Good morning Slightly less early morning today. That's good. I Apparently did it meant to scare all of you away with statistical mechanics on Wednesday Today, we're gonna be much more biological. We're gonna go back and look at protein structure. Remember the this whole approach I mentioned that we started out with biology and then we did a little bit of hand-waving physics and Then we went more in slightly more into the hydrophobic effect at least and then we did the hard-core statinic And today we're increasingly gonna go back to protein structure But now we will interpret this protein structure and the features we see in terms of proper free energies entropy and enthalpy Before we jump into that, let's follow up on some of these study questions I'm not gonna ask you to explain EHSFG, but if you do not know what those are This is the time to start reviewing it Because although today some of the things I say today you will need it for but increasingly throughout the course both Lucy and Burke will assume that you know what we're talking about when we talk about enthalpy for instance So as a small check up here, what is the difference between E and H energy and enthalpy? Yes Yes, exactly.  We're doing. Yes. Yes. Sure. Sorry potential and kinetic energy Normally, we don't care so much about kinetic energy and the reason for that is that the kinetic energy Room temperature is roughly fixed in biology, right? This is very rare that you will have parts of the system suddenly being at 100 Kelvin So the kinetic energy is super important But it kind of ends up being an orthogonal degree of freedom We have the kinetic energy there the kinetic energy is what makes it possible for us to occasionally go over these barriers But it's very rare that we're going to consider the difference of this particular protein whether it's a 100 Kelvin or 300 Kelvin There are actually rescues where we might have to do that for instance if you're freezing it in crye or so But in your body the kinetic energy kind of it's fixed. It's a distribution around 300 Kelvin according to Maxwell Boltzmann But it's a you're right. The kinetic energy is included me too and then here, too I can just apologize for a few generations here because We know that it's everything except the work But we don't really care the works and then we just call it energy and then we happily intermix e with h and everything Sorry about that. I wish the world wasn't that way, but I would be lying to you if I presented that it wasn't And then similarly F and G that too because we don't really care about PV right the PV the work term That's really the only difference between F and G and for that reason, too You will see us happily using interchangeably if you're a chemist you will say just will just say f and obviously it includes PV But thank God we work with proteins, which means that we don't really have to care about it anyway What is the partition function? Yes, so yeah, sorry go ahead Exactly so that The Boltzmann distribution only gives weights right and but to turn weights into probability you have to sum that you have to Normalize that over all weights So why in earth do we call that function? Well, it turns out that as you saw some of the slides for at least hand-waved a bit This sum ends up being a way that you can also calculate many things such as free energies Central peace and everything properties of the system So the partition function is not merely a normalization this way It turns out to be if you know the partition function, you know everything of your system literally you can calculate absolutely any physical chemical property from the system if you know the partition function and That's awesome. If your partition function has three degrees of freedom or if you've started doing the first hand-in task The reason why we have this super simple hand-in task We create a partition function that only has a handful of states and if you only need those handful of states You understand everything and then the curse and beauty in biology is that we have a partition functions with maybe six million states Well six million degrees of freedom hundreds or trillions of states. I Might not have covered question three explicitly, but that's an interesting one. What is stability versus instability? there is a simple and complex answer to this Mm-hmm. Yeah, that's a long description. I expect you to know that by heart not If we start to look If you don't know anything Think of your free energies and your energy landscapes. We can reason about most of those things So let's draw a free energy landscape here. So which state is Stable here or let's start which state is the most stable the lowest, right? So that makes sense because if you are here Sure, we might have some thermal energy so you might vibrate here a bit But it's stable in the sense that you're not going to go away from it So in some sense in the physical sense you can talk about stability being the global Minimum of the free energy and that's what we're going to come back to a stable states of proteins the native states Yes, we're getting to that now you could talk about these states are kind of metastable, right?  So I cheated here. This is some arbitrary degree of freedom, right? But what is the Y scale? No No Free energy, right? So here's the dirt and I know that sorry about that that we so this is a Delta G But there are no units here So I will completely arbitral now say that the height of this particular barrier if that is 0.5 K cal for more and we are at room temperature. How stable is that state going to be? Why? 0.6 kilo cal 2.5 kilo Jills, I know that sorry about that that is great with standards, right?  So it's a 0.6 K cal So you're gonna fly this is just gonna swoosh by so with hope so it's like it's might be metastable for a millisecond and That's the type of states that in chemistry. We happily just ignore them. So it's not stable at all But assume that instead that it's five Mega calories promote. It's a brick wall, right? Or is it your physicists some of you in principle, there is no difference here, right? It's just a matter of time. So here's the complicated thing with biology that In physics is just a matter of scales the same rules apply ever but in Biola we do we have these absolute times that are related to Processes that are realistic or not your life is a hundred years.  It's the same thing as stable So we occasionally talk about these things as kinetic stability. So the real thermodynamic stability That is true. That has to do with the free energy minimum But remember that we spoke about kinetics and barriers, right? You can be stable in a kinetic sense that means sure Strictly formally, you're not stable But in practice the energy barrier is so high that for the timescales. We're looking at you are effectively stable And that's the case for most of the things that happening in your bodies Are well at equilibrium. We're all dead. You might have heard that And that is the challenge is the stability versus instability is not necessarily that simple Instability in a way. It's easier that on the timescales where here. You're definitely unstable. You will move down there Here you might be metastable But how stable are similar will depend on the energy barriers and the timescales you're looking at So that's why I said what biology is actually more complicated than physics We spoke a little bit about phase transitions. What characterizes a phase transition You can say this there are strict physical definitions, but I don't care so much about the physics here Yes, and it should be something that's abrupt, right? So there should be some sort of all or none transition It's not a continuous transition and then there are is first and second order. This gets really complicated in physics But abrupt change. I think it's a biological reason. We would accept that And I already I think we already covered a little bit about how free energy barriers are Related reaction rates so that the likelihood that you will go over the likelihood that this reaction will happen depends on that energy barrier, right? So that will of course depend on how likely is it that I go from this method stable state up to the energy here And in principle the Boltzmann distribution will tell us that They were on the other end the Boltzmann distribution will not tell us Exactly how fast the process happens But if I have two energy barriers that I'm comparing if this energy barrier was one or 2k cal and the process would be roughly the same I can use the Boltzmann distribution to say about relatively how fast would they go and the difference then normally you're Normally you end up with the Boltzmann distribution Well, the Boltzmann distribution is normally e to the minus delta g divided by kt, right? But that's the likelihood of being in a state if If we're instead Sorry, and if the time it will take to go up here that will of course be related to the likelihood So that would be a minus sign in chemistry where you usually talk about how fast something happens And that's a rate the rate corresponds to one over the reaction time So how many things happen per unit of time? So when you talk about rates then we divide this do it one over this quantity And that's where you usually end up with a plus when we talk about rate instead, but that's just because you've divided Then I spent Quite some time although I hand-weighted a little bit that we had to understand what is the most Unfavorable way during formation of helices or sheets So why on earth did I end up spending all that time trying to quantify that peak state? How much time will I spend there? Am I stable here? well It has to do with that I What is the derivative? Yes, so in principle, I'm stable right, but it's like balancing on a knife's edge So I'm dynamically unstable if you have epsilon motion, I'm gonna pull down So I'm static stability by dynamic instability So the fraction of time I'm gonna spend here Is zero because there will always be some noise that pushes me while you're at it So why did that? Why should we bother about that state? Exactly right to go from that state to that state. I have to cross that barrier But who cares if I'm not gonna spend any time at the barrier, why should I bother about it? So it will determine the reaction rate that I just told you right that is the delta g value I know if I know how high that is I can estimate the barrier and then I can estimate how fast things will happen So it's not that I'm interested in the state, but I'm