Today I am going to depart a bit from the physics, so there are no more Equations today, but we're going to use the stuff we learned on Monday and Tuesday about free energy And now we're going to look at protein structure again, but we're going to look at it with slightly different eyes So I'm going to try to come back and interpret what we see or what we don't see in terms of free energy in particular So everything we see in these slides the reason we see the structures we see is because they are favorable from a free energy point of view Yesterday I was opponent in Orhus in Denmark and a poor student. She passed actually awesome thesis But I kept tormenting her for two hours about free energy And the funny thing even on the PhD level, right? She's a PhD in medicinal chemistry understanding how new serotonin related drugs work against depression and The way these drugs are designed they target a number of different transporters and iron channels that Lucy will talk a little bit more about on Monday But what determines how they work is what receptors are they binding? What are they doing to the receptors and all those things can be interpreted in free energy? Stabilization determining what binds what and why it's stabilized in the right binding posts It's the stuff we went through earlier this week is not just a theoretical exercises the foundation for almost everything We do both in my lab and a whole lot of other biophysics research groups Not to mention experimental studies, of course But today we're going to be looking at real proteins not just more secondary structures But the real workhorses the building blocks that do things in your body.  I'm not gonna I think I will I'm not gonna spend time on this slide But I'll go through them piece by piece there are Broadly speaking three big classes of proteins the ones you've probably seen most at least more Small proteins that work in solution like hemoglobin binding oxygen in your blood or an antibody or something I'm going to go through them the second half of the lecture today the first half of the lecture today I'm going to talk about seemingly boring proteins the building material a large circled fibrous proteins that form large structures such as skin bone and other things and it's By sheer quantity it's by far the most abundant proteins in your bodies So you need to know a little bit about them, but from a biochemical and From my point of view biophysical point. These are more interesting and Then on Monday, I need to participate in a plan s seminar about publication in the European Union So then Lucy sadly gets to give the really fun lecture about our research topics about membrane proteins And I'm jealous about that, but I bet she's gonna be a great lecture And this is the really fun stuff because then you're then you're literally looking at machinery Determining how Ios go through membranes and everything. It's super related to nervous signaling and everything There are a number of different tools to study proteins. You remember that movie I showed you by Cyrus Leventhal that they created the first way of actually visualizing proteins in three dimensions today you can just go download and there are a number of Programs, but I would particularly like throw a little fact in one called V&D that is developed by close friends of ours at University of Illinois, Urbana, Champaign There are also slightly more than the 300 proteins that were available when I was your age So that today if I would like to say a particular protein hemoglobin These proteins are represented with small four letter codes So if I'm going to tell you that you should look at a particular structure I might tell you to look at one HPD and then you can just go to a database And then you enter this short code and then you will download a very simple text file that just contains all the coordinates of this protein I'm gonna Or Lucy actually will give a lecture about bioinformatics to later on in the course And then we're going to show that there are similar type of databases for sequences and everything and a whole lot of them making sense about things It's starting from all these millions of sequences and being able to predict this type of structures from sequences Why do we want to predict structure from sequence? So why can't we do it? two questions Why do we want to do it? What was the central dogmouth molecular biology? Sequence to structure to function right so the function is determined by the structure on the other hand We also know from Christian Anfield said that a sequence will fold into a unique structure Right, so if I know the sequence the information about the structure is encoded in the sequence It's just it's not obvious that we will know it But why don't we just determine the structure instead?  Why do we determine the sequence? Well, so the difference is determining a structure in particular of a difficult protein can easily be a two-year project And in some case you can't do to remember that 100,000 protein sounds like a lot But those are all the protein structures that have been termed since the early 1970s and it's not a whole lot by those standards We probably determine well of we but the genome centers worldwide probably determine more than 100 through thousand new genome See as sorry protein sequences per day So that determining new sequences is something you do with 24-hour turnaround There are even some genetic rare diseases in particular with newborn babies and everything if you don't know what if you don't know What's wrong with them today we sequence their blood? Take a blood sample and within three or four days You can have their entire genome sequenced and they can scan through and hopefully find a mutation and realize what the disease is And of course in that perspective you need to do it in mere days So that it's cheaper and more efficient to do our misquences So if we if we have good ways to determine the structures from the sequences we can understand For instance if you have an alan alanine instead of a valine rescue What disease will that lead to in the protein with this cause it to bind something else for instance the serotonin receptor? You might get a chance to play around with this a bit in the labs or not and that depends on what Burke is gonna Have you do there? The reason why we get the amino the proteins we have is of course due to the amino acids as we spoke about All amino acids are not created equal we need all 20 of them But there are some that are more different than others for instance alanine or isoleucine or leucine They're all small and hydrophobic and the differences are not that large But there are a handful of them where you need to if you even if you're just scanning through a sequence You need to be able to react in real. Oh, that's likely significant Proline was one of them if you have a an alpha helix prediction and you suddenly see a proline You realize that proline is going to break the alpha helix why? Because proline can't create the hydrogen bonds that is necessary So if you put a proline an alpha helix you will get a kinky on the helix that will break it right away Glycine is very small so it's going to be common in tight turns and there are a few other amino acids that are good to have a gut feeling about There are a bunch of properties A few years ago my father is a professor of my well A few Jesus time flies 15 years ago. I wrote a paper together with my father as a professor of medical microbiological and he's now retired And then I wrote a lot of sequences that what are all these letters so even Even one generation ago everybody used explicit three letter codes for every amino acid So if you were if you see the Sanger paper that I had earlier