane proteins that have been previously crystallized. The first group to perform this minor miracle won the Nobel prize.
  • So if you can not crystallize the protein and you can not cut it up in pieces to study it, what do you do? Thankfully, computers are used to model proteins and determine which parts are found within the membrane. In most membrane proteins the amino acids found inside the membrane are nonpolar. So hydrophobic portions (around 20 amino acids long) suggest that that piece is found within the membrane.
  • You might remember from Biochemistry that proteins have nonpolar regions in their core that help keep them folded correctly. Would this skew the results? Actually, no it does not. The nonpolar amino acids that are found in the center are from many different sites on the protein. In other words, while the amino acids in the center of the protein are close in tertiary and quaternary structure, they are not close in the primary structure.
  • So a computer can normally model proteins, but does this always work? No, that would be far to easy. A major exception is porin-a relatively simple protein channel that allows charged and polar molecules into the cell. Porin's structure does not include the nonpolar regions in the primary structure that would be expected for a membrane bound protein. Porin's structure was determined by x-ray crystallography (with a detergent). Porin has alternating nonpolar-polar amino acids in a beta structure called a Beta Barrel. The nonpolar amino acids face the cellular membrane and the polar amino acids face the inside of the channel where charged and polar molecules travel.

Sisterhood of the Traveling Proteins[edit]

Syndai virus cell fusion.jpg

Membranes are fluid and the proteins can rotate like a top or travel all over the membrane. The only position retained is the internal versus external. A major experiment was conducted to prove this.

  1. Antibodies were created to recognize human cell proteins and mouse cell proteins by inducing an immune response in a laboratory animal and extracting the antibodies created. The antibodies are then tagged with fluorescence or radioactive isotopes or other markers.
  2. For a control you treat human cells with both antibodies and a mouse cell with both antibodies.
  3. The mouse and human cell membranes are then fused together and treated with antibodies.
  4. The results show that the proteins become distributed throughout the fused cell in a couple of hours.

This experiment will be reviewed in the next lecture.


Contact me via email if you need help. Thanks, April

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