Today Membrane proteins Hydrophobicity plots Transmembrane domains Inside vs. outside So you've all heard of various genome project. Very popular and important these days. You wind up with a sequence of zillions of bases of DNA and then you've got to interpret them. So first you figure out where the introns and exons are so you can find a reading frame. But for now, we will just concentrate on the proteins. One important class of proteins are membrane proteins because they are involved in signaling. We already had a mention of some of the signals (action potential, receptor potential, synaptic potential), but there are other signals that involve membrane proteins. Can be involved in cell-cell recognition and also in recognizing small molecules on the outside of cells Membrane proteins come in several classes (page 486 of steinbock book) Some of these membrane proteins penetrate through the membrane - transmembrane proteins. They have a domain of the protein that has to satisfy the environment through which it passes. Area that passes through the membrane needs to be hydrophobic. Some proteins go through the membrane several times. Proteins which are incorporated into the membrane = intrinsic proteins. In order to get these proteins out, you need to destroy the membrane. There are other proteins which fit this definition because they are bound to the membrane by a hydrophobic tail that gets stuck to the membrane and cannot be removed without destroying the membrane. But these proteins do not actually transverse the membrane. Proteins can also be stuck to the membrane with phospho-lipid parts, but can be separated from the membrane using phosphodiase (don't have to destroy the membrane to get it out) Other proteins get stuck near the membrane because they bind to proteins that are already in the membrane (transmembrane proteins). These proteins that get stuck to other proteins are called membrane-association proteins, or non-intrinsic. The bonds which stick the non-intrinsic proteins to the membrane protein are non-covalent bonds. You can dissociate these membrane associated proteins from the membrane by changing the strength of these non-covalent bonds. Different side chains of proteins have different degrees of hydrophobicity Alpha helix - 3 dimensional structure helps you to fit the protein through the membrane, along with the fact that it is composed of a stretch of hydrophobic side chains. Alpha helix properties: Uniform diameter and you just translate as you wind. Right-handed helix. 3.6 amino acids per turn - amino acids form hydrogen bonds in order to form the helix structure The nature of this alpha helix if you have hydrophobic side chains, then you don't have to worry about the NH and COO wanting to be near water if they are hydrogen bonded to each other. This is the way polypeptide chains are arranged in membranes most of the time. How do we know? x-ray crystallography Crystallizing membrane proteins is very difficult because they are made to be around lipids, and when you take them away from their lipid environment they get very unhappy. They do not want to crystallize next to one another. Channels in the membrane are made up of several alpha helices. The nature of this alpha helix that is happy when it goes across the membrane is 20-25 hydrophobic amino acids in a row (adjacent). If you see regions in the genome that have 20-25 hydrophobic amino acids, then you probably have a trans-membrane protein that will be made up of alpha helices. Graphs - hydrophobic is plus, hydrophilic is minus. Transmembrane domains show up as a large peak of just hydrophobic (positive peak). If the graph shows several peaks of hydrophobicity, then there will be several trans-membrane helix-shaped domains. Another way in which proteins can transverse membranes: beta sheet. Instead of having a helical array, you have a straight chain, and the hydrophilic reaction between the carbonyl and the NH make hydrogen bonds to a neighboring stretch of polypeptide chain. Called beta pleated sheets - much harder to predict from the amino acids sequences (only 10 amino acids long) In order to study further the aspects of the protein, one of the things we want to know is, if we have a protein that is predicted to be a transmembrane protein, but how can it be inserted into the membrane? Is this end inside the cell or outside the cell? You can get a cue by looking at the changes made to the protein after it is made on the ribosome. Di-sulfide bonds tend to exist on the outside of the cell If you look at your cystines and see which are bonded to other cystines, you can make guesses from that as well. If proteins have carbohydrates on them, the carbohydrate bearing domain is on the outside of the cell. In order to figure this out, you need to purify the protein (do protein chemistry on it). To do that, it is really nice to have a rich source of protein so when you are trying to purify these membrane proteins from other membrane proteins, the task is easier. Membrane proteins are fairly hard to separate from each other, in general. Certain biological sources in which certain classes of interesting membrane proteins (voltage gated channels, ligand gated channels) are found in high quantities - electric fishes called Torpedo. This fish has his eye on Matteucci, and he's thinking, "You bastard. When I get out of here, you're in so much trouble." Torpedo has ligand-gated channels in its membrane. Manages to produce this huge aggressive response of electrifying other things by having a whole bunch of these ligand gated channels. So it is a good source of ligand-gated channels for testing. There is an eel that is used to get voltage-gated channels, because they have a whole bunch of those. Animal toxins are really interesting - they affect other organisms so that the animal can eat them. These toxins are incredibly specific. The binding constant of a toxin called alpha Bungarotoxin is 10^-15 molar. It is a very tightly designed protein that sticks to the acetocholine receptor. The snake that makes this doesn't want to do himself in, so that is why this protein is so specific - if the snake just makes his own receptor immune to just this one protein, then it can use the toxin effectively. If it was less specific, then it would have to make greater changes. Having a very specific ligand like that, then you can use it on an affinity column to pick out acetocholine proteins. Then put the acetocholine proteins in an oocyte (which have no proteins of interest) and see which way the protein orients itself in the membrane. Three different ways to determine which way it is threaded through the membrane: Labeled ligand which binds covalently to a particular amino acid, usually to the side chain of Lysine ((CH2)4-NH2) This reagent is very water soluble and cannot penetrate membranes. If you take your membrane which has got your acetocholine in it and apply this ligand, it will only bind to amino groups that are on the outside of the cell. Then you isolate the protein and chop it up into bits, look at the sequence of the protein and see which lysines in the whole chain have their amino groups modified, and that allows you to tell which parts of the protein were on the outside of the cell (and therefore able to be modified). To check that you've done this correctly, you break the membrane to allow the reagent in so you can see that all the sections of the membrane should now be modified on the lysines - this is to make sure that there weren't some sections that could not have been modified at all. Use a membrane impermeant protease - you have a reagent which can affect the part of the protein which is on the outside and will chop up only those parts of the protein which are on the outside, and not the ones on the inside (until you break the membrane like in the last example, just to check) Antibodies - big proteins and don't get through membranes. If you have a labelled antibody, you make it specific to a small part of the sequence and ask whether the antibody can see it on the outside or whether you have to break the membrane to get reaction with the protein. Another way of looking at proteins in membranes is looking at Freeze fracture - a technique which is used for electromicroscopy, specifically for looking at membrane proteins. Principle is to take the tissue that you want to look at the membrane of and you freeze it to liquid helium temperature (very cold) and then the hydrophobic bonds were are holding the two leaflets of the membrane together will be broken and you can push apart the two leaflets. Then you look at this structure by platinum shadowing - beam of atoms from the platinum source will go at an oblique angle which allows you to see projections. You see little bumps which are the proteins associated with different structures. Image of a pre-synaptic terminal, looking into it, and there are rows of proteins that are the voltage-gated Calcium channels which control synaptic transmission.
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