in the course, they will spell out every amino acid you will say Pro dash ALA dash Gly dash met dash ILE dash low Makes sense, right? The only problem there are so many sequences today that we don't have room for that So what we've all converted to is are we using this single letter codes and then we don't bother with dashes So we just write straight letters that also makes it much easier We can compare the letters in columns and see how they align to each other I will come back to that in bioinformatics. There are a couple of tricks here I don't expect you to know these by heart, but our Genine for instance that are most of them are the first letter, but all of them all of them The other thing that you might see here already what you have out here in the right most column is the delta G of salvation and In the second right most column is the abundance. That is how frequent they were. Do you remember what determined this abundance? Do you see that some of them are more much more common than others? Why is Sistine so rare? While for instance Serene is much more common or Alani Or Lucy for that matter Exactly so this depends entirely on the codos not whether it's good or bad to have them in the protein This is encoded in the genetic code This delta G of salvation What could you use that for? Do you see some there? Arginine for instance Roughly minus 60. What does that mean? What should you compare it to? So salvation is the free energy change you delta G salvation is a free energy change We would get if we put this in water, right? So we take it from vacuum or a salt and put it in water So minus 60 means that you win free energy. That's a favorable process So that's kind of happens spontaneously. Is it good or is it it's a slightly good? Okay, or insanely good when it's minus 60 What do you compare it to? Why? Guessing is not enough So what there is a number you need to compare that to yeah. Well, yes Actually, that's not it you could argue that that's very bad But in particular if I change the temperature, what is our temp? What is our scale of energy determined by? This is of course the eight spawns are relevant to what's the energy of an eight spawned Two to five K cows or so, but what is what is our energy scale? We can say that the units is K cal but in biophysics sits or physics for that matter It's in it's instructive to think in a temperature-dependent scale KT, right? Because you always say e raised to the power of that energy divided by KT or minus an energy So what is KT? In cake house or kilojoules It's one of those is virtually guaranteed to appear on the extent or if not directly indirectly Two point five kilojoules or point six K cows And I cannot gonna kill you to say point five But you need you need to have the gut feeling that that the second something is an order of magnitude larger than that It will always happen the reaction will never go in the opposite direction So the likelihood of taking an arginine and moving that to the inside of a membrane It's not gonna happen. I would eat my left shoe literally and Same thing if you take something very hydrophobic, it's never gonna be a water. It will always be on the inside So many things in biophysics in contrast to physics where you want to calculate things exactly that the gut feeling or the hand Waving things are actually way more important. You can in there. Will it happen or not?  I Don't think we're gonna talk specifically about protein structure or two But you might at some point see you remember that I said that there was a C alpha and that's the central carbon That's what we call it alpha if you just keep going out the side chain Well, if you're if you're native Greek you have an advantage here because you just keep enumerating the carbon alpha, vita, gamma, delta, epsilon, zeta So that the further out you go in the sides and you just increase the enumeration of the letters in the Greek alphabet But that's also kind of sidetracking a bit Cysteine is a residue that I'm gonna talk a little bit about later today So that I will bring up. Cysteine is seemingly a normal residue, but it has a sulfur here so normally you normally you have an SH group but these can oxidize to form a Dysalt fiber it's sulfur-sulfur and that's a real covalent bond not an ionic bond or anything and not a hydrogen bond and That literally means that there is now a new bond linking two chains together And the second you've done that the protein can't move those can't move aside because you've literally formed a not a knot But glue them together so what you would have once one protein chain on the left and one on the right But now you also literally have a chain across here that can Occasionally be good if you really want to lock something in But it's of course a fairly dramatic reduction of entropy to But the reason why would happen has to do with the oxidization here that it's so much more favorable to form that bond Why would you? Possibly want to do that? I'll show you an example We've looked a little bit about a protein and protein structure. Does this like like look like an interesting protein structure This looks like a shoelace somebody just threw in water or something, right? Looks completely unintelligible You can add all the atoms if you want and it doesn't really make more sense it just looks random But now I'm gonna add show you the cysteines and in particular the solvers Do you see that there is? One two three bridges across there, right? So although this doesn't have any secondary structure But it has a bunch of cysteines that still locks the chain together and that means that this is actually a stable fold It doesn't look like it This is a toxin from a Taranto life, I work with correctly. It's called Hana toxin. It's actually has a fun story It was Kenton Schwartz to discover this Then it couldn't come up with the names he named it after his daughter and it apparently made his wife furious First but ten years later when the daughter is 15 years old was 15 years old. It's pretty cool to have a toxin named after you So why would you have that So this is a type of protein that should be super stable, right? Because it's course in a very toxic environment the toxin itself might try to destroy other things You want something that is difficult for the the other animals and Antibodies or something to break down so you literally want something that's so super stable that no amount of Unfolding or hydrogen bond or temperature should be able to destroy it And that's usually a characteristic of the things like toxins that they should be hard to destroy and disrupt And here too we can fill it in and getting all the surfaces and everything and here too you might see this is the reason I actually had that link to VMD. There are multiple different ways to visualize a protein And how you should visualize depends what you want to get out of it Do I want to I actually I really like to just see the backbone potentially disulfide bridges because they're just Understanding the topology is much more important to me than seeing all the details or seeing all the atoms If you look at these I would say that the far least instructive But that is the least instructive one for me because you can't see the forest roll the trees. There's too much information The other amino acid I spoke about was proline Proline is actually not an amino acid. Everybody's been lying until the formula proline is an amino acid And if you're a chemist that is a really important distinction But we are physicists, so we're gonna call it an amino acid The big difference is proline is that it doesn't have the nitrogen there is not free And that means that it can't form as many hydrogen bonds for a any particular that means that