 So for those that haven't found a seat, there's actually 270 of us. This room holds 300. So you might want to climb over people. There should be like a few seats available. Almost ready to get started. All right, we're ready to get started. Is there any questions from the last lecture? We went through the last part pretty quickly. So there's office hours. All your TAs are available for office hours by appointment. Just send them an email. I'm available on Thursdays after class for office hours in Sydney, Frank Hall. Yeah, so if there's any questions, maybe that's a better format. Also, sections start on Wednesday, which is tomorrow. So you can get your questions answered there. We were thinking that once we get into the sapling problem set, probably the more effective forum to ask questions about that is when you have conference sections. You have a little bit more time. You can have discussions on the questions. We do have a lot to cover in class, so it might be challenging. Hi. Conference starts tomorrow. Yeah, conference sections start tomorrow. Remember, these are not required but strongly recommended. You can go to any conference section. You can go to all of them. You can go to some of them. They're Brown students. You have choices. So whatever you want to do, we recommend you do go to at least one because we find them helpful. Also, SCAS has asked for me to solicit a note taker. So maybe after class, if anybody's willing to share the notes, put on some of those still print out PowerPoints and write stuff, and that would be really useful. So just let me know after class if you're willing to do that. Okay, so today, the first lecture, we began to learn the language of protein starting with the words, which were amino acids. We just began to put those words together into sentences forming AMI bonds between the words. So today we're going to be, so I already made this announcement. Oh, and you should also check your clicker. We've tried to input the response that you clicked into the Canvas grade book. And so if you don't see any response and you feel like you did vote, it'd be a good idea to talk to Alex after class. Also, there are cases in class when you click and like nothing happened. You probably might freak out or just panic. In those rare cases where you're having problems with your clicker, please take out a piece of paper, write your banner ID number, not your name, and put the response, you know, the letter response, and hand it in to Alex before the end of the class. So if you could do that quickly and then move out of the way, there's usually a queue start forming because it's a large class and lots of people might be having problems with their clicker. But please don't abuse this privilege. Bring your clicker to class. Try to see if it turns on, I guess, before class. Any questions on that? So I think there was probably how many, 70 people didn't, we didn't get a vote from? Only seven of the total students didn't vote. How many students were unregistered? So we got a clicker vote and you hadn't registered. We've stored your vote, but we can't give it to you until you register. And there was probably like 100 people that didn't, 80 to 100 people that didn't vote. So hopefully everybody's got their clicker today. Okay, so let's move on. So today we're going to be talking about the sentences of amino acids, proteins. We're going to be looking at different types of protein structure. We have some practical aspects to today's lecture. How do you work with proteins? How do you study them in a lab? And so that's today's topic. So remember I introduced this concept of different types of ways of thinking about protein structure. So the simple amino acid sequence from the end terminus to the C terminus is one form of structure. So this is the primary structure. Now it's not three-dimensional. It's going from the end to the C terminus. Secondary structure is where we have defined types of structures like a helix. You'll see a sheet today and you'll see a term. There's also a secondary structure that is structure-less. It's called a random coil. And that's useful to connect elements of secondary structure. The tertiary structure is where for those proteins that only have a single polypeptide, it's the arrangement of all the elements of secondary structure into a three-dimensional shape. So you might have different helices, for example, and you need to orient those relative to each other. The ordinary structure is for proteins that have more than one polypeptide. So here's a picture of hemoglobin. And you can see there's four different polypeptides illustrated in sort of surface contour mode. And so the arrangement of each of those polypeptides in relation to each other can be considered the ordinary structure. So we introduced the concept of the rigidity of the partial double bond in the amide bond. And so you see that we have these lone pairs here and you can have a resonance form where you have partial double bond between this carbon and this nitrogen, the carbonyl carbon and the amino nitrogen. And there's hindered rotation. In general, the substituents around that partial double bond are in the trans configuration to maximize or to minimize steric flash. And so, but this is very important to understand the structure. And remember I mentioned that there is one important degree of freedom here and that's rotation about this alpha carbon. So today we're going to talk about how we define the relative orientation of each of these planes formed by each amino acid in relation to each other. We're going to define some angles, phi and psi, define what they mean by looking at pictures and begin to build up into secondary structure. But at first we just started the amino acid. If you have any questions at all, just shout them out at any point, please. And so here's another way to look at this planar structure and so you can see here you have the alpha carbon and this is the trans configuration. In other words, you have the side chains or the alpha carbon is here and then the second alpha carbon is here and they're most distant from each other. If this amide bond is in the cis configuration, the amino acid side chains are bumping into each other. Now some amino acids side chains are rather small. Perhaps those amino acids, so we know glycine, for example, is one. Other amino acids have restrictions on some of these angles that we're going to be defining such as proline because we have that secondary bond to the amino nitrogen. So in general we're thinking about a trans configuration of this partial double bond. And so here we have a polypeptide and you can define two angles. And at first you might think, well, if I have two planes, isn't the only thing that's important, the angle between the two planes? That's not everything that's here. You do have two planes, but you also have a side chain. So if you just define an angle between these two planes, that's insufficient to tell the orientation of these planes relative to the side chain. So that's why we need these two angles, phi and psi. Our convention of naming proteins and thinking about proteins is from the end to the c-terminus. So from the left to the right, you go from the end to the c-terminus. So the way you remember which angle is which, it's an arbitrary thing, right? We just call them phi and psi. So phi is the first angle you come to, going from the end to the c-terminus. And psi is the second angle you come to. It's totally arbitrary. But these angles are not only telling you the angle between the planes, but also the angle between the planes and the side chain. Okay? So phi and psi, so far we've just defined them. But what about assigning values to phi and psi? I think this is a very confusing topic, because you have to imagine these things in three-dimensional space. So by definition, a phi and psi angle of zero these two planes are totally collapsed on top of each other. So you could imagine that this is just not going to happen