interested in how long it will take to go through it And in principle, it's not enough if I find one state here that could always be a lower state So if there wasn't even assuming that there was another path between these two It would could always be even faster But if I find find one way for this to happen, that's a possible way Then there might always be something even better And that's why we're case at least I had to hand wave a bit that this was also likely the best possible way the lowest But I didn't prove that So related to that, what was then the hydrogen bond pattern in the most common helix form? This is repetition, but it's important to know Mm-hmm, but what is the hydrogen bond pattern there? I to I plus 4 right? So the reason why I brought that up the other day because that is what gave is that you have four residues that are stabilized by two hydrogen bonds And that's what causes this extra initiation term that is effectively the barrier we have to go over And then we at least hand-waded it if you think about in terms of free energy what components favor versus this favor alpha helix folding So what that is that favorable or unfavorable? favorable So what is this favorable? Entropy so that putting the helix in this particular confirmation Sorry the I said I miss that Favoring the favoring was the hydrogen bond they particularly the enthalpy of the hydrogen bond right actually the hydrogen bond involves entropy too But the fact that you can form these hydrogen bonds between adjacent residue is very favorable for the alpha helix So that as we're forming more and more hydrogen bonds you're gaining enthalpy No, but they're they're slightly stronger. They're more polar and everything And in this particular case the water because the alpha helix is are typically not hydrophilic Sorry, not hydrophobic that the waters are quite happy around them too So the water is not going to lose that my entropy But it's a really good question because that the hydrogen bond per se is not a purely Entopic effect right as we told the other day There's both enthalpy and entropy involved in the hydrogen bonding too So I may be the best way of saying this really the free energy component here Primarily has to do with the hydrogen bond as a whole But overall the commonality between 8 and 9 I want to get here in general for all these things when you're forming something To us it looks nice to have something ordered and regular nature reports that But the beautiful regular shape of an alpha helix is horrible because that you're taking something that was very free and Forcing it to be restricted and that costs entropy. It's really bad On the other hand then we must have something else that compensates for that, right? And that's usually then enthalpy. Well, it has to be enthalpy or some sort of other entropy that gets even better There was a great paper in biophysical journal this week actually about this Arguing that you're gonna see later on in the course when we I already showed you some example there You see almost like a funnel going down that the protein finds the minimum and it looks nice It's narrowing but that's all good at all the narrowing per se is actually bad because you're losing entropy as you're going down And that has to be compensated for But the reason for having both of these what's the difference between helix and sheet No, I think both both of them benefit from hydrogen bonds, right? And both of them lose the entropy But if you think about the alpha helix the alpha helix is a very local structure So it's a matter of turning you orient to local amino acids close to each other And you also form the hydrogen bonds local to each other when it comes to beta sheets the hydrogen bonds are global You're gonna get the hydrogen bonds to some other strand that could be quite far away in the sequence space And it's also the orientation here You're gonna take two very different parts of the chain and put them together to form the beta sheets So both the entropy and enthalpy terms of the sheets are more global while for alpha helix is there a local And that's also this global nature of it or that the beta sheet is effectively two dimensional That's actually why I didn't have time to tell you that once actually you can show that the beta sheet is actually a proper phase Transition while alpha helix in the alpha helix is the coil and the street can coexist, but then we're getting into deep parts of physics Yes no, but No, it's a no that has something to do with the favor of it But with the sheet with the alpha helix you will gradually as soon as you get over that initial barrier, right? The helix will gradually grow There's a beautiful example that they'll come back to later today When it comes to the sheet, it's very much all or nothing So that the sheets can take a very long time to form when they actually form they will grow rapidly and suddenly boom They're stable. So they have this abrupt nature that we talked about phase transitions. I Spoke a little bit about helix dipoles, but I covered that already earlier this week. I won't repeat that here Oh, sorry in that and this was also the the sheet phase transition this had to do with that the helix is one-dimensional Why is the range of formation rates for beta sheets much larger while that's not really the case for alpha helices? so that had to do with these kinetics and the Energy barriers right and I show that the speed is going to have to be related to this peak and For alpha helices the height of that barrier was really only due to the hydrogen bonds And that's not really that different between two different residues. Some hair residues will be slightly happier But that's mostly going to influence the stability not the rate of the formation But beta sheet since you actually need to form this entire hairpin the two strands for the beta sheet to even start forming That means that the beta sheet formation barrier depends a lot on how stable a number of residues are in beta sheets So if you have residues that are very unstable in beta sheet, you can have a gigantic barrier But eventually they the entire sheet can still be stable. So it ends up depending a lot on the stability of the residues I think we already covered microstates and macrostates. I realize that's a lot of work. Well, actually I'll repeat that So what is the difference between microstates and macrostates? I think that's a beautiful beautiful explanation Multiplicity is a great keyword because as always everything depends on definitions, right? If you're a quantum physicist, you might say we need to do this on the quantum mechanical level including electrons and everything We ignore that we look at the proteins But at some other microstate has to be unique So whatever whatever way we use to define how things are different. There is always the microstates are unique There is no never any multiplicity inside a microstate While the macro states that's some sort of way of lumping things together And you could argue the way I treat this of course in my protein state I have lumped together a lot of electron states, but I don't care about the electrons I still just consider that one state on the other hand in so in chemistry I'm I think the stuff I observe in the lab is one macro state But this is in principle entirely up to you to define and and again That's as always the curse of statistical physics You have all the freedom in the world But when you have all the freedom in the world to define things yourself, they become fairly abstract and fuzzy But multiplicity is a great way of thinking about it. So what is the entropy then? What states? The logarithm of the number of Microstates within each macro states, right? And then we have the Boltzmann constant too So it's literally just a correction factor so that to be able to use all our equations But focused on macro states then we need to count the number of micro states in it There's purely a mathematical course right to help with this Yes Hmm, I'll use the helix Remember that I said that we had all these dipoles right the in the peptide bonds So that when you take lots of small dipoles and line them up That's going to correspond to a gigantic dipole going from minus to plus The start this is the start of the helix and terminus. We always throw them blue and The that's all but the dipole will go in that direction But if you have a gigantic dipole that corresponds to like having a small minus charge here and a plus charge there And the capping that means if you have a plus charge here What type of amino acid would you like to put here to stabilize that and negatively charged amino acid would be really happy there Right because it's going to be like the negative charge finds the positive one and similarly a positively charged amino acid is going to be really happy here So the reason why those things are used you actually see these patterns in biology and you can use that Today we wouldn't use that but you could use that to predict where our helices So if you see these patterns just in the sequence that are super cheap and easy to get from genomics We can actually start to use them to define structures Lucy will come back to that a little bit of bio bioinformatics a long time ago. We did this today We have so much data that we we use machine learning and computers is that we don't try to apply physics to find sequences And as I also mentioned a little bit the reason for being up this particular feature of the beta sheet range of Informations is that that's what leads a bunch of these aggregation diseases that people even think that's Alzheimer's and the whole of diseases Has to do with this bit gradual building up of plaque.  