it's it can't sit in a helix Because in a helix you depend on the hydrogen bonds being formed by every single residue So it's simply this loop this five-member ring here takes too much space This you can use if you're trying to predict secondary structure if you don't know what it is and suddenly you see a proline in the sequence That is an exceptionally strong indication that there can't be a helix here Or if there is a helix and you wonder how long the helix is the second you see a proline the helix will stop So this is something 20 years ago. We used information like this to try to predict protein structures What do you think we use today? Deep learning so there are so many proteins known So we just train computers on available structures And it particularly used the fact that we have thousands or tens of thousands or hundreds of thousands of related sequences And that makes it possible to see very very weak patterns, but Lucy will come back to that in the biofumatics lecture And the final promise the finalist amino acid I'm gonna measure it's tryptophan tryptophan is a very large and bulky amino acid So it has one five-member ring and one six-member ring where the five-member ring has an NH group It's slightly polar actually and they this is a completely hydrophobic ring This is so large That it's almost like two amino acids and in particular this large and non-polar thing is definitely not going to be a water So that's something that you would like to turn to the inside and this was a there was a famous paper in 2002 it says there when they Managed to create an artificial protein So it's because at some point if you just take two three amino acids and pull them together in polypeptide chains At what point does this start to be a protein? If one amino acid is one amino acid you can talk about two amino acids and at some point If there are multiple peptide bonds we like to call it the polypeptide chain But one this when does a polypeptide chain become a protein? So first it has to fold into something well-defined not every sequence will do that Just because it's just like any random parts won't form a car But the other thing that we like to say that it has to fold into something well defined But there is some sort of buried interior that does not have access to water So this TRP kates this super small protein in the bulb. It's under 20 residues Where the buried part is really a big tryptophan residue here And I'm gonna show you a small movie made by colleagues of ours from 10 years ago And this can be fairly fast when you see this starting from a stretched out chain We're now going to see that you don't sir. There's actually water all around this But I removed the water because if I showed the water here, you wouldn't see the protein So let's see what happens when we pull this in water This takes a few microseconds so that this is still collapsed a bit but there isn't really any stable structure and at some point you see that starting to turn to the inside and I think it will flip out again and then it will flip in and then eventually we're gonna form some sort of Do you see that it keeps flipping in and out? It's not really sure whether where you want to go and I think that's the stable state So this is characteristic of how proteins fold and very much related this Leventhal paradox that it's it's kind of searching see my randomly, right? But the one thing you did not see you did not see a single frame where the entire chain What stretched out from the left all extended to the right because that is just one single state and the likelihood of seeing that is exceptionally low Then of course the second we start to form some favorable interactions say that we turn the hydrophobic residues to the inside That is a very favorable change in free energy Because now basically the hydrophobic effect right like turning the oil drops to each other the second I've done that It's very unlikely that I will take a step back Formally theoretically I can but again the likelihood that oil will spontaneously spread out in water The likelihood is so low that in practice in chemistry. We say it doesn't happen Which is a lie in physics it can happen But that also means that based purely on probability If there is some sort of built-in arrow here that we tend to reflect you eight We diffuse back and forth But in general there is a net progression towards the folded states and that's what you saw here, too But then occasionally we realize we were heading in the wrong direction and we unfold and we refold again But the closer you get to the folded state The more favorable interactions we've formed and the less likely it is that we're going to disrupt all of them and head all The way back although it does happen now of them. I Spoke before about polar charge residues my only reason for repeating that here is it's important that just as Hydrophobic residues will turn to the inside all this polar residues will be seen on the surface And I also mentioned a little bit how they stabilize and versus the beginning versus the end of the helix and I also mentioned that they Can change the charts. I'm not going to spend time on that. It's not so important so I Think that's oh actually there is a small part what you're seeing here This is a small movie of one of our iron channels It's a very small part of this iron channel where we're actually seeing when we're changing the pH value That changes the chart state of a few amino acids and when we change this chart state It will actually cause the entire channel to close here And if you want to try this at home tonight You can actually have a glass of wine or two or a beer just for the sake of science, of course But just to confirm that this works in your bodies, too It's exactly the same processes although I think the effect with the beer is going to be the opposite that you will open these tents so the net Outcome of that. I'm not don't worry. I'm not going to ask you to learn this table by heart, but The take-home message from all these proteins amino acids tend to occur in places where they stabilize protein structure Or conversely protein structure We tend to form the structures that are stabilized by the amino acids So which is it? Is it the amino acid that causes the structure or the structure that causes the amino acids? We touched upon that in an earlier lecture So in principle, it should be easy for you to say what it is Sure. Why? So yes, so here's the point you could argue that if I have a large protein and it at this and at certain position Say that alanine should be the best one, right? And let's say that all your genomes have alanine, but then one genome has to try to find there is that So that individual but that's just one amino acid It can't really be that unfavorable for one individual to have a tryptophan in this protein They should still fold right they likely will fold actually, but if you just make one amino acid the only problem is that For everything that tryptophan can do in that position alanine will likely do it better For instance, if it's well alanine tryptophan is a bad example. Let's say that it's on the surface and we replace Arginine with an alanine so arginine is better. You can put an alanine there It's not an extremely hydrophobic residue, but it's slightly worse than arginine And the problem here is that through billions of years and every single individual having an alanine there I can do everything you can do but I can do it slightly better And it always creates a small advantage and this small advantage will be amplified due to natural evolution And then you can argue that at some point there is a second random mutation in this protein Now the individual that has two mutations