in a protein. It's impossible. You have this hydrogen atom and this oxygen just totally on top of each other. But how do we think about assigning values to phi and psi? So I found these animations online. I think they're very helpful. So let me orient you to this. So here is the alpha carbon. See? Yeah. Here is the alpha carbon. Here are the two planes from the amide bond. And this yellow, orangeish orb is the amino acid side chain. And this is the hydrogen atom at the alpha carbon. Okay? And so for phi, first we're going to define phi angles or not. Oh, that's most unfortunate. Cool. Let me bring it up, because we're going to be lost without this. Okay. Okay, let's start that one more time and hopefully this thing gets out of the way. Okay, so you see as phi increases that plane on the amino side is rotating towards the side chain. So that's going, let's do it one more time. So you see this? The side chain is the yellow and you're rotating 90. So that's going to be the amino acid side chain. So you might imagine, okay, that might not be optimal. Positive values of psi might not be optimal because you're beginning to have an interaction, a steric interaction between the plane and that amino acid side chain. Now let's look at psi. How do we define psi? And I'm not going to ask you just, you might be panicking now. He's going to ask me to draw phi of 45 on a test. Just sort of, there's actually an easy way to remember phi and psi angle, what tends to happen in proteins and it's this Ramakandran plot we'll get to and that's the easy way to remember these angles. So this is just to get you oriented. So for the psi, so this is looking at different values of psi and so obviously we're going to rotate the other plane and you can see with positive angles of psi you're actually rotating toward us away from the amino acid side chain. So positive angles of psi you might guess are going to be favorable because you're not having this unfavorable steric interaction with the side chain. I hope my movies are good. Oh, yeah, sure. Yes, there is, I suppose, yes. There's not, well, at the end in the C-terminus one of those angles is not defined. Actually Alex RTA is a master of structural biology so he could answer these questions as well. So yeah, there is a phi and psi, every alpha carbon is defined by phi and psi and if you knew the phi and psi angles for every alpha carbon you would have the three-dimensional structure of the protein because you would know the orientation of all these planes. So we've defined phi and psi of zero arbitrarily. It was where this is a closed-down configuration. Phi and psi of 180 is the complete opposite where things are highly extended. So in general you might imagine that that's a favorable orientation, much more favorable than phi and psi of zero. So phi and psi is a bit tricky. Any questions so far on phi and psi? We'll come back to that with the Ramacandran plot. Yeah, so right. So remember there's not just the orientation of the two planes in relation to each other, it's the orientation relative to the side chains, which unfortunately they're not shown here, but they are flat. So the planes have rotated through angles where they weren't flat until they're most distant from each other. So you can sort of think of it as distant trans. So phi and psi of zero is akin to a cis-closed-down confirmation where psi and psi of 180 are the most distant. You could think of it as trans. Hi. More questions? You'd have to look at that. You'd have to think about it. Let's see. It's a good question. So 180 would be when one, if so, if phi was 180, this would be pointing up and this would be pointing down. I think you can get yourself very quickly entangled in thinking about phi and psi. And I'm not going to test you. I'm not going to say draw. The only thing I might ask you is draw zero and draw 180 and 180 for both. That would be the only question. And then I might ask you to tell me where some of the secondary structures tend to occur, what the phi and psi angles that tend to occur. And you can just look at this picture and put it in your head and you'd be able to say that. So it's tricky. Okay. So let's move on. So the thing that holds the secondary structure together is the hydrogen bond. And there's two things about a hydrogen bond. It has characteristics of a charge-charge interaction, but it's also directional. So the strongest hydrogen bonds are linear. These are the types of hydrogen bonds that occur in the organic large molecules will be studying this semester. And so you can see in this orientation you have a linear orientation of these hydrogen bonds and that's the strongest possible bond. And so there's a dipole. The oxygen is partial negative charge and the hydrogen is partial positive charge. So there's a directionality and there's this charging part of this dipole that's going on. So the hydrogen bond is the force that holds together secondary structure. And the first secondary structure we're going to look at is the alpha helix. And so the alpha helix has a certain number of turns. So these planes, remember each of these amide bonds is a plane. Each plane turns in relation to the neighboring plane by 100 degrees. So it takes about 3.6 turns to have a full 360 degrees. The distance between the different parts of this alpha helix are 5.4 angstroms. So you have 3.6 residues and when you get to 3.6 residues you will be 5.4 angstroms away from where you were before. The forces holding this together are solely the hydrogen bonds. So you have the hydrogen bonds in the polypeptide backbone. So what kind of things can hydrogen bond in a polypeptide backbone? We have the carbonyl groups and the amino groups. And so in this particular picture we have the red circles. That's the carbonyl groups here. And it's making a hydrogen bond about 3.6 or 3 to 4 amino acids further along in the polypeptide. And so the amino group there is making this hydrogen bond. And these forces stabilize this structure. So this is called the alpha helix. Linus Pauling was the one that worked this out. He actually took pieces of paper in his imagination and he turned the paper. He was like, what could possibly happen? And just based on simple modeling he said, well this is probably a likely structure because he saw when you turn it just right you are going to have some good hydrogen bond possibilities. The orientation of the carbonyl groups and the amino groups is optimal for hydrogen bonding. So let's think a little bit more about this. So remember I said there's about a 100 degree turn between each neighboring amino acid and that works out to a fine psi angle of 60, negative 60 degrees and psi angle of around a negative 45. Now these are not rock solid. There are examples where there's slight variations in these angles depending on the other forces applied to the alpha helix, but generally the angles are very close to this. If you don't have these angles close to this you're just not going to be able to make this turn and form the necessary hydrogen bond. So phi and psi angles are defined and restricted. There's restricted entropy. If you just had a polypeptide on a solution, they could have lots of different fine psi angles, but when you put it into this alpha helix you restrict the entropy of the polypeptide. Right. Now what we're doing is we're taking that alpha helix it was just up and down. We were looking at the hydrogen bond. We've turned it and now we're looking down the shaft of the alpha helix. So you can see these purple circles are the amino acid side chains. And do you see how they're perpendicular to the axis of the alpha helix? So the alpha helix is literally coming right out at us and these amino acid side chains are optimally placed tangentially to the axis of the helix. So they have relatively, you know, a good amount of space. One of the other forces that stabilizes this is the lack of negative asteric interactions between these amino acid side chains. They're pointing out into space. So