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14_15 Biochemistry Dr.

Elliot 01/06/14 9:00-10:00 AM Note-taker #29 Protein Structure and Function (Part 2) Slide 37: Good morning Dr. Han, how are you today? Is this too loud? Sounds awfully loud to me. I got several questions on email. 1) Do we have to memorize amino acid structures? Be familiar with them, memorizing them, no. I wont ask for any structures. 2) Do we have to read assigned material? Its recommended so that you have a good background, when we talk we focus on aspects and not the entire chapter. Im not going to give you a book report. Were going to focus on little aspects of things, flexibility and lack of flexibility of bonds will cause only a limited number of conformations possible (amino acids to protein). We are piecing things together to get a structure-function relationship. Proteins are insanely dynamic structures; they are not rigid in their conformation. The next ten some slides are all neurophysiology. Were going to talk about things on a structural basis. Slide 38:

This is your basic model of a membrane. Points at phospholipids. They are made out of two fatty acids, phosphoglycerate [?] to some sort of polar head group. Points to integral protein. This is a protein embedded in the membrane. There are some proteins that are attached to the surface, they are peripheral proteins. Some of these are protein-protein interactions, some are protein-lipid interactions. Some require additional chains to be added. This is hugely important for signal transduction. Whats the purpose of a membrane? Protection, it separates cytosol from the rest of universe. Compartmentalizing things to sequester working systems in particular

environments to optimize function. Also, biological platforms, enzyme activity, transporter activity. Without membranes, there would be no life. Slide 39:

Structural features of some of these proteins. Here is an amino acid chain starting from exterior to interior. This is glycophorin A, very commonly found in red blood cells (RBC). Its a membrane bound integral protein. Its a single alpha helix with four rotations. The side chains are more hydrophobic. The axis has hydrogen bonds stabilizing the proteins. Side chains are sticking out perpendicularly. Leucine, isoleucine, glycine are all hydrophobic, so they are embedded in a hydrophobic membrane. Therefore the alpha helix is stable. The boundaries have charged amino acids. The polar head groups are attracted to them, the membrane interface has a few charged amino acids engaged in ionic interactions with the polar head bodies. So lots of stuff happening to stabilize the proteins. Inside the cytosol there are polar amino acids, these are targets for water to bind, for signaling like phosphorylation. That changes protein structure and function. We will come back to this in signal

transduction. The external part has branched chain carbohydrates, complex oligosaccharides. They engage in hydrogen bonds with water. This organizes a whole shield of molecular water. The water acts as lubrication for the exterior surface of RBC. The cell membrane is non covalent (held by random forces), you want to limit shear forces so you lubricate it with water. This is vital to maintain the integrity of blood cell. Slide 40: This is a member of a 6 or 7 membrane helix. Its bacteriorhodopsin. Helical bundles are the main point of this slide. These bundles have helix-helix interactions. How would that happen? Hydrogen bonding, ionic interaction, disulfide bonds, all of the weak bond strengths. All of these are participating to keep Helix A close to Helix B. Maybe you have Leucines on one and on another allowing the Leucines to zip together making it a Leucine zipper structure. This is just a model to show you that helices will interact and form bundles. Why is this important to you? Slide 41: Lets talk about the Na/K-ATPase pump. How many helices are involved? 7membrane helix, transmembrane protein. Sodium binds from the inside, it changes conformation so that an asparate residue is exposed and phosphorylated. It reduces the affinity for sodium with conformational change. Once the sodium departs it creates a binding domain for K. There is a reorganization causing affinity to decrease, the phosphate hydrolyzes off and gets released. Transient covalent bond drives a conformational change. Proteins are flexible. You are using this pump to create an electrical potential across a membrane to drive nerve and muscle function. Three positives go out [Na], two positives come in [K]. The membrane at this point becomes a capacitor predominantly, but also has resistor functions. A capacitor that can be discharged with a change of conformation. Water and chloride and bicarbonate are discharged, to regulate osmotic pressure. Regulates cell volume, regulates pH, if I have a muscle cell here and Im generating metabolic acids, I will pump out protons and bring sodium in. Sodium also drives Ca activity. Slide 42: If you go on the blackboard site, there is a page and a half of this is included underneath this picture. The Na+-K+ATPase pump is a monument to controlled reversible structural dynamics of protein sub-domains. The net effect is to transport or pump both 3 sodiums and 2 potassiums against their concentration gradients at the expense of ATP hydrolysis and generation of a transiently phosphorylated form of the transporter/pump.