will die because that protein won't be able to survive But the other individual that already had that already had arginine they can withstand one more mutation So there are these very very small and mild effects But due to natural evolution nature is fairly brutal in the sense that it weeds out anything that is not optimal and From that point of view that unless an amino acid stabilizes a particular structure There will always be random mutations and many random mutations will sadly lead to the fetus dying because it's not viable But in nature will constantly try trial and error in every single position It's in your bodies too I bet that you have a bunch of mutations that are more favorable than either of your parents and conversely you probably have some mutations that are less favorable And this will go on through generation and generation and generation and the likelihood of you having less offspring is of course It's a minute of a it's epsilon right but epsilon multiplied by a million generations starts to become large numbers So with that I'm going to spend a little bit talk about fibrous proteins They're not quite as I've it's I'm fair to call them boring and these are the structural building blocks in your body They're less specific and the reason why they're less because if you have to repeat them and nature runs out of imagination Because you need to repeat them until you get structures of macroscopic Extension as nails fibers hair shells claws these things right so that they can be millimeters or even centimeters of size And you can't have a protein that contain a billion different residues So you need to have those more just as we had alpha helices and beta sheets We need to have building blocks here too that we repeat And There are frequently lots of hide-and-go's one of the simplest ones you've touched silk silk is virtually pure beta sheets So beta sheets are smooth and nice. So it's roughly 80% anti-parallel beta sheets lots of glycine Can you imagine glycine being related to something in silk? It's an extremely smooth and flexible structure, right? So these are again, they're they're far better than they look together There is also it's packed in hydro every single side is hydrophobic in there The one is hydrophilic, so you tend to have hydrophobic hydrophilic hydrophilic hydrophilic That also packs them very well If you head out into a an expensive Hairdress or something you can probably buy. I'm not sure whether it's so popular But a few years was very popular to shell sample and everything that they contain silk protein And then I guess they put that on the label to be able to charge more for it Silk protein has nothing whatsoever to do with actually well It does have to do with silk, but it's not extracted from silk silk protein is just a protein engineering and synthesize amino acids like look like this and then you add it artificially completely artificial It probably it costed it Probably a few dollars per ton It's dirt cheap But of course if you say a silk protein on the bottle you can charge a couple of dollars more per bottle But it absolutely nothing to do with real silk apart from the high good So that this is almost like a crystal beautiful and repeated and everything is a super simple structure Collagen is another structure that you might not have heard. This is a super special helix And I don't expect you to I don't expect. Let's see. I think I can move. Yes I can move this is a very special with lots of proline, so it doesn't really the individual Amino acid chains here. They don't really seem to form anything that stable But when you put three of these together, do you see that they're forming hydrogen bonds to each other and waters? So this leads to very long. You see it's glycine poly it's tons of poly in it So normally wouldn't form anything that stable, but in this case it does This is roughly a quarter of all the protein in your body all the boring stuff bone teeth skin and everything It's kind of important to have. Yes Yes, all these fire all these large fiber our fibers and that's it they have macroscopic extension and it's typical They might have three to six amino acids that are repeated So they need to be small because otherwise if there was a billion residues here It would take forever for these to fold and you can imagine the amount of these proteins that your body needs to Produce just to keep up with the turnover, right? So they have to be small and simple to be able to produce them, but then we're gonna need a ton of building blocks Simply to reach macroscopic sizes So they start to be fairly long there can be a well millimeters long There is They in turn these chains in turn will form an even larger chain here. So it's super chain. I think I can move that too So here you see this triplets of chains they're now paired I think it's into six or nine chains here So do you see this pattern again you have first have amino acids and then they form a larger structure three chains now These chains can be seen as a sort of super chain the super chains We pack nine of them together Because again nature doesn't have a whole lot of imagination and we can't have because if you had too much imagination It would take forever to build it so simple building blocks that we repeat and repeat and repeat and Once you get to this size you can actually see it in an electron microscope So this is a dentine five bills and in your tooth if you take Your genome and mutate the glycine there remember what was the property of the glycine? Very flexible and we need that to compensate for the polines if you mutate that glycine to x and x is a way of writing any Other amino acid if that glycine is mutated to anything. That's not small and super flexible you get brittle bone disease So then these structures are no longer stable and that's when you're that but you well bone breakages from your teeth fall apart and everything There's a very minute. It's basically why it's basically one amino acid in one gene. That's changing And I say because it's 25% of the protein in your body, right? There are similar effects with normal alpha helices Alpha helices are we this far we've usually only looked at alpha helices one at the time You will see a coil coil helices that you can take two alpha helices and then have them twist around each other like two arms Like that. I will show you a better image of that in a second This is exceptionally important in fibers for instance in your muscles in your muscles fibers You have actin and myosin so that you basically have one fiber and then basically Gripping another fiber and walking along this fiber. You can you can find movies on this online I should have borrowed one of those movies and showed you here, but I forgot But the way muscles construct is literally you have two of these fibers Literally moving along each other and that again it happens in a millisecond I don't well. I know how it works, but I'm still amazed that it does work These helices are Frequent as you remember. Do you remember how many turn how many amino acids that was perturbed in a helix? 