here you can see the side view and down the alpha helix. So this is the orientation. Now it's a cork screw. So if there's any carpenters in the room, you know that there's two different types of screws. There's a right-handed and a left-handed screw. And you need to define these somehow in the way you do this. So here's an example of a screw. Similar helix. So in general, the alpha helices that occur in nature are right-handed helices. And so starting from the interminous down here and tracing the track of the helix going to the c-terminus, putting your thumb in the direction of the axis of the helix, if your fingers trace the track of the polypeptide with your right hand better, then that's a right-handed helix. You can see this other example starting from the interminous to the c-terminus. Now this is sort of a cartoon-tuned view of the polypeptide. It's just a ribbon and arrow. And so you can see with the left-handed helix your fingers wrap around in the correct orientation of that helix. Left-handed helices do occur in proteins, but in general they tend to be very short helices. They're much less common than the right-handed helix. So everybody's got their hands out figuring this out. Okay, one really cool thing about the alpha helix is it has a massive dipole. And the reason is, look at this, isn't this cool? So we have an electronegative atom here and a not electronegative atom, so we know there's a dipole between that. Generally I think of dipoles as pointing to the electronegative atom and the not electronegative atom, but in the case of the non-magnetic atom, the dipole is the same. The dipole is the same. The dipole of this carbonyl group and this amino group are in the same direction. They're additive. So you can see here throughout this polypeptide every carbonyl group and every amino group is going to add more and more dipole. This builds up an unfavorable accumulation of charge that turns out builds up a partial positive charge and the C terminus builds up a partial negative charge. You can use this in enzymatic catalysis by pointing this dipole at a chemical reaction or you can stabilize this helix by strategically placing certain charged amino acids either at the end or the C terminus. So at the end term, if you have a partial positive charge to neutralize that, you would want to have a negative residue, an acidic amino acid. At the C terminus, you have built up of negative charge so you would want to have some kind of positive residue, a basic amino acid. If you don't want to use that dipole to help catalyze the reaction. So it's this neat arrangement that is necessary. You cannot hold this alpha helix together without the hydrogen bonds. And once you make the hydrogen bonds, you set up this wicked dipole that you have to deal with. Okay, does that make sense? I almost always ask questions about this on the exam because it's what's at the end terminus sort of questions. What's at the C terminus? Okay, does that make sense to everybody? Okay. All right, so there's other things. So the hydrogen bond network that holds the alpha helix together is essential. Anything that tends to disrupt a network will tend to disrupt the alpha helix. So if there are hydrogen bond donors or acceptors close to the shaft of the alpha helix, you can exchange productive hydrogen bonds within the polypeptide backbone for hydrogen bonds with amino acid side chains. So for example, with aspartic acid, that's close enough to begin to affect the hydrogen bonding network that holds the alpha helix together. So in general, that particular amino acid is disfavored in nature in alpha helices. It would tend to disrupt this structure. Glutamic acid, on the other hand, has that one additional methylene. Remember, hopefully we remember some of these amino acids from the first lecture by exam one. Hopefully you remember more. So glutamic acid has one more, it's very similar to aspartic acid, but one more methylene. It's hanging out a little bit further, and that's too far to now make to interfere with the hydrogen bonding network that holds the structure together. Glycine is a free spirit. It likes to just rotate everywhere. Its amino acid side chain is a hydrogen. If you stick Mr. Free Spirit into a very defined, fine sighing, don't move, there's a very high entropic cost to pay for that. It's thermodynamically unfavorable because the glycine in alpha helix tends to disrupt the alpha helix. Proline has a fixed firing of minus 65, which is close, but not quite exactly what you need for the alpha helix. Remember, we needed a minus 60 for the alpha helix. So it's close. You might squeeze it in. We'll see some examples of special helices that have proline, but generally the normal alpha helices do not have proline. So they're firing angles incompatible. Okay? Listen to me so far. And so you can also have favorable interactions. For example, we can have two amino acid side chains that help to cement the structure in place. So here we have an aspartic acid. Three amino acids away, you have an arginine. You have a negatively charged amino acid, physiological pH, and a positively charged amino acid. So these could have favorable ionic interactions that hold this structure together. So there's certain amino placements of amino acids that are disfavored in alpha helix and some that actually stabilize the alpha helix. And we already talked about strategically placing basic or acidic amino acids at the ends of the alpha helix to neutralize dipoles. Any questions so far on this? Okay, we're going to move on to our next structure. So our next structure, luckily, integer version six has finally corrected this figure. And so this is a beta sheet. And normally you think a sheet on the bed is flat, but this is something called a pleated sheet. So think of origami, we have folds here. So we go up and down and up and down. But look at the arrangement. What is the force holding this structure together? Don't have it too quickly. You guys drink here. What is it? Blue atoms and red atoms, right? So these hydrogen is on. The same exact forces that we're holding together, the alpha helix, hold together these beta sheets. And so here we have a segment of polypeptide. And then we have another one. And what gets people confused every year is they're not connected, confusing. We'll come back to that. But for now, we just have two stretches of polypeptide that have made hydrogen bonds. And look at the angle of these hydrogen bonds. Do you see how they're linear? This is the reason this thing is pleated, because if it weren't, the hydrogen bonds would not be linear. The fact that hydrogen bonds are directional causes this thing to pleat up. This is referred to as the anti-parallel beta sheet. And so that means that the orientation into C-terminus alternates between each stretch of this beta sheet. So here we have, remember, we draw from the arrow from the end to the C-terminus, right? So here's an amino group, right? And then we're going to the C-termis. That's in that direction. This next segment is anti-parallel, going in the opposite direction. And then the other direction. Okay? So there's about 7.8, or 7 angstroms between neighboring amino acids in this structure. So you can imagine that's a longer distance in some of the distances we were seeing and the types of angles we were seeing and the alpha helix. So the phi and psi angles by intuition are going to be larger for the beta sheet. And you can just look at this thing. It's more spread out. The other cool thing about this is the amino acid side chains, again, are perpendicular to the track of the polypeptide backbone. So here the amino acid side chains are in purple. Do you see how they're pointing up? Then the next one is pointing down. Up, down. So this is really optimal. So neighboring amino acid side chains are very distant from each other. Lots of space here to work with. Now, you think about, okay, well, so what happens in between these? A lot of things can happen. But here's