[For the explanation of the pump, I copied and pasted what was written in the Notes section below (numbers 1-7), since he mentioned it, and I incorporated whatever Dr. Elliot added to the explanation] 1. The transporter/pump has a central pore open to the cytosol. In this conformation there are 3 high affinity sites for sodium. As the sodium ions bind to their sites the protein complex changes conformation slightly to activate a catalytic site. This site transfers the terminal gamma-phosphate from an ATP to an aspartate group adjacent to the central pore opening. This forms a high-energy phosphoanhydride linkage with the aspartate residue. 2. The phosphate group negative charges induce a conformational change in the transporter core by charge charge repulsion with other aspartate and glutamate residues stabilizing the sodium-binding pocket. The phospho-anhydride is temporarily buried in a more hydrophobic environment allowing it to stably exist. The altered conformation is open to the extracellular fluid outside the cell. 3. The new conformation has a lower affinity for the 3 sodium ions and they are displaced into the extracellular space. With the sodium gone, the binding site rearranges to precisely accommodate 2 potassium ions. This binding site has a high affinity for potassium. Why is that? Remember the periodic chart? Where is potassium? Below sodium. Potassium is much bigger than sodium. The reason that two bind is because thats all the space available for it. Those two bind in the space that could hold three sodium.

4. Potassium ion binding shifts the conformation of the central pore once again, and in this conformation the phospho-anhydride bond between the phosphate group and the aspartate group is repositioned (re-exposed) into a strongly hydrophilic microenvironment. Water hydrolyzes the phosphate group from the phosphoanhydride linkage regenerating free aspartate. You reduce the affinity for potassium and it leaves. 5. Without the phosphate, the regenerated aspartate is free to force the central pore back into its original conformation. 6. The original conformation has a low affinity for the 2 potassium ions and they are released into the cells cytoplasm. The vacant central pore opening now has a high affinity for 3 sodium ions and the cycle repeats. 7. Net effect: 3 sodium ions out and 2 potassium ions in. Generating both a concentration and electrical gradient used to power cellular molecular physiology. There is a lot of flexibility in this. That particular mechanism in the brain takes up about 1/3 of ATP. Thinking is exercising. Slide 43: Here is the actual structure for the Na/K-ATPase membrane protein. Here is a cartoon of the helices and the phosphorylation binding domains. Im not interested in you drawing this cartoon, but know that its mostly alpha helices and flexible. It can be poisoned by a couple of different things. There is a specific binding domain for cardiac glycosides. They affect your heart? Has anyone heard of Ouabain? Its a cardiac glycoside. Ouabain is a widely used rat poison. Put it in some bait, it binds to these pumps, the gradients break down, and the rat goes into respiratory arrest. What are we doing? Some doctor decided to give some to patients. It doesnt completely inhibit that activity? No it brings it down a little bit. You cant add too much because you will kill the patient. You knock out 15% or so of the pump, breathing may slow down. Put in just the right amount, sodium levels come down, so less calcium gets pumped into the sarcolemma. The contractility will increase. The patient with heart failure, give them a little bit of this, bring down the sodium gradient, so that it cant pump Calcium back into the sarcolemma to the sarcolemma reticulum where it is stored. A lot of these toxins are found in beautiful plants in nature. For example, Foxglove. Slide 44: So another transporter. Here is a glucose transporter like one in the GI tract. It requires a symport. Glucose binds to GLUT transporter and sodium drives the uptake of glucose. This is secondary active transport. Other types of transport do not require sodium. Glucose transporters are 1-8, GLUT-2 is found in all cells all the time. Diabetics with exercise can produce GLUT-2 expression via altered genetic expression. When diabetics exercise, GLUT-2 is expressed more and so they can lower blood glucose levels just by exercising. This will keep blood glucose down. It