3.6 usually there's 100 if it's an easier way to remember that is one every amino acids is a turn of 100 degrees You can look instead of looking like he looks that this way you can look at them from the top and if you look at these from the top What if we take this a prime and D prime and the A and D residues these residues A and D if we make them hydrophobic Then they're gonna love to form pairs with each other, right? The only problem is with 3.6 turns there's gonna be a slight offset every turn So that but if you take these helices and twist them just a little bit harder, so it's 3.5 turns That actually means that we can get them to line up perfectly in the middle So when you get hydrophobic residues, they're gonna love to pair up while you turn all the water liking residues to the outside We will see more about that in a second And that that might not seem advantageous there but the reason why that is so great that again if you look at the molecular structure of these helices Here it's much better to look at the surface You can literally define these ridges edges and then depressions between the side chains So the side chains sticking out will be either the solid or the dashed lines depending on whether and then there are depressions between them So if you now take two helices like that and turn them against each other And then you will have to rotate them a bit You're gonna get all the edges dipping into the depressions of the other helix and they will pack perfectly This was yes, so sorry here you can see it So you take one helix you turn it around and then you rotate it and then you see that how they line up And there is pretty much zero space here This was predicted by Francis Crick in 1953 pretty productive year for him He predicted protein structure and DNA structure but apart from that Why was this just as cool as the DNA structure? Remember when they for when they determined the first protein structures a few years later, right? So again, this is the cool part Finding this five years later after the protein structure no big deal predicting it before the structures are out very big This occurs in a very common Protein called alpha keratin that you might or might not have heard about so here you see two Helices called up what I marked in red here are leucine residues So leucine is a hydrophobic residue just like I mentioned And if we place them with roughly seven so in this case I don't do 3.5 But I do it every two turns so I placed every seventh residues a little leucine or at least hydrophobic Do you see how they're gonna pair up along the helices? I will need the helices to twist slightly So there is even a name for this leucine zippers So when you have these folded that the second the second they say the two lowest leucines here are in contact I have pretty much forced these to be at least very close to each other And now they're gonna probably gonna jump together and form a hydrophobic contact But that in turn will cause these pairs to be relative close to each other And then they're gonna form a contact there, too So it's just literally just like a zipper you're pulling up This leads to very stable structures and the second they've coiled up like this. They're never gonna release again You might also have a fair number of residues being cysteine and then you might even have disulfide bonds between them And then they will be even more stable Can you imagine where this occurs? An important part of your body Hair, this is hair And it's more again, it's not as simple as that because that so you take the alpha keratin helix and then you form pairs of these helix But this helix is still tiny right we're talking about something that's a few nanometers across So then we're gonna need to form a filament of maybe eight such pairs of helices then we're up to 16 helices We're still talking about 20 angstrom at 20 nanometers or so that's still small So then you need to form a matrix and a macro fibril And you're just pairing up more and more and more and more of these helix at this point You might probably have a few hundred or maybe a thousand of them and then eventually you get up to the point where you have Real large extension you might read the extension say 0.1 millimeter or so and at that scale It's actually what we have in the hair and if you've ever Well the problem being there and if you create a permanent wave in the hair, what is that? Permanent Swedish so that you can do chemical treatments in the hair to make it stay in a certain shape, right? And there's a two-step process can you imagine what they do given what I said in the last slide Systeins so you first add a chemical that reduces all the systeine bridges in the hair And now you give the hair is very floppy and then you form the hair to the shape you wanted to be it Actually can form it first and then break this it But then you add another second chemical that reforms the systeine bridges in the shape you want them to be and then you create To the hair that is you now get the dial the systeine dial sulfide to lock in in a new structure the structure that you prefer The hair to be in and the permanent wave in the hair that might be a fun parenthesis But this is used in a ton of other features too actually Hair grows by roughly 10 turns of alpha helix per second So from that you can even use that to get a rough correlation with the folding times of alpha helices And it's going to be in the right ballpark compared to the theoretical reasoning you can do There are plenty of other places where you use this for instance If you're going out and buy a shorter thing is very common with iron free products, right? Iron free products works the same way. It's not really disulfide, but there too you can Basically adding chemicals that first releases the hydrogen bonds and then forms new hydrogen bonds so that you have a structure That is for very simple biotech, but it's all based on things that are related to physics and free energy I think this is the final example. I have it's one called elastin And that's an very elastic fibrous protein. It's roughly similar to collagen, but it also for its very long matrices This for it's very common to say you're a work tower any type of blood vessels or anything And there's a problem here because as we get older these becomes more Brittle and everything and if you get an or to aneurysm you pretty much die On the other does look entirely easy to transplant things like this too So what you normally do for instance if you have a heart attack or something you tend to take a vein from the leg or something But in the future we might actually be able to do artificial material and there's a lot of research in this case It's an elastin biomaterial or to here Which is completely different from the stuff that they've been trying to do it's a karyliska with Paolo Macerini and everything So they're not not just based on what you can but if we could create artificial materials We could make them way more biocompatible because the problem is that your entire body is trained to get rid of anything We put into it any metal or anything or it's even worse if you have any say a heart Donated heart or something your your entire immune system will recognize that this is a strange Individual that we should try to get rid of we should try to kill this heart And then you need to take suppressant rags for the rest of your life But if we could artificially construct proteins and in particular make sure that they are compatible with your immune defense They're not recognized as foreign material. This would basically be an infinite toolbox of spare parts for the body I think that's all I'm gonna say and oh, we're doing great on time here So I'm gonna spend a little bit of time talking about the globular proteins the rest of today Globular proteins are much more fun than the fibrous ones and they're colorful beautiful pictures And I think I'm gonna start to speak a little bit about the beta sheets Here too, there are a ton of different ways we can draw them and I'm not gonna interrogate you in different There are lots of different structures and again It depends on do you want to see the large-scale shape? Do I want to see the surface? Do I want to understand say the binding sites? Or do you want to see this in a more schematic level? Do you want to understand what are the helices? What