Why wouldn't evolution take care of this? Make sure that why should we have these proteins? What on earth can be good with falling ill? So we don't know and little bit of what can speculate So how does natural selection work? But when do mutations happen and when does the selection happen right in our offspring? And when do we typically get offspring? In particularly historically you would be 20 when you had your offspring today. It might be 30 35 So diseases that you get when you're 60 70 has his story So first mankind historically man didn't grow to be 60 or to 70 to 80 years old So historically there has been virtually no evolutionary pressure for anything that happened at that age And even if it was you would have had your offspring long before this type of diseases happen So that is very bad for the individual But for the reproduction of the ray for the reproduction of the DNA the cell and everything it doesn't really matter It does matter to the individual but And that's the strange thing that then happened with mad cow disease and everything if you then start to eat brain Whether it's in hamburgers or in cow food Then we might reintroduce some of these things so that rather than getting the plaque to read the critical level when you're 80 You might suddenly have started having a critical level when you're 20 and that is bad Today I'm gonna jump back and look at real proteins There are some classification but this is gonna be much easier. We start looking at proteins There are three gigantic classes of proteins and I'm a bit sad because I would have left the most fun to lose sick But that's that's all right There is a fairly boring class. That's by far the most common in your body It's these large building material structure keratin fibrous proteins Skin nail etc And then there are all the water soluble proteins ever called globular which roughly has to do with the shape Most of them are circular and that partly has to do with these droplets of oil and the hydrophobic effect Then there's this really cool classes of proteins that exist inside the cell membranes The membrane itself is hydrophobic and the reason why we love them virtually all our research It's highlight lab in my team is on these proteins and that they're effectively the windows and doors into the cells Any 70% or so of the revenue of modern drugs are targeting membrane proteins It's a super important area and one where Sweden in particular and Stockholm is world-leading. There's all my colleagues at Stockholm University There are a ton of different tools to study proteins You will see some of them in the class later on if you want to download one There is a program called Vmd that is developed by colleagues at Urbana champagne in the US But the point here is not so much. I think you will be looking at this viewer But my point here is to stress that there are different ways of representing a protein to you can choose to look at Every single atom, but if you were to look at every single atom things rapidly become very complicated, right? So here too depending on what you're doing We can start to look at just we can't plot just to see alpha so you get some sort of backbone trace Or we can draw the secondary structure or we can draw the secondary structure But keep all side chains and in this particular case we use a different color for a group that's bound This is the so-called heme group that's binding the oxygen and there's none of these representations more or less correct But the reason we use them is kind of to stress the regularity of the patterns If you look at one of those two top examples, you don't really see the helices, right? And in a way the helices aren't there the helices is just a sort of regularity in the pattern But for me, I much prefer I tend to use representations like that because I don't want to be I don't want my focus to be Disturbed by seeing all the side chains and everything. I want to see the big The grand scheme of the protein The reason why things fold into this patterns is entirely due to the amelior acids as already mentioned with Christian Amphinsen and Cyrus Leventhal These structures are unique if you put aside if you put a chain in water It's always gonna fold into the same thing with very few exceptions And then I already hinted to you that there are some examples here probably and it's exceptionally rare in alpha helices Because it can't form the hydrogen bonds. It will break the alpha helices Glycine doesn't have any side chain at all So it's just very free in this Rammachandran diagram and that means that it's beautiful to have if you need to make a very quick turn and go back The helix capping we just covered means that some residues are more common that helices ends and Then there might be differences inside versus the surface of the proteins. What is the difference? We expect to see there. We covered that the other day hydrophobic inside yes And that has to do with these classifications of proteins If we draw up all of them there this abundance it came from the genetic code remember that there is no fancy biology here The percentage of each amino acid here is entirely due to the genetic code So this is the fraction of the various building blocks we're gonna have when we create proteins and The last column here is this Delta G of salvation that literally describes how easy this is to solving water is minus 10 k cal per mole a lot Minus 60 if it starts or minus 80 So first what does the minus sign mean? It's a favorite process and the process of salvation is when we take it from gas or some sort of interface to water So minus remember I said what was KT? 0.6 we talk about minus 80 compared to 0.6 This is gonna be so downhill that there it's actually difficult to measure it because there is so little that will not be in the water So if you have a protein if I give you a protein model and say that look I predicted the structure and I think that there's gonna be a lysine right in the center of the protein You're gonna say that you're wrong That it won't happen those things those things are impossible in practice in chemistry While of course minus point 76 that could go out of the way we can Do all these amino acids any possible way I already mentioned that glycine is super flexible alanine is also fairly flexible It just as a CH3 group at the end and alanine can go either way It's okay to be in helices, but sheets are also okay Glycine on the other hand hates to be in helices and sheets. Why? It's small. It doesn't have any side chain. Why should glycine hate being in secondary structures? So what was favorable versus unfavorable when you put things in secondary structure? sorry Well, but glycine can form hydrogen bonds. That's no problem loss of entropy right So any of these processes remember that you can't just look at one state You have to look at the before and an after and what is the difference? So you now have a rescue that's exceptionally flexible This is gonna be super happy out in water or so and if you take this rescue This is gonna be happier in water than the other residues because it's so small and flexible And if you take this rescue and move it into a helix Relatively speaking glycine will lose more freedom by being forced to be in a structure than some of the others So for glycine, it's actually the entropy that costs us There are a bunch of longer hydrophobic residues Just to get to know that C alpha is the first atom and then we typically then we know I don't have more imagination that we continue along the Greek alphabet So the beta carbon the gamma carbon that's if you continue out the side chains Cysteine is another small cool residue. We have a sulfur atom here Cysteines are special because these hydrogen under some conditions You can actually do these and form if you have two cysteines that are close to each other They can form a bond a cross link. I'll show you how that works So if you have one chain here and another chain here those two cysteines Can form a bond between the cysteines and then there's literally a covalent bond between the two chains And at first sight that might seem like a small quirk or so But that is generations of biochemists are using that if you want to find out if two parts of a protein just a sequence You don't have the structure and if we want to find out what these things are Let's try to insert two cysteine residues That's very easy to do in a sequence and then you try to create this type of bond and if a bond then forms You knew that they had to be within roughly one nanometer or so in space. Otherwise that bond couldn't have formed Covalent bonds, do you know anything about the strength of those? They're very strong. So I'm going to show you another small protein This does not look like a protein, right? This is just a random piece of coil thrown together. I Can show you all the side chains too It's still not particularly interesting and Then I'm going to show you two Cysteines here No, sorry, six systems. So there are three pairs of systems.  So that was discovered by Kenton Schwartz And he actually named this after his daughter Hanna, so it's called Hanna Toxin Which apparently made his wife curious But as Apparently or so when you're 16 year old it's pretty fun to have a toxin named after you We already spoke about proline and the key thing with proline has to do that the Since you've looped back here We have lost you see that the carbons are binding the nitrogen there, right? So the nitrogen will not be able to participate in a hydrogen bond once it's part of the helix There is a reason why I'm going through all of these for you So if you give me a little bit of patience Tryptophan is a big and bulky amino acid. It has two rings One of these rings is quite hydrophobic and the other one has an ability to form a hydrogen bond there There are all sorts of if you're an organic chemist There are all sorts of funny way of classifying these prolinus strictly an amino acid The tryptophan ring is an indole group, etc. Forget about it that We care about the physical properties here. It's not an organic chemistry class But here you can probably all see it the high the hydrogen bond forming part There is going to be quite happy to interact with solvent, but the big aromatic ring there. That's hydrophobic And that's not going to like to interact with water. So that would like to be turned into the inside and Some 15 well slightly more almost 20 years ago now. This was used to create one of the world's smallest artificial proteins So at some point there's a definition. What is a protein? Well Anything that's a collection of amino acids. We can call a polypeptide chain to be able to say that it's a protein we wanted to adopt some sort of stable and unique structure and That's not really enough because you could argue if it's just a single amino acid that has a structure So typically we say that there should be some sort of inside There has to be some rest use that are not exposed to water so that there is an surface outside And there's an interior your inside And this TRP cage is literally a cage. We put a trip to fan on the inside And I'm going to show you a small movie of that. So when even 20 years ago We used a hand way about these things this particular structure was I think determined both by NMR and it might have been x-ray since too But these things are small enough