a simple, this is an actual anti-parallel beta sheet from Nature. And you can see we just have little short connecting segments between each of these segments of the beta sheet. But they don't necessarily have to be near each other. You could have a segment that goes all over, all over, comes back and makes another part of the beta sheet. The alpha helix is contiguous within the primary sequence, whereas the beta sheet is not necessarily contiguous. So you have one part. You set start a beta sheet and then you have the next part. And then you have something in between. Then you have the next part. So people get that a little confused. Does that make sense so far? This is the second structure that we've looked at. So if we can have an anti-parallel beta-plated sheet, we could have a parallel beta-plated sheet. Here's an example. What molecule is this? Are there any immunologists in the audience? Can you guess? Something is being presented. MHC. So this is a beautiful anti-parallel beta-plated sheet. And you can see in this example, you have stretches of randomness, random coil, connecting each of these strands of the beta sheet. And you have a nice comfy home for Mr. Immunological Peptide to hang out and be presented to T-SELF. And you have alpha helices that are also connecting these things together. So here's a parallel beta-plated sheet. The first thing you see when you look at this is rut row. These angles are not optimal. They're not linear. So these hydrogen bonds, this is a stressed out secondary structure. There's just no way you could squeeze this thing and it would be so pleated that it would be impossible. So now you have these non-linear bonds. The repeat distance is shorter because it's trying to make these directional hydrogen bonds linear and it's just not quite getting there. So here we... And in the parallel beta-plated sheet, you have an orientation-intensity terminus going in the same direction for each segment of the sheet. Again, it's confusing. How do you connect them all together? It's like, well, there's something in between. So here's an example of a parallel beta sheet. And you can see here you have one strand and then it goes off and it's a sort of... And then eventually it makes it all the way back here and has another strand. So each strand of the beta-plated sheet is not at all contiguous in the amino acid primary sequence, but these structures form. They form because of the favorable hydrogen bonds. All right. So that's our third structure, sort of second, 2A and 2B. Any questions so far? So we have one more structure to cover and that's the beta-turn. So this is a very simple structure. You have four amino acids and the idea here is come in one way and come back out the opposite way, right? And there's some serious angles that you need to achieve to turn yourself around in four amino acids. Because of that, prolings in one type of beta-turn, prolings are favored because they have the contorted sort of angles that you need, but they're not your average prolings, not the trans prolings. These are cis prolings. So we'll see that in the next slide. Glycines tend to occur in a different type of this beta-turn because glycine, although it pays a high entropic cost to be restricted into this confirmation, it's like a yoga amino acid. It can adopt any kind of contorted position that is necessary. So cis prolings and glycines are favorable to make these radical turns in direction. So let's look at these proline things. So here you have trans proline. Look at the track of the polypeptide backbone. You're coming in here, you have a little jog, and then you're coming out in the same direction. Not helpful to turn us around. The cis polypeptide cis proline, you come in one way and you're coming out the other way. There's an enzyme, a protein, that causes the isomerization between cis and trans forms of proline. You can't just rotate this, right? You need an enzyme to help. Here's another view of that. Trans, you come in one way, you jog, and you come out the same way. You're not turning. Cis, you are making this turn. So this is a simple structure. The first two are more complicated. So let's see. You can also see what holds it together. Hydrogen bonds hold it together. In this case, just this one. Plus the particular amino acids selected. And now I'm going in the wrong direction. All right. So it's a beta turn. Okay. So if we look at all the beta turns, all the alpha helixes and beta sheets that exist in nature, we can look at what is the propensity for each amino acid to occur in these. This is nature speaking to us. But we make hypotheses about why nature is saying these things. Why are these trends? Do these trends exist? And so we've talked about this a little bit, like with alpha helix. Certain amino acids are less favorable. So pruning and glycine are not favored. And we can generate a hypothesis to say why nature would have not favored those. And you could do the same with the beta turn and the beta confirmation. Or the beta sheet confirmation. Questions? Yeah? Okay. One has a pruning, one has glycine. You can have, so now that's a nice segue to the next. So you can take these secondary structures and assemble them into a tertiary structure. Start connecting them together. So you guys are already starting to imagine, okay, this beta sheet thing. Like how am I gonna, you might say, well the anti-parallel beta sheet, beta turns awfully handy for that because you could just turn around. That's exactly what you need to do. You don't necessarily have to have a beta turn between each segment of sheet. You could go up, loop all around, and then come back. Whatever is necessary for the structure of the protein. So we're gonna begin to assemble the secondary structure into a tertiary structure. But before we do that, we're gonna cover Ramakandran plots. So we actually had a Ramakandran in class like five years ago. It was the most exciting thing I've ever had. It was wonderful. Because he had a plot. I don't know if there's any relation, but this is a very handy and extremely confusing plot. But once you get it, it'll help you to remember phi and psi angles. So you can think of this as a sphere. So Christopher Columbus said, and he said, well, people, you could just fall off the edge of the Earth, right? The world is a plane. You just fall off. But it turns out it was a sphere. Ramakandran plots are a sphere. Plus 180 is equal to minus 180. Do you see this little shading here? That goes with this. But what is the shading? The Ramakandran plot is used to plot the angles at each of the alpha carbons. So you have a particular protein. Say you have 100 amino acids. That means you have 99 phi and 99 psi angles. You don't have ones at the very end. And so each of those angles, each of those atoms, those alpha carbons, those angles can be plotted here. So you can make a dot plot. And you can assemble that into a contour plot. And as it turns out, when you do that for a lot of proteins, you begin to see some trends. There's not much happening over here. One of the reasons is because of the types of amino acids. So naturally occurring amino acids are in the L configuration. So if they are in the L configuration, that side chain is in a certain place and that's going to tend to affect the favorable phi and psi angles. Remember to go back to that movie. If you put that side chain in the opposite place, then different phi and psi angles would be favorable. But these sheets and these other hydrogen bond structures, the structures held together by hydrogen bonds, have very restricted phi and psi angles. So you'll see the alpha carbons within those structures pile up in certain regions in this plot. And so here we have the right-handed alpha helix. Those have these particular angles. There's a bit of flexibility in them. But in general, in reality, when you look at a real-world example, they tend to cluster very tightly. Then we have the anti-parallel beta sheet and the parallel beta