are the beta sheets? In this case if you saw this as space filling you probably wouldn't even see that there are beta sheets here and helices there, right? So here By drawing in this way I can see that all these beta sheets are parallel and then there are helices on the outside So you have one sheets go down and then the helix going back and then the next sheet go down and the helix going back And then the sheet goes down and the helix going back, etc And if you really care about you could do it to pull it your cartoons, but they're not very popular anymore So I would if I were you I would offer so much about that One of the simplest structures here is beta sheets actually because beta sheets remember with that they have they actually they look like They're super complicated. They look like something like this, right? But forget about all those loops. The important thing here is to look at the shape So you see a sheet there sheet there sheet there sheet there and they all had it and bond together Yes, they just like a paper you can twist it a bit and they usually have to be twisted due to their geometry But the pattern here is the important not the exact shape of the loop between them Same thing here You have a sheet sheet and there too you can probably see that it's gradually twisting as you go out The reason for this twist has to do with the Ramachandran diagram It's a very close to 180 degrees, but it's not exactly 180 degrees If the if the amino acid preferred to have the peptide bond and the alpha Sorry in the five-side torsion in exactly 180 degree would be flat, but in practice like 178 or 175 So if the structure is pretty much that you have sheets of paper What is the simplest structure you could imagine well you could imagine having one sheet, right? But that's kind of like trying to build a house and only building one wall It might look beautiful and it's certainly a wall, but one wall. It's it's not particularly useful because you can always walk around it So there too, it's Just one floppy sheet. It's not going to do anything in your body So that the easiest thing you can imagine is pretty much two sheets and Then there are only two ways to put them you can put them orthogonal to each other or parallel to each other And that's what nature has done So again, if you look at this on the atomic level, you wouldn't see the tree at the forest for the trees But look at that schematically. There are only two simple ways to create very simple proteins Both of these are used The this beta cylinder is something called fatty acid binding protein I'll come back to that in a second and this is an immunoglobulin that occurs in your immune system The reason why why would you imagine it could be useful to form these two layers? I'm kind of cheating it. It's a little bit more than two layers There's two layers, but I have some things on the side, right? So you can not sure whether you can see it But if you believe me for a second we can kind of prop things up to the left and right and top and bottom So you actually have an inside and an outside and I agree it's perfectly visible here So you'd remember property of the beta sheets that you turn the residues they alternate They point to one side of the sheet and then the next rest you point to the other side and then you're back to the first side So if you take any sheet They will look like this if you put one rest say if you put hydrophobic residue here Hydrophilic residue there hydrophobic hydrophilic hydrophobic hydrophilic hydrophobic hydrophilic That's going to give you a hydrophobic side and a hydrophilic side Just like we had for the silk protein If we now take two such proteins then you can Well, if you have an hydrophobic and a hydrophilic side and you put this in water, what would you imagine it's on the inside? The hydrophobic part will be on the inside so we can turn the water like the rest used to be outside So that's like a small pocket Let's see. Do I have a No, I might not But this small pocket can actually bind a small fatty acid that has to be transported elsewhere in the body Why would you need to bind a fatty acid to transport it? Because the fatty acid is not soluble in water, right? It's fat. It's oil and just when you're boiling pasta If we want to transport it we have to transport it through the blood But by binding it to this protein, this is essentially the same thing as Detergent if you're washing clothes or something we could take the fat bind it on the inside now It's water soluble. We move it to the place where it should be and then we release it And this is our large part of the fatty acid transport in your body works I'm gonna just gonna give you one more example of this and this is a protein called gamma crystalline And that's what your eye lenses are created of Also very simple protein and you have a bunch of loops up here And these loops are frequently created to somehow try to close the interior or something super simple small structure Do you see the pattern here? So that I have a small loop That goes in one direction and then a long loop that goes in the other here too I have a small loop that goes in one direction and a long loop that goes in the other This was discovered by Jane Richardson in 1977 and it's called a Greek key and I'm gonna give you a break in 30 seconds here And this is exactly the same type of pattern you see in a Greek urn or something and the reason why this is common It's a pattern that you can you can draw it with a pen without lifting the pen Which is exactly the same properties of these patterns, right that you don't have to lift the pen But that sounds fairly stupid. It's not like we're drawing proteins. So why what would the use be? So we want these fairly long loops because if we only had the very short loops You could imagine just going up down up down up down up down and having short loops everywhere But if we only have short loops, it would make it very difficult to close the protein at the top and bottom So we want some of these slightly longer loops to have enough material to close things so that we actually create the pockets That is excluded from the water So there's a bit of a parenthesis, but it's one of this it's classical result And it was I even think there was a Greek urn on the cover of nature in 1977 when they published it So this if you heard your word Greek key, that's gonna be it This is a good point to take a break. I Stole two minutes. So let's meet here at 17 minutes past 4 p.m. And then I'll try to finish and let you go five minutes early today Good, I will continue the last 40 minutes here. So I spoke about gamma crystalline And these Greek keys. Oh and they actually do have a small movie of the fatty acid binding protein So here you see the small fatty acid bound inside the fatty acid by a binding protein An x-ray crystalography structure of it For this protein to you can almost see that Well, actually here there it's not a Greek key But you see that you're fairly long loops here that try to close it up a bit, but might not be a great visualization so the common part of this is that there are I Think these is way to sum this up is that you need stable structures to form stable structures We need amino acids that stabilize those structures But that in turns means that amino acids will occur in the places where they stabilize and favor the structures So again, you have this mutual relationship that Amino acids create the structure But the structure will also create a bias of what amino acids mutations will tolerate then there are some things that are rare and Most of the things that are rare are rare because they're not disadvantages And if they're not disadvantages, they tend to be weeded out by evolution