13 14 biochemistry elliot aminoacidsproteinstructure-function continued 1-3-14

8/12/2019 13 14 Biochemistry Elliot AminoAcidsProteinStructure-Function Continued 1-3-14

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13_14 BiochemistryEliot

1/3/1411:00 am-12:00 PM

Notetaker #28

Amino Acids and Protein Structure-Function Continued

Slide 25 : So lets analyze these things. We are being very redundant here. We arebeing very redundant with hydrogen bonds stabilizing the alpha-helices. Hydrogenbonds are probably stabilizing the helix-helix interaction. Electrostatic Bonds again,helix-helix interactions. Hydrophobic interactions, all core amino acids will tend tobe hydrophobic. But, that doesnt mean I might have a g lutamic acid with a negativecharge but as long as it is satisfied with a lyseine over here, with a positive charge,as long as I have a strong electrostatic interaction and the rest of the protein isshielding water away from it, it is an extremely stabilizing interaction.

If I heat the system up, making the water more molecularly active, and that heat isconveyed into the protein, disrupt sthat salt bridge, causing the protein to lose itstertiary structure. So what happens if I lose a structure? I lose function. A. Theslightest little thing can make you lose structure, and mess up the function of theprotein.

Slide 26 : Quarternary structure, hemoglobin is an example. A quarternary structureis an assembly of tertiary structures. One enter n-terminal to c-terminal lines up ,forms a tertiary structure. Those tertiary structures start interacting like inhemoglobin. In hemoglobin I have an alpha-subunit, beta-subunit. That beta-subunit can interact w/ 2 alpha subunits. Each beta subunit can interact with 2alpha-subunits. An alpha cannot interact with an alpha and a beta cannot interactwith a beta. So there are surfaces on the alpha that allow it to interact with the beta.There are surfaces on the beta that allow it to interact with the alpha. As a result, itforms a structure with an opening. This is what the structure is, 2 alphas and 2betas. Each one of these subunits has a heme.

Slide 27 : I through this in for Dave, Dr. (Leisenberg?), so you can see what anantibody looks like. There are a number of different components to a very complexantibody. Here is the heavy chain, 2 of them, light chain, 2 of them. There is thevariable region which gives you the antigen specificity, and the constant end thatallows you to interact with other things. Less stabilizing for each structure thatthere is a whole lot of alpha helical content for each of the monomers, but when youstart forming the tetramer, you are going to get some intrasubunit disulfide bonds.Covalently linking these strands together which is unusual for quarternarystructures.

Disulfide bonds usually stabilize tertiary structures, and its unsual to see themstabilizing quaternary structures. It is essential in this case. You have disulfide


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bonds stabilizing the tertiary and quaternary structures. So there is a whole lot ofsulfur chemistry in antibodies. Antibodies tend to last a long time because they areexceedingly stable. Enzymes, dont redox antibodies like they do insulin.

Slide 28 : So Structure function of myoglobin and hemoglobin. Myglobin, 8-5 is alpha

helices. Its got 7-8 alpha helices all folded with hydrophobic pockets, like a hand orbaseball glove where heme can bind away from water. Here is hemoglobin, with 2alpha and 2 beta subunits, 4 hemes. It has got heme bound in there as well. What isunusual is that they both like going for that iron. Well, myoglobin, binds to oxygenand low parital pressures of oxygen, and doesn't give it up till extreme levels, inanaerobic conditions.

So myoglobin holds on to oxygen in cells, until the oxygen is extremely low. Onceyou get rid of all that oxygen, you start unloading all that oxygen from myoglobin.For hemoglobin, its 4 globular molecules, with 4 heme residues. You run thesethings through lungs, hemoglobin from red cell goes to lungs, saturates with oxygen,

and lets go of oxygen at higher levels, about 24, 25 atm of oxygen pressure. Somyoglobin holds onto the oxygen very tightly, until the oxygen levels are nearly allgone. Whereas, hemoglobin holds onto it for a little while but is much happiergiving it up. It is the same heme, have 4 globulin proteins instead of 1 globularprotein.