doesnt require secondary active transport. It works down the glucose concentration gradient. If there is more glucose inside the cell, its reversible. Thats the livers job. The liver stores glucose as glycogen. If blood glucose levels go down, glucagon will get released and release glucose from glycogen stores in the liver. It will raise blood glucose in circulatory system. Its a dimer protein with a number of alpha subunits, rocking horse in its shape. Slide 45: Synaptic vesicles, here comes a depolarization event. It stays in the membrane; it doesnt jump across the membrane. All these vesicles have a neurotransmitter ready to go. Calcium floods out and binds to proteins related to protein kinase C, like synaptotagmin. Acetylcholine gets released and binds to receptors via diffusion. Slide 46: There are five anchoring proteins in the Acetylcholine Receptors. All are heavily alpha-helical. They roll as bundles together, they occlude the opening. They roll apart and open when there is a signal. The signal is the Acetylcholine binding to two of the domain sites. This binding causes reorganization, and rotation. The channel opens. The channel is not specific. This shows up on the step 1. We have two gradients, potassium trying to get out, and sodium trying to get into the cell. Sodium wins the overall diffusion gradient. That causes depolarization of the membrane. The depolarization is localized. I dont need a whole lot of sodium to come in, you can move anywhere between 3-7 sodium ions, its negligible. This helps explain why depolarization can occur so quickly and so often. Slide 47: Four bundles, each has 6 alpha-helical components. They come together, a good number of these have hydrophobic amino acids, to embed into the membrane stably. The central core, has electrical charges for binding. What stabilizes the alpha helix? Hydrogen bonds going in the same direction. So the structure is voltage dependent. You can organize the dipole moments in one direction. They will assort into thermodynamically stable structures according to what satisfies each alpha helix. Slightest changes in voltage potential will change the protein structure. Slide 48: When this is open and we get flux coming through. There is a random coil associated with an alpha helical ball, it plugs the channel. Were going to resort into the original conformation. Slide 49: Fun stuff. I dont recommend Fugu fish, because if this structure is not totally denatured, it is detrimental. The symbiotic bacteria makes this toxin, the fish has sodium voltage regulated structures. Only its binding site is mutated and different from ours. If we eat a little bit of this, it binds to the site on the protein and disturbs the Na voltage regulated channels. Your respiratory rate dies down, you throw up.

Some people are very sensitive to this, others just experience profound food poisoning. Slide 50: Heres another one, Red Tide. You couldnt go out clamming for a full 2-3 weeks after a red tide. Red tide is caused by dynoflagellates. It has a similar structure to tetrodotoxin. It gets into and binds to the sodium voltage regulated channel disrupting its activity. One part of this toxin and serotonin receptors like each other. There are stories of people who take these toxins as powders, if you hit it just below toxic threshold; it makes you very compliant to suggestion. They act look and behave like zombies. Hence Voodoo zombiesm. Slide 51: CFTR: Cystic Fibrosis Transmembrane Conductance Regulator. Very large, multiple domain. It transports chloride and bicarbonate outside of cell. This is to electrically neutralize the sodium gradient and fix the osmotic imbalance. Those are the two main functions. The chromosome damaged is 7 on the long arm along band 31.2 there are at least a 1000 known mutations. Some are upstream, some are in introns, and so they dont actually become proteins. There are point mutations in exons, with missense or insertions or deletions. Some have no effect on functionality on protein. Some have profound effect. SNP: single nucleotide polymorphism. Theyre trying to change it to SNV: single nucleotide variant. This is because variants are more specific and polymorphisms are very vague. Use them interchangeably. Here is a missense mutation, there is supposed to be a glycine at position 551. But it is D, aspartic acid. Whats the difference? Glycine has no side chains, asparate has a long and charged side chain. What are we trying to transport? Chloride. It is negative. Aspartic acid has a negative charge and negatives repulse. So there is a physical and electrical barrier. Delta means deletion. The F is for phenylalanine. Phenylalanine 508 is a deletion, it is coded for by 3 nucleotides. A triplet deletion. This obliterates all activity. This is the most profound version of CF. Slide 52: This is where you can find where the CTFR is. If you look at your blackboard PowerPoint in the notes section, it has it numbered and written out. What you can do isN: nucleotide M: Messenger RNA. You can dig up any gene and find out what mutational analysis has been performed. With a target that large you will accumulate mutations. Here is the mRNA, the hash marks are exons. They run about 200-30 nucleotides in there. Each one is a functional domain. It helps the entire protein fold up on its self. It needs chaperones to do this. The hash marks are the exons. Down here are all the known SNPS, the blue highlighted are disease causing and green highlighted is major disease causing. What CF ends up doing isbasically its a hydration disease. Under normal conditions, you have sodium coming out of the cell. You hydrate the external component of the cell. You want to hydrate to get nutrients and oxygen to diffuse through and about. There is a cycle that works through here. You pump out chloride to match the sodium. In a CF airway, you dont

have the chloride pump. The chloride doesnt go. So a lot less water is outside, and so less nutrients and oxygen. The mucus layer is inviting to bacteria especially pneumonia. Chloride doesnt get out, you dont get water balance. You get a viscous material outside the cell.

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