sheet. So why does parallel beta sheet have smaller angles? Because they're trying to make those linear bonds. They're pushing the angles a little bit further so they can try to remember they don't get there. It's more pleated. So, unpleated would be like 180 degrees. Thing is fully extended. So they have smaller angles. We have the left-handed helix, very rare type of structure. But logically, it makes sense. If you're going to corkscrew in one way, if you want to go the other way, you have to go through the origin. You transpose those angles to the origin to get the opposite twist. So this is the Ramakandran plot. Remember our definitions. Zero, phi, and psi. And 180, phi, and psi. So nothing is happening right here. You will not see dots at zero, phi, and psi. You tend to see them at the larger angles. And because there's L amino acids, you tend to have this quadrant being very active. If you had D amino acid, this quadrant would be more active. So the majority of amino acids are in the L configuration, at least for us peoples. Any questions? Ramakandran plot is confusing. This is right next to this. It's a sphere. So take this thing and wrap it around. But then you get like, how do you get the corners together? I don't know. It sort of doesn't work. But it's connected. Maybe it's a cylinder. All right. You can dream about that. So here's a real world example of that very confusing plot that helps you to remember angles. So just visualize that on the exam. That's where each of those structures exist. So here's pyruvate kinase. Here's its structure. But if I just had this plot, and I didn't have this structure, you could guess the type of secondary structure that exists there. You've got this nice like blast of helix. So you have a bunch of alpha carbons that are in this helical set of angles. And so you could guess that this protein probably has a fair amount of helix. And then you have a little bit of alpha carbons with the angles that are more appropriate for extended sheet configuration. And so there is some sheet here, especially up top. So this is a real world example. Each dot is the fine-sci angle for a single alpha carbon. Okay. All right. So we're going to move on to tertiary structure. Is there any questions? Conference section. All right. So tertiary structure is where we orient segments of secondary structure in relation to each other for a single polypeptide. And here are some examples of the tertiary structure of some proteins. So here we have a contour. And inside of it is a skeleton of polypeptide backbone, these ribbons. And so you can see for different structures you have different composition of secondary structure. So myoglobin is predominantly alpha helical. Whereas lysozyme is a combination. And ribonuclease has maybe a little bit more beta sheet. So you can see, look at the orientation. They put little arrowheads here. So these are anti-parallel beta sheets. They're going in opposite direction. Remember, always the direction is from N to C terminus. So this is what tertiary structure is. And this is the structure necessary for whatever reason for these proteins to catalyze chemical reactions. So this myoglobin is just holding. Its reaction is to hold something. It's a hand. But other enzymes are actually catalyzing chemical reactions. And this combination of secondary structure and tertiary structure positions things in a way that you can aid in the catalysis. We'll see examples of later on in the class. So we have a little bit earlier clicker. Are you pulling, sir? Pull away. Now remember, if you clicker isn't working, you've got your clicker. Please try to bring it next time. But piece of paper, banner ID, and your vote. If you can't click. Yep, it's not line of sight. All right. So this question you want to pick the best to answer. You guys, everybody voted? How many votes do we get? 241, so 30 non-clicking people. So okay, you can stop the polling. We'll get to the advantage of the class. Okay, what is the correct response? This is where you say something. Yee. Yee. Yee. I don't know. Who has it? The Yees or the A's? Yee. All right. That is correct. That is indeed. Yee. Okay. All right, so now we had our break. We're all regenerated. Ready for more. You want more? Come on, bring it on. Okay. Now I have to capture your attention. I might start dancing. All right, so let's think about tertiary structure. We just began to get into it. I just couldn't help myself. I had to ask a quick question. What is a tertiary structure? It's actually a very limited number of tertiary structures or motifs that can exist. So a motif is just a certain structure that a polypeptide adopts often attached to other motifs by some kind of flexible linker. In some enzymes, different motifs catalyze different reactions. So in some enzymes, you have more than one thing going on right from one area of the protein to another. So there is, in general, some correlation between the motif, a particular polypeptide is adopted, and the type of chemistry that is catalyzed. So these are referred to as super-secondary structures. So the tertiary structure would be all the science triangles, right? The orientation of everything. But these motifs are aggregates of secondary structure, right? And so there's a certain combination of alpha helix and beta sheet and turns and so forth. And so it turns out, I don't know what's the current number, Alex, in terms of motifs. It's sort of how you define, this is different from something else, but what Leninger says is hundreds on the magnitude of hundreds. So perhaps that's a pretty... You would guess, why not? When you think about this, if you have 100 amino acids, there's tremendous amounts of possibilities of those angles. And to only have really 100 super-secondary structures, that's amazing. So nature is doing something here. Nature is finding out this structure is useful to do something. And it might be useful in a different... That kind of motif or fold might be useful in a different protein. We also can refer to these motifs as domains. So if you have one polypeptide with multiple motifs, we can call these domains of the protein. All right. So let's look at some oddballs. So this is the freak show of a protein structure. Here we have a coiled coil. And so this is about, I think, 25%. Oh, no, that's the next one, the triple coil. So this is at least a large percent of the protein in your body are in these sort of structured coiled coils. They have a lot of rigidity. So you have an alpha helix, and one alpha helix is orbiting the other. All right? So this is a coiled coil. So transcription factor, like a leucine zipper, has this coil coil, and at the bottom it opens up and inserts into DNA. And you might imagine, okay, it is an alpha helix, but it's orbiting another alpha helix. So obviously the phi and psi angles can't be exactly the same as you saw in a normal alpha helix, right? These things have slightly different phi and psi angles, and they have a different number of residues per turn. They have to be wound a little bit tighter because they're making this orbit around the other helix. So we have, in general, we have right-handed helix, and the coil coil is also two right-handed helixes. But these right-handed helixes are orbiting each other in a left-handed orientation. Okay? And you might say, why do they do that? Why does the Golden Gate Bridge not fall down? The same reason. If you have this cluster of helixes and then you cluster those helixes in the opposite orientation, that provides structural strength. If everything was right-handed, you would have less strength. So the purpose of these, in many cases, is to have strong structures. Okay? So we have two right-handed helixes orbiting around each other in a left-handed orientation. All right? So look at the way they twist around each other. It follows your left hand better. In between these helixes, there's not much room. Normally, in alpha helix, it's just floating around in space. And so the side