with a few exceptions Because in one one protein a hundred it could be something that actually is good and one such example is that Proteins virtually never contain knots so you can think of proteins are topology, right? That's looking at a very high level and in general a protein can never take let's see we start from blue You normally start calling the n-terminus as blue and then we go to light blue Life blue and you see how this protein continues here is green and then yellow But then it actually goes inside the green loop here and the red and out there It's literally a knot if I were to take the blue and red parts and just pull them I would have a knot here that can't happen The only problem is that in biology in contrast to physics to every rule there is an exception And this is an exception. This is a small protein called pepsin Pepsin occurs where you could almost guess by the name It's in your stomach So this is a small protein involved in breaking down amino acids Breaking down large proteins into amino acids and that's important because that's how we get the building blocks for all our proteins You can't synthesize amino acids all the amino acids that we use to build proteins are what we have eaten in terms of food But the problem is now that now we have a super acidic environment and everything that the entire the point of this entire Environment is to destroy proteins and in this environment. We need some proteins to help us destroy proteins Those proteins had better be super stable, right? Because otherwise we're going to destroy them too And I don't know exactly but I would imagine that that might actually be a reason why we created pepsin like this This possibly makes the protein exceptionally stable and harder to degrade. It can't just unfold as easy So the pH in your stomach is basically pure sulfuric acid. It's exceptionally hostile environment And you can continue there are there are lots of examples where there are things that virtually never occur There are there are a few things. These are common structures. There's called Greek keys You have this classical what you call beta meanders And the meander is the concept that the rivers meander when they go back and forth through nature and that can occur in beta sheets But these are pretty much the only three common Confirmations you have and for the rest of them we hardly ever observe them. We don't really know why they're just also advantages So natural selection has killed them I already spoke before when I spoke about prions and everything that there are cases where you can have multiple beta sheets in particular form pairs of structure Super important if you want to form large things. Can you imagine just the second these two proteins are just close to each other? I would bet you would likely have 10 hydrogen bonds between them here and the second you form those 10 hydrogen bonds They're never gonna. Let's go again. And with that I think I'll wrap up the beta sheet structure I realized there are lots of structures here, but I want to give you somehow a gut feeling for the diversity of nature And I promise I'm not gonna ask you to you will only have to learn how to be structured by heart. No It's the concepts that are important here So I'm gonna go back to Alpha Helis in a second But beta sheets are really characterized. You have these non-local hydrogen bonds that I mentioned This part of the sequence is not necessarily close to that part of the sequence This could be a rescue 1 through 10 and this might be a rescue 320 to 330 So there are lots of other parts here and that means that it might take quite long for them to form The strands themselves are quite flexible and then you form a ton of hydrogen bonds between them So once they form they tend to be stuck and there are lots of constraints the second you form these large sheets They can't really move a whole lot and Alpha helix on the other hand there you also you have roughly the same number of hydrogen bonds But here all hydrogen bonds are local so they form a hydrogen bonds with the rest of you four units further up in the helix So that actually makes the helix itself is very rigid But it's only rigid in the sense that it's isolated this part forms a rigid element This entire helix is fairly free to move around because it doesn't form hydrogen bonds with other parts of the structure Multiple helices can pack but you don't form hydrogen bonds between two helices at least not a whole lot of them So in contrast to beta sheets where you have these fairly large extended sheets Without the helix's we have a helix and then we pack many helix's and that's slightly different To might be a movie I'm gonna skip that in the interest of time Depending on how we pack them then you can add like well you might have a hemoglobin carrying oxygen in your blood This is a small ion channel Lucy will talk more about that on Monday And this is a small protein called hemorrhytrim. I can also buy an hemoglobin and There are in principle similar there the helix's tend to be roughly parallel or they form some slight angles to each other But you would probably agree that they are They're a bit harder to classify exactly what the differences are I'm not sure about well, I can say that that's a membrane protein and that isn't but beyond that It's a bit harder to say whether they are orthogonal or parallel or so and just to show you more of this diversity Let's look at something very simple. We can take Four simple here small proteins. They all consist of four helix's But they're gonna have very different functionality Cytochrome C the tobacco mosaic coat virus protein and the hemorrhytrin binding oxidant protein Which one of these do you think is most stable And which one looks not very stable the middle one looks strange, right? That's like it looks like a bit of a shoelace thrown around That's the most stable one Anytime you see things in nature, there is usually a reason And so I didn't say that it but in this case when they are these bundles They always go down and then up down up down up. So two adjacent helices. They always go in different directions The one on the left here cytochrome seafolds We worked with that when I was a postdoc at Stanford These are very common in there in the electron transport and they can bind heavy metals And at the time we studied a whole we were interested in understanding how simple proteins fold and understand going after these very small structures is Interesting and lucrative because with four helices that's simple enough that I can almost simulate it in a computer I can least predict it much easier than the large proteins So when we started this with bioinformatics methods, it turns out that one of them Organisms that has by far the most cytochrome seafolds or cytochrome domains is a small bacterium called 7-ela on a denses mr1 And that's a heavy metal bacterium Not so much in the music sense, but it definitely likes to bind them We had a we've been had a grant from this from DARPA, which is the US defense research agency We lost that grant two years later. Unfortunately due to the rock war that happens in the US. Suddenly they realized that they need the money for cruise missiles is that Why on earth with the US defense be interested in this So this type of bacterium are possibly candidates for binding radioactivity Because radioactivity is frequently heavy metals like uranium and things like that So if you could somehow get the bacteria to just spit out the bacteria the bacteria would bind the heavy metal from the surrounding And then we could somehow dispose of or collect the bacteria in another method You would potentially have a way to clean up radioactive contamination Which is sad, but