that when I was a Student your age we dreamed of being able to use physical rules to predict proteins And the cool thing is that we can do that today. So this is a protein if you throw it in a computer We can simulate how it folds So what happens? Let's see here if you turn this on these are colleagues of mine a bit of pandas team I did that do you see here this going super fast that it's exploring all typical Possible confirmations in space. It's moving across these and energy barriers Every time you see it be not moving It's kind of metastable right and then it crosses another energy barrier a few millisecond Well, if you know a microseconds later and then eventually you end up in this state Which is actually the lowest free energy state and that corresponds to the experimental x-ray structure So these are horribly simple Representations is using the type of force field we showed you there is water all around this But based simply on physics these things hold Which is quite cool because it's really a reconciling things with Anthony's and and everything and it's also showing that Despite Leventhal's reservations One way or another these molecules are able to find each other in the range of milliseconds. There's no biology here We don't have any ribosome. That's helping us to find the structure. We're not guiding it We're literally just throwing a small chain in a virtual Water box and then we're using the loss of physics and the loss of physics will guide it to us And that's also the reason why I go where I went through all these slide with different residues, right? The what gives us this uniqueness is that all these residues have slightly different properties at first sight You might think that some residues are better than others and in in some ways They are it would be difficult to form a protein that has 20 percent proline residues in it because it would be They're too big and too bulky But in this particular case this ball big bulky difficult rescue was really would stabilize the protein and In other cases you might need a hydrophilic residue or a hydrophobic residue So the difference between say super simple plastic bags that are polymers and proteins is that this heteromeric nature of proteins That's what gives us the uniqueness Why specify this particular fold is stable for the protein? But if I change that trip to fan to another rescue the protein you just so would of fold So there are the other pattern if they already mentioned these well you mentioned these patterns that polar residues They are frequently present in rare regions close to the surface. They love to have form hydrogen bonds with water Charged residues, they virtually only occur in surfaces or if there is some sort of internal active site Some of these iron channels I showed you there the charge residues can almost appear in the membrane But they're not really in the membrane. So you effectively create those more Water cavity or something so it's effectively a water environment even though this water environment then goes through the membrane And then was this was this helix capping that I spoke about. I also mentioned earlier This or last week that the charge residues It's a bit sloppy to call them charts because anything that's charts in chemistry has a pKa value And that is the pH value where this charge will either well the rest you will either leave a service it will either donate or Take up a proton from water. So that when we talk about charge residues I typically think about what is the charge at neutral pH pH seven Which is might not strictly be what you have in biology, but it's again. It's good enough in approximation So that all these residues that have very high pKa values are in the lysine that typically have a charge of plus one Glutamic and aspartic acid low pKa values virtually always minus one and then histidine as I mentioned in biology There's always an exception to destroy our beautiful rules six point five. Why did it have to be six point five? Because this is so close enough that it can go either way Depending on what residues you have around it If you have lots of negative residues around this histidine it can actually be good for the histidine to be charged So all bets are off with the studies just as the backbone cis trans for polling. Yes Do you remember At some point you likely calculated chemical reactions Do you remember equilibrium constants in chemical reactions? Okay? So that the K a that's really the equilibrium constant if you have x8 plus 8 to 0 on one hand And then this can go over to say x minus plus 8 3 0 plus so that the x group here would donate the hydrogen to something and this is of course a process that can be either further to the right or to the left and Depending on the process the balance here will depend on the equilibrium constant But it gets very complicated to talk in terms of equilibrium constants and everything as chemists It makes much more sense. Well, we want to know at low pH It might have at low pH. It might take upper proton or vice versa. So the pKa value It's literally the same way when we go from the concentration of Hydronium ions H3O plus to pH and here we use instead of having talked about the k value We can talk about this would correspond to the pH value where this reaction is exactly 50 50 So it's just a simple way to think about what is the pH when you will start to change your proton Rather than having to worry about the specific concentration The way you should think about this is just remember that By default, you don't want charged residues inside proteins But under some conditions things can become non-charged You can deprotonate them or protonate them to make sure that they're neutral and then they're slightly easier to assert But you might have to spend energy to achieve that The reason why that is important I hinted about last week earlier this week to that charge is a very nice way To exert force on something if you have a charge and put it in an electric field, it will move So it's the way we use all these small channels Let's see No, sorry. That's the wrong type of way If you have a small channel that should either for as a close the There's a water pour here that would normally go through if you have hydrophobic residues here at some point when this moves in You're effectively gonna cut off the water pour. Do you see what's happening here? That the water is gradually breaking and around two microseconds or so now you really cut off the water here So now this channel will no longer be conducting So if you have lots of hydrophilic residues, you will have water inside a pour and if you then move them in But most of them are hydrophobic you can cut off that flow Similarly, if you have these large No, that's both ligand gated if you have large Oh now I know sorry this particular channel pH gated That's why so normally you have would have things charged But when you change the pH from 4.6 to 7 the rest use loads their protons and When they're no longer And we have in there when the rest use are no longer charts, right? They're not going to repel each other other as much and then they can get in proximity and then you will close the channel Sorry, I was thinking about voltage gated channels voltage gated channels I think I mentioned the other day right voltage gated channels They need to open and close and we achieve that by had literally having small pistols Just like it was an engine move up and down in the cell membrane where we change the voltage across the cell membrane So nature likes to use charges to do things biologically There are tons of different ways of Classifying things and depending on these properties. They're going to be more common in main chain side chains Whether they are in healers or sheets. I don't expect to know these things by heart But over decades we've learned quite a lot first to understand things in terms of physics but these are also patterns that nature is used and You would imagine that a simple way to achieve things that we could use all this physics to predict what proteins looks like And it's a really great idea people did it 40 years ago But what we have since realized is that no matter how smart we think we are You are not the smartest 4.3 billion years of trial and error So what we're increasingly doing now is that we're just letting the computer sort out the sequencing and let the computers Find something that's similar to something. We already know we don't try to understand the physics But this is the reason for the similarity Proteus can't swap out risk use anyway Proteus kind of have to swap out residues to maintain these patterns You can't take a hydrophobic residue and replace it with a hydrophilic one because then you would likely no longer have a stable protein But nature is unfortunately smarter than we are than it comes to the physics. I already stole three extra minutes here So let's take 15 minutes and reconvene just after that's 18 minutes plus the hour or so and then I will take you through a few more actual structures so that Overview of amino acids brings us to the actual proteins. We're gonna have a look at And the first class here is what we call fibrous proteins and that's that leads us to with the nature of fibers Something things that are long their large Their structural building blocks to tell the truth. They're at first sight as proteins. They're somewhat boring But they are important One of the classical example of this has to do with silk fibroin So it's a very small and the acid repeat is really alanine and glycine that are very flexible and then combined with a bit of serine And this still turns into gigantic anti-parallel beta sheets So if you feel silk and everything you're just especially you're just feeling beta sheets This has a very peculiar and there are the reason for the for the beautiful Luster here said that this simple repeats of the beta sheets is very well packed and they're mixed hydrophobic hydrophilic It's almost like a like crystal, but not quite If you go down to the hygiene a lit, you know There's a supermarket or something you have these shampoos and everything where they claim that it contains silk protein and everything And it's completely artificial. It has nothing whatsoever to do with silk But they've generated artificial protein that it looks just like this This is another very peculiar structure that I'm just gonna show it to you've seen it It's also a very small repeating pattern glycine prolin prolin normally you would never believe anything could be