So what is the difference? Cooperative bonding. If I start putting Oxygen into thesystem, myoglobin suck up the oxygen quickly. But it takes a while for oxygenconcentration to get high enough to force its way into hemoglobin because thetetramere is different. This makes it hard to get oxygen to stick. It is easy in thelungs. So, hemoglobin can give of oxygen at higher pressure, because thedeoxygenated form is more stable. Myglobin is a lot slower. Remember, the onlydifference is that one is a monomer and ones a tetramer. What this should mean toyou is that subunit subunit interactions, the more stable an object is.

Slide 29: Spontaneous Protein Folding

Slide 30: We start to form various stable structures. We start with a sequence ofamino acids, it just happens to form an alpha helix. So how does it do that? If your astructural biochemist (?), you get a sequence of the primary structure through x-raycrystallography, You say, oh well amino acids, 39-50 will definitely be in the alphahelix because this sequence of amino acids has been shown over and over again, toform an alpha helix. Then you start building other structures around it.

How it forms in nature is you get the most structurally thermodynamic stablestructure first, and they form this thing called a molten globule where it pries itselfout. Now I like this structure is thermodynamically stable, If I put this structurenext to this other one, its more thermodynamically stable, but if I just it somewhat,they are even more thermodynamically stable. There is a lot of testing going on


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until you get the native confirmation. This is highly reversible, active process that isgoing when while proteins are being synthesized.

Slide 31: **Shows video of the three stages in unassisted protein folding, &narrates**

video can be found under Electronic Reserves Tab under the MedicalBiochemistry course on blackboard. It is called GCSF- Protein-folding- illustration movieNarration: There is your amino acid sequence. The primary structure forms withthermodynamically stable regions coming together preferentially. They will formalpha helices first, and then the alpha helices will mix and match until they find whatis thermodynamically stable confirmation. Adjustments will be made. Here is youramino acid, now primary sequence then tertiary sequence, all the side chainsinteracting with each other appropriately in the most stable thermodynamicstructure. That structure, will now interact with a receptor on a cell surface andhelp stimulate the T-cells.

Slide 32: Now what that showed was here is a small poly-peptide chain, withessentially 1 domain, tend to spontaneously fold up into the mostthermodynamically stable structure. On the other hand, there are some proteinswhich are huge. The cystic fibrosis protein, for the Cl- ion channel, has over 14,000amino acids w/ multiple domains. These larger proteins need help folding. Theyneed to determine what structures are important and how to fold up into the finalpermanent structure. The bacterial model here is here. Very similar things arehappening in the human model. Here is the ribosome, the ribosome starts to makeprotein, well go over this in a lot more detail later. As the protein emerges from theribosome, a certain number of polypeptide chains, and residues, start to form asystem that is a target for a chaperon protein like a heat shock protein. Heat shockprotein is a type of chaperon protein, I use the words interchangeably because thekind of do the same thing. Heat shocked proteins take denatured proteins andrefold them into their native confirmation. And they do that with the synthesis ofbrand new protein as well as thoroughly denatured proteins.

Here are chaperon proteins, that help fold proteins, and cart them around like a taxicab service. They help speed things up. They also take newly synthesized proteins,and fold them up and deliver them around. There is a lot of work being done inheath shock protein and chaperon proteins.

This chaperon protein, takes energy, takes ATP. In bacterial they have amulticomponent protein, that if carried by the heat shock protein, it will help it findits most thermodynamic form.

These proteins are emerging from the ribosomes, one amino acid at a time, from then-terminal end, to the c-terminal end. They can fold up spontaneously or they canget assistance from chaperon proteins.


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Slide 33: Now the most famous experiment with this is the Anfinsen Experiment.Anfinsen picked a very stable protein, RNAse A. This is often found on the surface ofyour skin. If it comes in contact with any RNA, it cuts it without discretion(nonspecific). How do you know its working?