chains are pointing tangentially to the axis of the helix. But here, you can see that the helix is the axis of the helix. But here, you're orbiting at the point where these things are touching, they're orbiting each other. There's not much room. So there's going to be some restrictions to the type of amino acids that occur at this interface between the helix. In general, it's a hydrophobic interface. This is called a leucine zipper. One type of these coiled coils is a leucine zipper because there's leucines that are interacting with each other, and there's a hydrophobic interaction at the interface between these. Okay? No questions? Yep. Does it retrieve? I don't take your microphone, so the button is on the bottom. Is it? It's a danger, danger. I don't... No. Well, let's see. Let's just click random things until something good happens. Yes, this would be a dead mic. All right. So I'll stand close to you. So, here I should add. Yep. Does a motif describe the adopted shape of one polypeptide or multiple polypeptides? Right. So a motif is sort of, in general, these motifs occur within one polypeptide, but you can have the non-covalent interaction between two different domains from two polypeptides, that's possible. Motifs can interact that aren't a part of a single polypeptide. And then the difference between tertiary structure and super-secondary structure. So tertiary structure is the fine-tie angle for everything. That's the whole enchilada. A super-secondary structure is just a motif, right? So it's a segment of structure, the arrangement of, for example, different helices, but those might be connected to other parts of the protein through a flexible linker. So sort of the definition of super-secondary structure is anything that's not a flexible linker. That's the association of polypeptide with itself and not a flexible linker. So it's a somewhat arbitrary definition, right? You say, well, that looks flexible. That's a different domain. Any other questions? Yes. Hi. Are the super-secondary structures typically function-defined? They're defined just purely based on this. No, they can also. You see, a super-secondary structure are domains. It's synonym. So domains generally are associated with a function. So if you have cartoon later in the class, we'll get into sort of the genes and code for these proteins. And you have certain domains. And each of those domains like bind something, or catalyze a particular reaction. You have like a tyrosine kinase domain. You have an SH2 domain. And that's something that binds to tyrosine phosphorylated residues, for example. So in general, there is a relationship between functions. That's why there's a limited number. Right? Because it's a toolbox. Nature has a toolbox of different motifs. And those are combined in interesting different ways to make different polypeptides. But we're combining some of the same functions. More than one protein might want to bind to the, for example, a phosphotyrosine. I have biased examples. Yeah. Because that's what I study. Yes. So secondary structure is flexible anchors or linkers? Our second... So secondary structure is just an alpha helix, beta pleated sheet, beta turned. That's it. Super secondary? Yeah. Super secondary can be anything. It's defined as some part, it's defined as a contiguous segment of structure that is not a flexible linker. So you have a blob, a line, and a blob. So the blob is a super secondary structure. Right? It's not all of the fine triangles, just the ones that form one part of the protein. Yes. Aha! Random coils. So this is a good thing. So remember in these cartoons, sometimes we have these nice sort of cartoony arrow things. And then we just sort of had things going all around. Random coil, you could also consider as a secondary structure. It helps to connect things together. But random coil is just a secondary structure, right? It's not a super secondary structure. It's a little confusing because it's absolutely arbitrary. It's just blob, line, blob. Yes. Yes. They're the same. They can be. Yeah, I think it's about the same. Yes? Yes. Yes, these are sub-segments of secondary structures that generally contribute a defined function. So this part of the sequence binds something. This part of the sequence catalyzes a certain type of chemistry. Those are different domains. All right, dead horse? All right, I think we're getting there. So we're talking about coiled coils at one point in today's lecture. Okay, so let's look at the most insane structure. Look at this thing. The triple helix. What are you saying? Let's get it rotating. Do you see it twisting? Or isn't this the coolest thing in the world? I don't know. I'm a geek, I admit. So we have in this structure three left-handed helices orbiting around their neighbors in a right-handed orientation. So 25% of your proteins in your body are this structure. So if you didn't have a triple helix, you would melt. You would just be a puddle on the ground. This provides the rigidity of your bones and the strength of your tendons and different connectors in your body. So this is collagen. Your skin wouldn't exist. You would just leak. So this is parallel left-handed helices that orbit around each other in a right-handed orientation. And this is insane. You think about it. They've got to wrap all one helix. They've got to wrap all the way around two other helices. So there's some serious yoga contortion going on here. And the interface between these, we don't have any just hydrophobic interactions. We've got glycine. We don't have room for anything, but a little tiny hydrogen hanging out here in this interface. And you say, well, wait a minute. Glycine is to favor any helix. Yes, unless the helix needs to use yoga, unless the helix needs to pack very tightly. So all these helices are very tight together. And at the interface between them are glycine amino acids. And there's 3.3 residues per term. Remember that alpha helix is 3.6. The coiled coil is 3.5. And this one is 3.3. So this is very tight helix. So this is an important protein structure over here. Triple helix. That's my favorite. And beyond having glycines doing yoga in the middle of the structure, you also have proliens in the alpha helix. You say, well, come on. You just said proliens are not in the alpha helix. Well, this is not an alpha helix. This is a triple helix. And in this particular angle, to get that 3.3 residues or amino acids per term, you've got to contort things. And as it turns out, proline does have the right angles once you need to contort into this triple helix. In addition, the hydrogen bonds that's holding this structure together are insufficient to keep these helices held together. You also need additional hydrogen bonding functionality within the amino acids. So for example, you have these hydroxyproline. So this is a post-translational modification. So the polypeptide is synthesized by ribosome, and afterwards we plop on using vitamin C and dioxygenase enzymes' hydroxyl functionalities. And these help to provide some of the additional hydrogen bonds holding this together. We also have hydroxylysine. So if you're a sailor and you don't have oranges, your proline and your lysine is not hydroxylated, and you begin to melt. And that's bad. Any questions? It's a little disturbing, isn't it? All right, so now we've pretty much covered all of the tertiary structure. We'll look at specific examples of the coordinate structure in our hemoglobin lecture, so we'll come back to that. But you might imagine, well, how do these structures come about? Where do they come from? Do they just exist? Is the fundamental question, is the primary sequence sufficient to define these structures? Is all the information contained in the primary sequence to make a tertiary structure? Or do we need accessory molecules? Do we need help in folding a protein? So ribosome synthesize polypeptides, but how does the structure come about? Where does that come from? So proteins, you'll learn, one of the key