stable with that pattern But and it doesn't but if you take three chains right next to each other It turns out that you can create a beautiful regular I think I'm moving of that. Yes, you can create a beautiful chain that have first some hydrogens inside the chains And then some other hydrogen bonds mediated by waters, so it's not just surprisingly stable It's very stable and this is the basis of bone teeth skin and it's basically 25 percent of what you are. We are sorry We are fairly boring This aggregates into gigantic ordinary structure, so here you have the amino acids in Chains three of them create the small collagen helix and Then you create a number of those chains into a gigantic super helix here So do you start to see this hierarchical thing it comes back nature likes to you a repeat and reuse building blocks So you only need a very small gene coding for those three amino acids and then by repeating it you can create gigantic structures And you can actually see this in the electron microscopes The first of you look at a tooth or something If you take that glycine and mutate it anything else Remember that I said that the glycine was fairly flexible and anything if you mutate that to almost anything else You create what's called a brittle bone disease, so the both become very fragile So the very and the reason why it becomes so severe is that you will have a ton of mutants in the structure But it's just an example of this hierarchy and how we can create for instance the skeleton Another very common pattern again that these patterns are somewhat boring because that's to create this super gigantic Structures, we can't use all the freedom in the world You can take two helices and just as you paired up to beta strands You can take this helices and just coil them around each other That is called a coiled coil because it's helix is already a coil, right? And then you have two of them. It's a coil that in turn is a coil. So it's a coiled coil It can be more than two, but it's fairly rare one of the classical example of this is in your muscles So that the fibers that contract it's literally two of these muscle fibers And then you have a small protein called myosin that They effectively have one protein that is walking along this fiber that causes muscle contraction Super fascinating. You can find movies of this on YouTube Although You might recall that I said a helices was usually 3.6. I mean as is perturbed.  It's not hard mark seven instead of 7.2 So if you have normally this should be say hydrophobic on the inside, but if it's exactly seven You can for instance take two residues here Make sure that either both of them are hydrophobic or make sure that they can form a hydrogen bond with each other So by having exactly seven you make the entire thing quite tight This is almost like having a little bit of glue between those residues This might not look great at all when you first look at it You're gonna have a ton of collisions there and that's both two and not two The easiest way to look at this is by using some of these alternative representations because yes There are there are atoms all over the place in a helix But if you forget about the sides just for a second and just draw the molecular surface here This kind of like ridges and valleys on the helix here and they're they're not really ridges They're just points where the side chains are sticking out But you can think of that as ridges going either along the solid or dashed lines here So if you now take a second such helix and Then you rotate the round and try to fit the ridges of one helix into the valleys of another helix Then they're gonna be at least two patterns where they will fit, right and this Here everything is clashing, but if you take these and rotate them roughly 20 degrees they fit perfectly So this is what you call helix packing So again that you might have seen this structure before it looked very irregular Why didn't we have beautiful nice parallel helices? Well, because if they were parallel their side chains would clash But just as the beta sheets slightly turn relative to each other if the helices are rotate if one helix is Rotated slightly to the next helix That is actually how we achieve packing so it's not just that it's suboptimal This is optimal having the perfectly parallel would not be optimal and this recurse We see this all the time in these coiled helices and that's why it's so useful that you have these coiled helices They're gradually twisting around each other. Do you see here that they're gradually moving and These red residues if it's now 3.5 per turn every 7th rescue if you make that a loose scene Which is a small real well mid-sized hydrophobic residue We're gonna have lots of loose scenes in the ceilises. That's a hydrophobic residue. So that does not like to be exposed to the surface But all these rest red residues they will now pair up against each other And because it's not exactly 3.6 or 7 sorry, it's not exactly 3.6 But 3.5 this gives them the slight turn so that the red residues per se is acting like glue and holding the ceilises together You can even have some cysteine bonds here They remember those disolved fights we talked about and this can make it even stronger. So this can become super rid of structures There is a common place where this occurs and that is hair So your hair is out just as your skeleton is beta sheets. Your hair is the first approximation alpha helices and It gets slightly more complicated. So you have these small alpha helices there They end up in coiled coils and then you're paired of these coiled coils And then you end up having roughly eight or so of them coiled up in larger prototype builds And then you keep adding up the size until you get to these Cuticles that have might have something in the diameter of 0.1 millimeter or so But it's literally just a ton of alpha helices lined up and again use hierarchical structures to reach macroscopic scales So if you would like imagine that you were a chemist and you would like to Reform hair or force glue hair into a particular structure. There are some tricks you can use here What if you were to take those cysteine groups and first we reduce them? So we break all the cysteine groups all the disolved fights And then you form the hair the way you want it and Then we add another chemical to reform all the cysteine groups So that will effectively that you can shape hair Permanently and that's literally what you do if you go into the hairdresser and get a permanent wave That used to be how popular in the 70s and 80s you're literally working with these disulfide groups to create a permanent pattern and In the department of almost useless knowledge, actually, it's not at all useless Your alpha helices growing at roughly 10 turns per second It's 3.6 residues per turn And if you ever forget how fast alpha helices form everything you can calculate it backwards that way and from that you can calculate roughly how fast your hair will grow and it It's it's surprisingly accurate Even if we derive remember last lecture where I had some hand waving about how fast alpha helices form We are right within an order of magnitude Just some very simple reasoning of physics and the hydrogen bonds the free energy barrier you have to get over there are a ton of other Things like elastin is another cool one. That is the Component that you have in blood vessels, which at least when you're young are very elastic and when you're sadly getting to my age or something that elasticity degrees For tons of reasons in particular because you have all this fat depletion inside But there is a lot of experiments today of designing biomaterials that materials that have biomimic properties so that you can create an artificial protein that has properties like a natural vessel But that's stupid.  Well, you can do anything you want with plastic right so that you have much more degrees of freedom in plastic But could you imagine any advantages to using amino acids? What will your body think of amino acids? It's happy. It looks like any any other material in the body You're not the second you're introducing methyl or something you have problems that your immune system will attack it Right, your immune system is not going to attack a normal protein that looks exactly like the protein should like So that they're biocompatible, which is a very nice feature I'm not going to spend more time talking about fibrous proteins because yes They're super important as building blocks, but they're not that fun in terms of studying structures of physics So I'm going to continue on to globular proteins that are these small sphere like shapes that can be Either helices or sheets or a mix of helices and sheets There are The fascinating thing with life size and life sizes develops much quicker than physics That's actually what I love with it But the challenge with that is that we're still in the middle of that development So when I was your age, it was super important to take look at small structures and classify them Exactly where do the helices sit exactly where do the sheets sit? I even think the Finkelstein book loves to draw them in different corners and classify what is the topology And we're going to look a little bit into that But this is less and less important because we have the computers doing this for us and to tell the truth This works for a small protein like hemoglobin or whatever. Yes, it's hemoglobin on the left But if you have a ribosome with 60 different chains, it's just too much information. We can't handle it So as proteins have become more complex, we're giving up on this way But it's you're likely seeing the regularities and the helices and sheets here, right? And all these gradual twists and everything had to do with the particular properties of the amino acids Beta sheet structures, they're simple, but Here's also where biology comes in. It's virtually impossible to have anything with just one sheet So physically you can have one sheet, but the problem is that you can't really that's having a Piece of paper you can't draw anything on a piece of paper. Well nature doesn't know how to draw yet So if I have one piece of paper, it can't separate anything both of it's accessible to the water, right? It's going to be difficult to have any function. It's also going to be super floppy So what you typically do is that you have at least two sheets and remember that I showed it at the acid binding proteins Because the second you have two you can have an inside and an outside and that's