Anfinsen would put in the protein into RNA. To see if it the RNAse A proteinworking, he would measure UV light because RNA absorbs more UV light as amonomer. If the protein has been denatured, the function is gone and thus itwouldnt chew up RNA. That means, the nuclear protein had been denatured. Thishappened because the protein structure was dissolved or denatured by adding achaotrophic agent (8M urea) and a disulfide bond reducing agent (5mM -mercaptoethanol). The protein unfolded and lost its RNase activity (structure =function). However, when the denaturants were dialyzed away over 99% of theenzymes activity returned. If the structure forming process were totally random,less than 1% activity would have returned. So, as long as the primary structureremained intact, the protein would refold spontaneously into its active tertiarystructure. Th is meant, that the whole primary acid sequence was its own functionaldomain, with all the information contained in the primary sequence.

If we go back to earlier today, DNA sequence = RNA sequence = protein sequence.Protein sequence then folds up spontaneously into a structure, which is what heAnfinsen experiment proved. PRIMARY STRUCTURE DICTATES TERTIARYSTRUCTURE AND FUNCTION.

Slide 34: And how do you do that? (ie.e what are ways you can denature a protein?)

You can heat it up. You can change the pH, like put an acid, you would createcarboxyl group sand break up any sulfate bonds. You can add chaotrophic agents(chaos inducing agents) that can disrupt hydrophobic interactions, which usuallystabilize globular protein cores. Examples include urea. Oxidation can also effectthe stability of disulfide bonds, and the degradation ofsome sensitive amino acidside chains (e.g. tryptophan).

Slide 35: Irreversibly denatured proteins. Kinetics has a role here. If you make aprotein a little bit at a time, so alpha helices are made a little bit t a time. Whathappens if you have all the pieces together at once, especially for a protein withmultiple functional domains? You will get irreversibly denatured proteins (theycannot go back to normal). You cannot get them back to their native form.

This usually happens with larger multi domain proteins. Alzheimers, Parkinsons,Cruetzfeldt-Jacobs (Mad Cow Disease), chronic wasting disease. These are allglitches where a protein, is folded up into its correct structure, and then somethinghappens in the microenvironment that causes it to refold into something much morestable. So the prion is all nice, doing what it is supposed to do, normal brain


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function, then something catalyzes the protein to form a more stable structure. Wellthat more stable structure, induces other proteins to form a more stable structurearound it and they start forming plaques of denatured protein crystals. These arenot fixed by the body.

Why do you run a fever? If you have a bacterial infection, why do you run a fever?You are heating the system out to denature proteins and the bacteria like DNApolymerases and RNA polymerases. So if you run above your body temperature,you are going to denature the proteins in the bugs selectively. On the other hand, ifyou are running a temperature too high for too long, you start denaturing your ownproteins.

You are looking at selective denaturing that is causing disease states. Proteins dontlast forever. Some are supposed to, at least for your lifetime, like the cartilage yourknees. Some proteins turn over rather rapidly. Like cyclins. They turnaround cell

division in nuclei. The cyclins are there for only a few minutes. They are made witha sequence that says eat me, destroy me, get rid of me as soon as Im made, be causeif you dont Ill keep going, creating a million cells, leading to cancer. So there arespecific sequences that say destroy this protein. Or there are specific denaturesstructures, that if the protein starts to unfold like in cystic fibrosis, the membraneprotein pumping ions, if it starts to unfold it is going to get targeted for destruction.This is the neural mechanism for getting rid of proteins.

But in diseases such as Alzheimers, they start forming denatured plaques. Thesestable plaques cannot be attacked by the system so they start to build up. Normallyas the proteins age, become damaged, partially denatured, youre going to turn them

over. Here is your primary structure rolled up into your tertiary structure. Theystart to denature, and exposing a domain. This domain is recognized by theubiquitin enzyme complex. This protein is called ubiquitin because it is ubiquitous.It is found in every single cell. It is found in every single cell because you take it andcovalently attach it to a denatured protein. The ubiquitin helps guide the denaturedprotein to S26 proteasome complex. Its like a pez dispenser. You put thedenatured protein and it gets chopped up into chunks in the complex. These chunksare then denatured to amino acids. They then get recycled and so does ubiquitin. Sosignaling sequences that say I need to be turned over, Im old, they get targeted bythis ubiquitin pathway and get degraded.

13 14 biochemistry elliot aminoacidsproteinstructure-function continued 1-3-14

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