repeating memes in the class is that proteins are dynamic. You look at a picture and it's like, it's frozen in space, but really these things are breathing. We'll see hemoglobin literally throbs in and out. It's breathing. And that breathing is essential for the catalysis. If these proteins are not flexible, then they wouldn't be able to carry out the catalysis that they need to do. So the atoms are vibrating and oscillating. There's different types of motions. And you can have domains. And you can think about, well, how does it fold? Did the domains fold first and then collapse into the tertiary structure? Could be. Different ways to think about it. And so the forces causing proteins to fold, the single most important force is water. Water is exclusive. Water loves water. Water does not like to make interactions with proteins. I think I have a very handy... So water pushes a protein down. Proteins can be described as globular. So when they're initially made, they're flexing all around, they take up a lot of space. And then the water molecules form a cage around the proteins because they're trying to make interactions with the other water molecules. And this is bringing down the structure into small and smaller globules. So that's a very important force that drives this. But as you form structure, the forces holding it together are all of the different non-covalent and covalent bonds between different parts of the protein. So you might have hydrogen bonds, electrostatic interactions. You might have covalent bonds in the form of disulfide linkages between cysteines. All of these things come together to help to stabilize the folded state. And so we have folding G. So we can think about this thermodynamically, but the most important thing is the force of this bucky ball of water squeezing down on the protein, making generally proteins are rounded, meteorite looking things. So the question is, is the native conformation the most stable conformation thermodynamically? The purpose of the folding of proteins is to make a functional protein, to be able to catalyze the reaction. So it's not necessarily the case that the competent structure of a protein that's able to catalyze the reaction necessarily has to be the most thermodynamically stable. In fact, you might guess that that generally would not be the optimal situation because if your chemical reaction requires some flexibility of the catalyst, if it's the most thermodynamically stable, it's not going to be, it's going to be locked in place. The other question might ask, one already asked, is the primary amino acid sequence sufficient to in and of itself determine the tertiary structure? Okay, and is the change in free energy as you fold sufficient to drive this process, all those new bonds that you're forming? Okay, so this is Christian Anson-Fanny. This makes me happy. I think he got a Nobel Prize, so he stoked. So he did a very simple experiment. He took a protein, ribonucleasease, an enzyme that cuts up RNA molecules into small pieces. This particular protein is just a single polypeptide. These yellow bars are disulfide linkages. So he broke these disulfide linkages and then he denatured the protein. He added substances in the protein that caused the protein to become unfolded. So in this case, he added a molecule called urea. And so this, after this, he had a completely denatured protein that didn't have any disulfide bonds. And then he just had that one denatured protein in a test tube and he just put it on the bench and said, go. And he wanted to ask the question, could it refold? And you might say, well, he probably had to have a really fancy NMR x-ray to figure it out. No, he knew the structure was right when the enzyme was functional. And active enzyme, if it's unfolded, it's not going to catalyze the reaction that you want. And so he looked for RNA activity and before he denatured it, there was that activity. He denatured the protein. All the activity was removed. He then allowed the structure to reform by first removing the urea and then adding or removing the mercaptoethanol. The mercaptoethanol was used to reduce the disulfide bonds. So when you remove that, you get reformation of the disulfide bonds. And lo and behold, it worked for this very small protein. And so I don't know how many you tried, but for this one it was able to obtain its catalytic activity. So in this particular case, absolutely, the primary sequence was sufficient to confer the structure of the protein, at least the structure necessary for the catalysis to occur. All right, yeah, send that, send that. But then when you think about it, let's think about a protein with 100 amino acids. And let's say each of those amino acids can have maybe at most 10 fine-sci angles. Now, in reality, it's just free rotation. You could have millions of fine-sci angles between two alpha carbons. So let's just say 100 amino acids in 10. And let's say, well, what's the fastest that we could have a little bit of movement here? Well, a vibration of a bond in a molecule. So it's about 10 to the 13 seconds, right? And so let's add that up. So how long would it take from just the primary sequence alone for this protein to adopt a particular one-defined structure? Well, it would only take three times 10 to the 79 years. Well, unfortunately, the age of the universe is 1.5 times 10 to the 10 years. So it would be orders of magnitude, many orders of magnitude longer than the age of the universe for something to fold. So it's just not enough time for this to happen. This is the coolest. He's got his shades. He's got the Vax or the teletype going on. But all he did to come up with this is just think about it. It's impossible that for all proteins, it's just the protein sequence alone would be sufficient. So there must be some sort of order to this process. There must be local thermodynamic minima, for example, the formation of a domain, a super secondary structure. So here we have an unfolded protein. And first you make this segment of structure in this. And then those two come together and begin to collapse into a more and more restricted state. So you're not sampling all of the possible positions of the alpha carbons. You are reducing the number of possibilities by forming segments of structure, even just forming a helix. That restricts the structure. And maybe once a helix is formed, it's less likely to unravel. So that's stabilized. That's a local thermodynamic minima. And so you can think about this thing in terms of a protein funnel. On this axis we have 10 to the 79 possible confirmations. And on this axis we have the energy. So if this were a progressive process where you first form a helix, and then a motif or a domain, a super secondary structure, and then you collapse that. Each of those would be a little minima. And you don't necessarily have to get all the way to the bottom. That's the absolute most restricted and lowest energy state for the particular protein to adopt a confirmation that it's allowed to catalyze reaction. So this is how we're thinking about this. This is probably how this occurs. But does it just occur spontaneously, or does it get some help? And so we can think about the thermodynamics. Is it a local minima? Is it a global minima? And we can also think about the kinetics. We might not have enough time to get all the way to the maximal or the minimal energy. So there's two forces here. And so protein folding, think about the context. In your cells there's extraordinarily high concentration of protein. So if a protein is just doing its thing sort of folding a little bit here, a little bit here, as it's folding it's revealing parts of its structure that aren't normally revealed in its native shape. So there could be sticky patches. The molten glottin general proteins have polar or charged residues on their surface, and the insides tend to be hydrophobic residues. This molten globule. And so if you were just folding an extraordinarily high concentration of other