likely caused by evolutionary pressure exactly why we don't know and Initially you might think that all sheets should be beautiful They should be perfectly planar that would make sense And I lied a little bit to you and said that if you just look at the sheets the amino acids are on opposite sides The problem is that it's not exactly 180 point zero zero zero zero degrees so on average It might be hundred seventy five or so for one amino acid it doesn't matter But if you then have lots of them that's going to lead to these slight twists if you if you now start Look between the strands right so the fact that they're twisting here That's not an artifact or that is not as stable as it should be all sheets will be slightly twisted because they are not exactly at 180 degrees There is no particular reason why the amino acids bonds should be at exactly 180 degrees and again If you do something very simple with two sheets There are I already mentioned that fatty acid binding protein The other way could be to put the sheets rather than having a crisscross orthogonal pattern you can make them aligned Immunoglobulins, it's very common super important molecules in your Immune system there is another one I'm not sure whether I have that protein called gamma crystalline with this a crystalline in the lens of your eye It's also beta sheets The reason why this works so well to solvate the fatty acid I already hinted over that you see all these red Green side chains there and if you take every single side chain make them hydrophobic every single one make them Hydrophilic you can literally create this think of a sheet of paper that has a property a on one side property B on the other side And it's a beautiful way of separating compartments or something. Oh, yes, I even had the gamma crystalline here That's what your eyes are made of On the other hand you see that in addition to the sheets you also need to connect these loops And that seems a bit stupid. It doesn't just go up down. It keeps jumping back and forth in some sort of strange way here That way at first sight it might appear strange But those loops are actually quite because having slightly longer loops makes it possible to close this structure at the bottom and the top So they're not unused. They're not junk. These turns literally help create the entire compartment Because otherwise you would just have a can without bottom or top right and that's great if you want to look through it But it's not great if you want to close it The first person to identify this was Janet Jane Richardson And this pattern you can see before they're called Greek keys And the reason why they're called Greek keys is that she noticed the similarity between these patterns and the patterns you would have on a Greek Earn So why on earth would you have these similar patterns on a Greek earn as you would have these things going back and forth? Is it a pure coincidence or? So what's special about the pattern on the Greek earn? You all know it. It's just that you're not thinking about it How many times do you have to lift your pen to draw it?  What does that correspond to in the protein? You can it's a pattern that you can create with one chain, right? Because if you had to add these tops on bottom and top separately you would need multiple chains expressed by different genes Again a gene will express one chain So this is nature's way of creating a small repeating pattern But still having these things on the bottom and top but doing this as part of one protein So it's not the coincidence at all. It's a very nice geometric feature. I think this will say nature paper in 1977 You might not have been born there, but I was five years old. It's fairly comparative physics. This is the fun part here It's new I think we see the fatty acid. Yes, that's the fatty acid in the fatty acid binding protein Nowadays we have x-ray structures here. So here you see An oleic acid that doesn't sound sexy to you But it's super sexy to me because this is what I've spent the last 20 years of my life on These are the chains that become the tails of the membrane of the lipids and your membranes So all the fatty trans fat transport and everything in your body, which is super important To understand both transport and a whole lot of disease This is actually another way that you might not think about it But your body is what you eat not in terms of proteins proteins are synthesized from your genes But your membranes are created for what you eat and this is how nature builds it uses the building books So the point is that there are not really that many different topologists or different patterns are generating things And that's simply because that these are complicated molecules. There are not that many things that are both regular and that can be reused You virtually never see mixed parallel and anti parallel beta sheets because it's you need a pattern, right? It's complicated to mix different patterns. It's easier to stick to one pattern, but there are always exceptions from everything and This all comes down to the properties amino acids and that's why I took you through all those different amino acids The specific properties of different amino acids will stabilize these structures different amino acids will stabilize different structures for instance alpha helices and in addition There will never ever be any knots in a protein They have if you take the two ends of a protein and pull the string should come out straight, right? There should never be any knot on a protein and Then there of course always an exception in biology pepsin This protein do you see that the chain goes? So let's see here the chain goes from blue over here and then the yellow part actually goes inside the green here So it has created a knot So why on earth would you like a knot here so pepsin have you heard the name? Where do you think this protein occurs? It's alimentary Your stomach So what's the pH in your stomach? Ha, you're not even close like one It's like it's really aggressive with the sulfuric with then the sulfuric. It's HCL Normal proteins the whole point of your stomach is to digest proteins And that's a problem because you now want the protein to work in an environment that digest proteins The only way to make it protein stable is making it super stable So the reason for this knot is likely to create a protein nature wants a protein that's super stable Here, too There are some topology features with C and others we don't see the reason why again the reason why this interest that Historically so we could use this to predict What the structures must look like because most of them never exist today? We let the computers do it so it's more of an historical interest that for whatever reason not every single way of putting these building blocks together are stable But what they can do that if you have two of these proteins Let's say this beta sheet. Do you see what they can do? These are separate molecules and This so the green one is a small beta sheet and the blue one is two But if you now take them and put them together you create an even larger beta sheet because you get hydrogen bonds all the way here, too This is very much related to what I said at the beginning of the lecture today how you form these large plaques, right? So they have one protein adding another protein adding another protein So you can form structures that consist of billions of proteins and the larger. They are the more stable They will be and that's roughly all I'm gonna tell you About small beta sheet structures Alpha helices on the other hand they we already mentioned that they had slightly different properties So let's have a little bit look about what alpha helical structures might look like Alpha helix says they look completely horrible in comparison the only real requirement here These helices need to be packed one way or another So remember with beta sheet that strand Dependent on the next strand dependent on the next strand to be stable That's not the case for helices each helix is roughly stable on its own and then you just need to find one rough way of packing it and The reason why this looks like it's just a bunch of helices thrown out in space that had to do with this not crossing each other Well, not crossing them exactly parallel. It's actually better because you want them to pack against each other. I Might have and they're here, too There are a bunch of different ways so far we might have something that's just curled together We might have them where they're twisting a bit in this case There's almost a hole in the middle of the podium, right? Can you imagine where this protein occurs if I didn't see the name here? If this is hydrophobic on the outside You can put it the entire thing in a membrane, right? And then we've literally created a hole in the membrane where you can push through ions. They're super common as I tell Or you can under some conditions you can put all of them nicely parallel. They're not exactly parallel, but they're relatively parallel Let's have a look at all these. They're simple. So let's start with them They're called if you have four helices and put them together. We just call them four helix bundles And there are a bunch of different ways we can draw that So if you look at these Which one appears to be most stable and nice? I would probably guess cytochrome, right? That one is horrible. Oh my god. This must have been an experimental error. We can never gotten that published And this has something strange in the middle here that whatever might not be the stable without the thing in the middle the reason for this diversity though is coupled to function and All these are stable, but they're stable under slightly different conditions In all of these cases the helices go up down up down up down So two helices that are right next to each other in sequences. They are anti-parallel. They point in opposite directions That cytochrome on the left It's very it happens it occurs in lots of different organisms But they're very diverse domains that are typically involved in electron transport You can't see that just from the shape of it But nature has basically created a nice building scaffold the structure and then by having specific side chains We can create things that can for instance can stabilize electrons in different states That means that they are frequently involved in binding metals Sometimes in higher organisms, but it's very common in bacteria. There is a small back there called chevonelau in a densis that I Forgot how many