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Membrane Proteins Overview Immunology/Microbiology > Introduction to the Cell > Introduction to the Cell Membrane Proteins Overview Immunology/Microbiology > Introduction to the Cell > Introduction to the Cell FREEZE-FRACTURE METHOD • Freeze cell and fracture it along cell membrane's hydrophobic interior • Proteins associate with either layer after fracturing • More proteins associate with cytosolic layer INTEGRAL PROTEINS • Embedded in the bilayer Transmembrane proteins: amphipathic, pass through both membrane layers • Single pass or multi-pass • Alpha helices: hydrophobic side chains • Beta barrel: multi stranded beta sheet (i.e. porin proteins) Monolayer associated • Alpha helix • Lipid-linked PERIPHERAL PROTEINS • Do not extend into the bilayer • Protein-attached: non-covalently bound to transmembrane protein • Oligosaccharide-attached: bound to carbohydrate head group of glycolipid Glycocalyx • Oligosaccharide side chains and glycolipids form carbohydrate coat on external surface of cell MEMBRANE PROTEIN FLUIDITY 1. Fuse mouse and human cells with surface marker proteins 2. Marker proteins mix on hybrid cell surface 1 / 6 • Conclusion: membrane proteins are fluid MEMBRANE PROTEIN FUNCTIONS • Transport ions, nutrients and other substances across membrane • Anchor cells to each other, to extracellular matrix or basement membrane • Transduce external signals to inside of cell • Mediate cell-cell recognition of glycoproteins on adjacent cell surfaces • Enzymatically catalyze metabolic pathways FULL-LENGTH TEXT • Here we will learn about membrane proteins, which are the "mosaic" in the fluid mosaic model. • To begin, let's experimentally examine the cell membrane. • We'll use the "Freeze-fracture" experimental method, which is commonly used to study the interior of the plasma membrane. • Imagine a cell frozen within a block of ice. • Now, imagine that we fracture the ice along the cell membrane's hydrophobic interior, which allows us to look at the inside of the plasma membrane. • So next, draw a plasma membrane in the process of splitting into its cytosolic and extracellular layers, (opening like a book). • Show that membrane proteins remain associated with either layer; they do not fracture into separate pieces. • Illustrate that more proteins remain associated with the cytosolic layer than the extracellular layer. - In an electron microscope, the cytoplasmic layer appears as a "mosaic" of bumps, each of which is a membrane protein. 2 / 6 • Now, start a table to learn the types of membrane proteins. • Denote that they include the: - Integral proteins, which are embedded in the bilayer. - Peripheral proteins, which do not extend into the bilayer. Next, let's draw each kind of protein within our freeze-fractured membrane. • We'll start with integral membrane proteins. - Show that we will distinguish them from peripheral proteins. First draw a transmembrane protein as follows: • Draw a protein with a single alpha helix that spans the length of the membrane. • Show that the C-terminus extends into the extracellular space. • Show that the N-terminus extends into the cytosol. • This transmembrane protein passes through both layers of the membrane once, and is therefore a single-pass transmembrane protein. • Now, draw another transmembrane protein with three alpha helices that span both layers of the membrane. - This is a multi-pass protein, it passes through the membrane three times. • Show that the N-terminus extends into the extracellular space and the C-terminus extends into the cytosolic side. - Notice that it has a different orientation than the first protein, but its terminal amino acids are still on opposite sides of the membrane. - This topology plays a role in protein function. Next, let's take a closer look at the alpha helix portion of a transmembrane protein. • Draw a phospholipid bilayer. 3 / 6 • Label the interior as hydrophobic and the exterior as hydrophilic. • Then, draw an alpha helix (of the transmembrane protein) that extends through the bilayer. • Write that all transmembrane proteins are amphipathic: they have hydrophilic and hydrophobic portions. • Specifically show that the alpha helix has hydrophobic side chains that interact with the phospholipid tails. Return to our freeze-fractured membrane. • Draw another transmembrane protein as a large barrel, which comprises a multi-stranded beta sheet that curves to form a barrel shape. • Indicate that beta barrels are often found in porin proteins. - They allow for the passage of nutrients, ions, and water through their hydrophilic centers. • Draw a horizontal alpha helix in one side of our bilayer, which is also amphipathic. • Show that both ends of the protein extend into the cytosol. • Label it as "monolayer associated." • Draw a protein on the cytosolic side of the cell. • Show that it covalently binds to a lipid. - Label it "lipid-linked." • Proteins can also be linked to the membrane via prenyl groups, which are hydrophobic. This concludes the types of integral proteins. Now, let's add some peripheral proteins to our diagram. • Connect a protein to the cytosolic side of our single-pass alpha helix. 4 / 6 • Indicate that it binds to the transmembrane protein by a weak non-covalent bond. - Peripheral proteins remain on the "periphery"; they do not extend into the bilayer. • Now, illustrate a glycolipid in the extracellular leaflet, which is a branched carbohydrate molecule, also known as an oligosaccharide, covalently bound to a lipid. - Glycolipids are only found on the extracellular side of the membrane. • Connect an oligosaccharide-attached protein to the glycolipid's carbohydrate group. • These peripheral proteins are only on the extracellular side of the membrane because they attach to glycolipids. This concludes the types of peripheral proteins • Now, draw oligosaccharide side chains bound to some of our proteins and extending into the extracellular space. • Indicate that these side chains, along with glycolipids, form a carbohydrate coat on the external surface of the cell called the "glycocalyx." Now that we've illustrated the mosaic of proteins in the membrane, let's experimentally investigate their fluidity. • Draw a circular cell with pink marker proteins on the surface. - Researchers use these markers to label membrane proteins, rendering them visible under a microscope. • Label it "mouse cell." • Draw another circular cell with orange marker proteins on the surface. • Label it "human cell." • Show that we fuse these cells to create a hybrid cell. The question is what will happen to the marker proteins: do they stay put or do they move? 5 / 6 • Indicate that after about an hour, the marker proteins mix on the surface of the cell, which shows that membrane proteins are fluid. - They move laterally in the membrane. Now, consider membrane protein functions; we'll learn their details elsewhere. • Denote that they: - Transport ions, nutrients and other substances across the membrane. - Anchor cells to each other, to the extracellular matrix or basement membrane. - Transduce external signals to the inside of the cell. - Mediate cell-to-cell recognition of glycoproteins on the surface of adjacent cells. - Enzymatically catalyze metabolic pathways. • Protein orientation in the membrane facilitates all of these functions. Powered by TCPDF (www.tcpdf.org) 6 / 6.
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