proteins, you're going to begin to make unproductive bonds to those other proteins. So we need a nice, happy, safe place to fold. Is there a question? Yeah, hi. Okay. It might not be optimal. Yeah, it might not be optimal. And there's the context of the cell to consider. And so we need some catalysis here. We can't just fold in solution in all cases. We need enzymes. So we need to sample all possible disulfide bonds. So we have this protein disulfide isomerase enzyme that helps to switch the bonds which systems are being covenantly bonded to each other. We have an enzyme that can switch a proline from a cyst to a trans-configuration. So maybe a particular orientation of a proline is necessary for a certain structure. So this structure, actually the first valedictorian of the Brown-Fleamy class is Art Horwich. And he, this is a big famous researcher at Yale that studies chaperonin. So this is a nice, happy place for folding to occur. This is the structure of a chaperonin. It's sort of a barrel. And inside of this barrel you deposit a protein that's folding and then it provides an environment isolated from all the other proteins in the cell. So here we have the protein. It's a barrel. That's where the protein gets put inside of the barrel. Here comes a folding protein. ATP has hydrolyzed the whole thing. Shift. And then he put a happy hat. And shifts back. And then it flashes. Magic things are happening in the barrel. And it's a double-ended barrel, right? So you have on the other side another protein comes in. ATP has hydrolyzed, change in conformation. When these things are twisted, Art actually gave a lecture at Brown. When these things, the ATP has hydrolyzed. You have this twist. Do you remember you saw that? You're actually exposing hydrophobic surfaces. And so that's allowing, it's providing a safe place to expose hydrophobic patches. So as you're folding, you might not have this globule state. You might have these unfavorable hydrophobic patches. So it's a way for these proteins to be isolated from their environment and also be free to expose hydrophobic patches. Okay, so that's a chaperoneum. So many Brown graduates end up in amazing careers in science. So many of you will become doctors. And so there are diseases caused by aberrations and protein folding. So a variety of proteins, when misfolded, confer a phenotype of disease. And so, for example, prions, a particular protein that has a normal configuration that's helical, goes through a transformation to a sheet conformation. And this transformation is unproductive from the perspective of the chemistry that that protein is supposed to be conducting, but it causes the sheet to aggregate. And these aggregates deposit on cells and cause the cells to die. And so changes in conformation that perhaps are unproductive can also cause diseases. And so if you're going to be a doctor, you need to think about protein structure and how proteins fold. So I talked about denaturants, many ways to denature a protein. You can change the pH. So that would change the ionization state of certain amino acids. You can provide detergents that associate with nonpolar residues. They disrupt hydrophobic interactions. You can warm things up. So if you have a folded protein, you warm it up at a certain point. It becomes unfolded. It begins to become denatured. You have chaos-causing agents or kaotropic agents. And these agents tend to make replace hydrogen bonds with these small molecules with productive hydrogen bonds that should be occurring in the folded structure of the protein. So for example, urea and guanidinium chloride are examples of this kaotropic agents. So we're lowering all these manipulations of proteins, lower the free energy, have a lower free energy that the unfolded state has a lower free energy than the folded state. Lots of examples. So here is a melting curve of a protein. So this could be determined by enzymatic activity. So as you warm a protein up, it's able to catalyze less and less reaction because it becomes unfolded. And they can define a half, a point at which half the protein molecules are unfolded. You can define that as a temperature or as a concentration of a kaotropic agent. This is a sigmoidal curve. Sigmoidal curves are indicative of cooperative processes. So as a protein begins to unfold, it collapses. Things run out of control. It's sort of like a chain reaction of unfolding. And we'll see lots of examples of sigmoidal curves throughout the class. And any time you see a sigmoidal curve, that's indicative of there's some kind of cooperativity here. So in the last part of today's lecture, we're going to think about different ways to work with proteins. Many of you have done biomedical research already, but some have not. And so there's a technique called chromatography where you can separate proteins. So here you have happy different colored proteins. And you're separating these proteins in this tube that has some sort of matrix or a material that separates proteins based on their size. So say you crack a cell open. Lots of different proteins. Some of them have happy colors. And you can pass them through this matrix and you can separate them out, those proteins out based on their net charge, their size, or other physical characteristics. So one example is gel filtration chromatography. And so also referred to as size exclusion chromatography. So in this process you're separating proteins based on their size. And it's the opposite of what you might get. So the smaller proteins take longer to pass through this material than the larger proteins. The reason being is that there's these small channels. And if a protein is too big, it doesn't enter into the beads. It just goes around and quickly passes out of the column. But if the proteins are smaller, they have a slower route. They wiggle their way through each of these beads. So you separate proteins based on their molecular weight with the large proteins coming out first from this column. You can also separate based on net charge of the protein. So we have what's called as cation exchange chromatography. So in cation exchange chromatography, you have negatively charged beads. And those negatively charged beads tend to bind proteins with a net positive charge and repel proteins with a net negative charge. So it's cation exchange chromatography. You separate proteins based on net charge. You can also have anion exchange chromatography where you would have positively charged beads. And those tend to slow down negatively charged proteins, proteins with a net negative charge. So you can separate based on charge. You can separate based on molecular weight using a SDS gel electrophoresis. So in this process, many of you have done this. You coat the protein molecules with a detergent SDS. This confers a negative charge around your protein. SDS also has the beneficial property of denaturing the protein, causing it to become unfolded. And so you have denatured proteins covered with negative charges. So because they're charged, you can simply apply a voltage, a negative potential and a positive potential. And you can draw the negatively charged protein molecules that are coated with SDS towards the positively charged electrode. And this material that the proteins must pass through is very viscous and thick. And so it tends to retard things. The larger a protein is, the slower it passes through this column. So you have the molecular weight going from small things that travel quickly through the gel up to large molecular weights. So you have one last movie that's showing this process. So you have your first denatured protein using SDS. And I like it because it gets real hot. Ouch! It's red. So you have a warmed up denatured protein. It extends out, gets coated with negative charges. And then you add it to the gel. And the gel is viscous and thick. And you pull it through with voltage, but the larger protein molecules go much slower through this. All right, so that's the lecture for today. Whee!