Cytochrome domains it has but it has like tons of the dozens or a hundred Which is a lot because the bacteria might only have a few thousand genes in total. I Spent about six to 12 months as a postdoc working on this And it was a grand that I can describe now second confess it even if it's a recording We were simply interested in understanding protein folding in general So we had a product that we wanted to map out all the domains of this small organisms And we to tell the truth we really didn't care that much about the organism, but this was in the US So the Department of Defense was super interested in chevonelau on a densis So we got a large grant from them for that reason Why do you think they were interested in chevonelau on a densis? Radio radio activity right said if you can get bacteria to bind radioactive metals You can essentially cause the bacteria to take up the radioactive metals and use this and nothing ever came out of this But so there are lots of potential Engineering applications of bacteria in general because these things they will interact with molecules around you They will typically interact way with way higher specificity than any normal chemistry experience it can do and again in physics You can separate anything, but if this material is now spread out all over nature very low concentration You can't take all the soil in Stockholm and purified with a physical method, but with biological methods. We can We're basically enslaving bacteria to work for us the second one did not start out seprotein actually it started out as this small black It's an infection on tobacco plants, which is not particularly important in Sweden But in large parts of the world we're talking about billions of revenue tobacco is a very important crop If you put this in an electron microscope You will start to see some sort of regular patterns here like small rods And if you amplify that even further you're gonna start to see things here. So this is 15 nanometers So here you're almost starting to see small domains parts of proteins So these small black things are actually the domains the T and V domains I showed you and at first sight they look just strange. There is no regular pattern at all to this Until you get an x-ray structure of this So do you see that the fact that this was kind of pointed? That's the whole point ha ha pun intended So that the small part here is packed on the inside and that has to be smaller than the part on the outside because otherwise They wouldn't fit as well, right? So here to evolution over billions of years has optimized the particular fold to stabilize this protein So this is a virus what do you have on the inside here the red part? RNA yes So what what will that RNA do? Hmm, so that RNA will infect The cells of the plant right? And it will enslave the cells of the plant. What does that RNA code for? What does that RNA so RNA? You store your genetic material in DNA a virus stores its mirrored genetic material as RNA But could you matter what what might that RNA code for? It just codes for that domain Just one for a little bundle nothing else And you think you're beautiful Sorry, we're fairly ugly factors. Can you imagine that the efficiency of life? This is a molecule that has been optimized for one person one purpose only you only need a code for one specific protein pretty much Well, you need a bit of a reverse transcriptase too But one building block and it creates a stable environment that protects its RNA from the surrounding It's so amazingly beautiful that I have no idea that it was a religious feeling that nature is You are we are slow ugly complicated features that expend a lot of energy to reproduce or anything It doesn't get more beautiful than this in nature. Although technically this virus is not really alive, right? It doesn't have any turnover process itself or so But if you define life as replicating genetic material, you could argue that it is like the person who discovered this is actually a Friend that you've met before Rosalind Franklin of DNA fame too this was her first real project that she actually got full credit for in contrast to the DNA and then Then we're gonna move to this a slightly more complicated structure. I haven't forgotten the third part and This is a much larger protein now hemoglobin. What does hemoglobin do? It carries oxygen and it's the reason we like that it was one of the first proteins ever to be determined It's easy to prove it. It's actually complicated because it's a hemoglobin because there's a four different groups So you have the same building block four times and then inside each of these you have this proto porphyrin group And the port proto porphyrin that binds iron we call a heme group So that's the group that is actually able to bind the very comb well stabilize an oxygen molecule Hemoglobin if you look at the single unit of it it has a bunch. I think it's six alpha helices and Then you need very special amino acids. It's typically a histidine Up on each side that are pointing into this region where you're binding this entire heme group So the heme group is typically not covalently bound, but it's a very nicely stabilized as part of the protein Hemoglobin and my there is another Sister molecule of this which is myoglobin and myoglobin only consists of a single molecule And I might come back to that. I don't remember if I'm going to come back to this later on in the class They were actually that was determined by Kendra almost in parallel And the reason why you have why on earth would you need two molecules in your body that both bind oxygen? That sounds like a waste Could we just get rid of it? Reduction cost savings fewer genes that should be more efficient make you more like a bacterium Yeah, but then you could just have one gene and express more of it So as if you look in your hemoglobin should hemoglobin be good or bad at binding oxygen good So you want it to bind oxygen really hard Why so you want it so of course if hemoglobin is good where hem where does hemoglobin gets its oxygen? In the lungs right so that yes if it's minus 20 k cal That's awesome. You're gonna be really good at binding oxygen Unfortunately now two seconds later your hemoglobin is now going to release the oxygen to your muscles and You have minus 20 k cal that molecule will never be released So the problem is that on the one hand you want the molecule that it should bind oxygen really efficient in your lungs But then it should release it really efficiently in your muscles So this is a fairly complicated interplay where hemoglobin has when the concentration of oxygen is low It has a relatively low affinity binding energy to oxygen But the more oxygen it binds the stronger it binds it So in the lungs when you have lots of oxygen hemoglobin will actually be good at binding it But in the muscles where the oxygen concentration is lower hemoglobin will actually change its mind and realize You know one second thought I should get rid of all my oxygen So then hemoglobin hands it over to my global so my global you have in the muscles while hemoglobin in the blood I would never have come up with that, but nature had slightly more time Remember we had six helices here, right? Remember that we also spoke about this eukaryotic genes Genomes that have introns and exons that not all parts of your DNA actually codes for things But that you have to take your DNA and stitch certain parts together and Based on that what are the parts? What do you think that these blue, green, red, blue and orange parts correspond to? The helices and this would have been such a great idea if it was not completely wrong There are three exons in the DNA, but they cut straight through the helices They have nothing whatsoever to do with the secondary structure We still don't really know where the introns and exons come from But I just just because it's so obvious to think that they corresponded the the introns and exons stitching happens much sooner than any folding So their exons have nothing to do with it, but it's important for other reasons And you can actually show that you can take just one of them and they will kind of bind the hemoglobin a little bit anywhere But that's parenthesis The reason for bringing this up is not just that I wanted to fool you But again to drive home the message that biology happens as we speak and when I gave this lecture a year ago It was really cool So this is just a year old that a year ago there were the first Well, there have been lots of it, but we're getting all the time new ideas What are the interactions role and what are they doing? So compared to physics where most things happen in the 18th century here things are literally happening now And that's what I love with the field. This is so not the ultimate results, but we're learning a little bit all the time I think I already mentioned this helix rids and grooves for you but if there are at least two different ways of Packing these you can either pack them that way right or that way Then you can start making statistics So what are if you take the axis of each of this helix here? Let's start to do some sort of informatics Just let's calculate what are the angles start there crossing 10 degrees 15 degrees 20 degrees and look through all the hundred thousand structures or so We know and then you can start making databases of this So what are the things that do occur and here too as we already hinted there are pretty much only two ways That these things can be packed We couldn't be reused that for protein design So if I now to if I then take a model I take two helices and I know that they should be either Roughly minus 50 or a plus 15 well plus 20 Let's try to build some side chains there that stabilize each other and see if I can can I create a small artificial Protein that is stable. That's a small four helical bundle Well, that's possible, but then I also need to stay stable So one way of looking at this if I now look at my two helices from the top So that's one helix and the reason why you get this pattern right is that it's 3.6 Residue hundred degrees freeze residue of 3.6 residues per term This sequence there might not tell you a whole lot But if you look at these two we have a loose scene with a small and hydrophobic imposition 5 19 12 13 and Then we have lots of live scenes and hydrophilic parts Here and there again in the rest there is no obvious pattern here But if you look at it here, do you see that it's hydrophobic hydrophobic and then water soluble on the outside So this will instantly cause these two phases of the helices to like each other And that's effective where you would have what you would have to do But you can have the computer help you if you want to design them We can do that there's slightly more advanced way because that if you only have two helis The problem is that you don't really have a whole lot of space here because it's going to be perfectly packed But if we do the same thing with four helices You effectively have an inside here And that you can use that to create small proteins with particular properties. I think this particular one is One that combines the heme girl Yes, I even say there this one combined a heme group and that has been used with the idea of being able to create artificial red blood cells So that you could have it's a pure chemistry There are no cells in this but they would have a molecule that had at least some of the properties of hemoglobin that could bind oxygen There are drawbacks with that it is not at all as fancy as hemoglobin in terms of this efficiency and releasing and binding oxygen But in theory it's something that you might be able to store and there are also lots of people in the world that don't wouldn't either they wouldn't accept the blood donation for Religious reasons and blood also decays very rapidly That it has a half-life of a few weeks or something and that's why they constantly need new blood donors So if we could artificially create this you would have a molecule that would not have all the complications with Matching different blood groups and anything, but it hasn't quite taken off. It is available on the market, but it's not been a tremendously success You could also use this way if you have some that's hydrophobic on one side and hydrophilic on the other one You can put fat on the inside So fats are nice because in particular in food they come they convey a lot of flavor So we want fat in our food, but if we have too much fat we end up being fat So you tend to use this as emulsifiers So they will be water soluble on the outside So the whole thing will be soluble in water, but the interior they can bind fat So you cannot having say even margarine where the margarine is actually only 15 20 or 30 percent fat But it can still bind all the tastes and everything There are some problems for this so There's one particular problem That some of you might have noticed if you're a poor student and trying to say fry in Low-fat margarine What happens if you take a low-fat margarine and try to fry in it? You can go home and do this experiment, but do it in a cheap pan because you might end up destroying the pan What suddenly it looks great at first, but in contrast to butter that would just melt suddenly you get something strange water like So what happens as you're heating the protein the protein falls apart And then your whole emulsion falls apart So you have a separation of water and fat and suddenly you have lots of water in the pad and Apart from not being able to fry in it This is actually quite a large industry in particularly Lund in the south of Sweden They have had designed various proteins and emulsifiers to be able to withstand high temperature So if you go out and buy say creme fraiche or something some of they will say that even the low-fat one that you can boil it So then they've designed it with specially multipliers that can withstand high temperature I'm not sure whether that is it might actually be proteins in that one particular one, too So the neat thing about doing this with proteins is what? Chemistry is what you have lots of fancy emulsifiers in chemistry. Why should you do it with proteins?  That's what you have in muscles anyway, so that it's perfectly safe to eat There are Why can't continue with this? There are certainly mixed alpha and beta domains to the one thing to be aware of there You typically don't mix healer's sheets, but you tend to them in disjoint domains And the reason for that is that if you look at a helix that helix can't really it doesn't have any free Hydrogen bonds to bind the sheets right and same thing with the sheet it needs hydrogen bonds to be stable So they can be right next to it You cannot have helix sheet helix sheet helix sheet in a sequence But what you would then would need to do so you can have a sheet going up and then the helix on the outside going down And then the sheet going up again and helix on the outside going down So these barrels they would have sheet helix sheet. Sorry helix sheet helix sheet helix sheet, etc Or you could have a Sheets straight heat sheet in the middle and then they go up and then down with the helix on the side And then go up again and the helix all the side this is alcohol dehydrogenase and if you it's Friday today so I would really praise it if you go home and didn't well either home or downtown you might be able to do an experiment tonight purely in the interest of science of course if you drink alcohol this is what breaks down the ethanol it's part of the whole process and certain parts of the population on earth actually have a deficiency in alcohol dehydrogenase and that's why so some of these things like it almost sounds like cultural appropriation or or racist and that same for said you took it like Asians in particular they can't drink as much alcohol but it's actually true so that certain parts of the population in Asia certainly not everyone they have a deficiency in alcohol dehydrogenase so there they become significantly more intoxicated from alcohol so you might want to test it's important to test whether you have a deficiency for this so please help me with that experiment the remember that ligand gated ion channel I showed before that is that is one of the channels where the alcohol will bind and influence your nervous system Lucy will talk about that on Monday so you will literally have the alcohol binding to that channel and change how it works Rossman fold is another example of this I'm not gonna go through all these folds but the point is that most of these things were discovered in the 70s and 80s where we were able to determine structures proteins and since I'm not gonna spend a lot of time with you the part to be aware of though is that most of these things in particular with the sheets on the edges of these things or between them this is where you tend to have binding sites so whether the binding because you have all the the edges of these rare of the molecules here or the secondary structure elements that will really correspond having free hydrogen bonds or so so they typically create beautiful small cavities where you can bind things such as an ethanol molecule or something although this particular protein doesn't bind ethanol and there are is a number of combinations do there I'm well I'm all the time but for once I'm gonna steal two minutes because it's Lucy and Burke is gonna be teaching you next week these are important in one class of proteins that are binding DNA and RNA so at first sight these small molecules like almost look unstable right and they are kind of unstable but when this floppy unstable molecule then finds a DNA it turns out that it fit it has evolved to fit perfectly in the large and the major group here so that the valley and the DNA sequence so these are typically the proteins used to recognize where we should start reading your genetic material and now we're going all the way back to the first lecture because then I just hinted well there are proteins that determined where to start reading and nature has evolved that through the genetic material we have evolved particular proteins to start recognizing the specific pattern in the DNA that would correspond to the start of the new protein and this how we find up this is sort of how we find the sequences in bioinformatics because we've learned to recognize those patterns ourselves too I already mentioned the toxin this is another toxin Brazilian scorpion toxin it's a neurotoxin and here too that you might have a something that appears so that the scientists screwed up a little bit in the structure determination I would guess that this is a structure that becomes a complete beta sheet when it's binding to its partner so that's up sorry here too it's important to consider before and after and everything all of these things have evolved to perfectly match the function so with that I think I have two more slides and I'll show you that there is an interesting discussion that will we keep discovering false and the cool thing is that we there is a very famous paper by Cyrus Schottia who died fortunately in December called a thousand folds for the molecular biologist and the idea is that despite the astronomically large sequence space it appears there are relatively few scaffolds there were relatively few shapes in the ballpark of a thousand that made your uses and reuses and reuses so did you remember how many genes you had in your bodies no that's base pairs but I mean genes roughly 20,000 sorry you're not that complicated there are only 20,000 building blocks but those 20,000 building blocks in turn use only 1000 building blocks there are only roughly 1000 different shapes of proteins in your body and that turned out to be a little bit of underestimation it might be 1500 or so but we're getting to the point where we're more or less discover these we don't know all of them but we know the vast majority of them so relatively few that are undiscovered and that has opened an amazing map for protein engineering we can actually design most of these things and in most cases things are uniquely defined even if there are small changes in them or so but there are a few cases that you can change less than half of the residues as they turn a beta sheet into an alpha helix or so but typically all these things have evolved to A have the function they want and B be stable so that they're not destroyed if I change one amino acid by mistake sorry for stealing a few extra minutes Lucy there are a bunch of study questions here that because it's going to be Lucy taken over on Monday do mail me about this I'm more than happy to do screen recordings and go through this in as much detail as you want and on Monday Lucy is going to talk about membrane proteins because membrane proteins obey all these principles and they use same thing but in addition they have this really nice functional things where they're acting like channels locks opening doors and windows and that's also why they're so pharmaceutical important but with that let's call it today and have a nice weekend