important for the US military apparently Tobacco mosaic virus was another classical discovery If you're growing tobacco this TMV as you can see it as more these more black deposits on the leaves And that's absolutely horrible because it's the leaves you're interested in you're growing tobacco So tobacco growers they lose for 50 well hundred years. They lost billions of dollars on this and When people started doing structure biology and had electron microscopes They could take the tobacco leaves and then try to isolate this black compound and then you can magnify it So this is a there's probable part of a few hundred nanometers or something and then you see these rods eventually literally in the microscope And if you amplify it even more You get down to this roughly 50 nanometers and the rods turn in some sort of material like this This was the first virus to be discovered in the 1930s So what does the virus consist of? Do you remember that from a secondary school? Not so much the membrane but RNA So what does I would I would actually Viruses is the most important the most beautiful discovery in nature is by far You can argue whether it's a life form or not because it's not alive It doesn't really have any turnover like a cell or anything, but it's pure genetic material, right? And it's not like the DNA itself doesn't have any any intelligence and it doesn't have any energy So you take our pure RNA And then the point what is the goal of RNA? Yes So i'm sorry to tell you but we are just a fancy way for RNA or DNA to replicate itself Without us our every DNA can replicate itself But the problem with RNA what did I say about RNA when we spoke about nucleic acids? It's fragile, right? It will break down spontaneously in the lab. We even need to put it on ice So if you just had the RNA in isolation, it it would very rapidly break down and they could not infect cells So you need something to protect the RNA around the virus and that's what you call the coat And that's typically not the membrane But via the protein So what does that RNA in the virus code for? Yes Yes, but what RNA has to code for something, right? So what does the virus has to code for? What proteins does the virus had to produce? Yes, so the RNA only codes for its code protein it only codes for itself And that's the reason why it's such a beautiful form is that you pretty much just have one protein you code for So the tobacco mosaic virus Just codes for one single four helical bundle and that's the bundle you saw And then this is repeated again and again turn off the turn off the turn And that's why you need this almost triangular shape that if you just looked at one of them It appeared to be disordered and everything But you need a protein that is very thin So that's why we can't have helices all the way in and then further out Just slightly more room and then you see you have thousands of these formed into these very long rods And then you have the RNA spiral inside here protected This was sold by Rosalind Franklin in 1958 I think I told you about this Slightly less noble way that Watson and Crick kind of borrowed her results It was this really fun meme posted on facebook the other day about the class Where apparently the teacher had asked the entire class So what did what did Watson and Crick discover then somebody says from the back of the class Rosalind Franklin's notes? So there was actually a third noble a second noble price in all they do that Wendell Stanley crystallized the tobacco macrosic virus in the 1940s something 46. I think it was and it got noble price for that the third example Actually, it's not too much for a little bundle, but it's related to the hemorrhage this is One of the hallmark globular proteins mainly because one of the first proteins to be crystallized hemoglobin And you probably know that this is the protein that codes that binds oxygens in your cells Hemoglobin is a bit of a complicated protein It actually consists of four molecules that are identical and each of these molecules as one of the heme groups or proto porphyrin bound To which you can bind an iron and that iron in turns binds oxygen As a bit of a freak of nature The two first proteins that we Determined the structure of where hemoglobin and myoglobin and myoglobin is pretty much exactly the same protein But just one subunit Do you know what myoglobin does? Sorry Myoglobin also binds oxygen, but in your muscles So why do we need two different proteins? That seems like a waste So this is a famous result by monovime and on changé. Uh, the problem here is that Hemoglobin do you want hemoglobin to be good or bad at binding oxygen? Who think it should be good at buying oxygen really strong? What would be good with that? There is something that would be good with that Well, in particularly you could at some point they you are in your lungs, right? And you need to take oxygens from the air and bind it if you're not good at binding oxygen You're not going to be very efficient at taking up oxygen and that's bad, right? So if you can bind oxygen really strong, that's great You will be able to take up more oxygen and have a higher oxygen uptake This is even something you measure in in athletics for instance demand v o 2 how much oxygen you can But the problem is the second you're out in the muscles Now you have the oxygen bound very strongly to hemoglobin You're not going to let go of that oxygen So you now you have the oxygen and hemoglobin, but that doesn't help because the oxygen doesn't do anything until we have it in the muscle So nature has solved this with this force of units. Uh, so what happens is that This molecule can exist in two forms, uh, what you call a tense and relaxed states. So normally When we don't have any oxygen bound it occurs in the state that is relaxed And then you start binding and when when you start binding a little bit of oxygen one of these subunits starts to move over So as you're binding the protein changes its structure a little bit And when it changes the structure a little bit, this increases its affinity how strong it can bind oxygen So this means that in regions where the oxygen concentration is high where I start to bind oxygen That's kind of like kicking me awake. So then I become even better at binding oxygen So then the second subunit starts binding oxygen to Now the remaining two subunits really really want to bind oxygen because they're left out otherwise So now you're binding a third and a fourth oxygen molecule two and that occurs in your lungs So that's we have lots of oxygen pressure in your lungs. So there we take up a lots of oxygen now you take this molecule and Transport it out to my muscles and the muscles. I don't have a whole lot of oxygen So that the first oxygen molecule will instantly be released But now I started to release oxygen. So now the others that's kind of like kicking them to sleep instead So that guys you too should let go of your oxygen because it's not very good to bind oxygen here So suddenly where you know out in the muscles the hemoglobin will It's like a Janus face that it's now going to change it completely You know or second thought I don't want to bind oxygen at all here. You can have it all myoglobin And that's how it works. You have high a molecule that has high affinity for oxygen in the lungs But low affinity for oxygen out in the muscles Pretty cool for just a few alpha helices, right? So each of these small subunits contains roughly six small helices that forms a binding site around this heme group And you have a histidine and everything that binds to the iron here I'm sorry, and I just explained a little bit about the binding between hemoglobin and myoglobin Myoglobin only being a single monomer It doesn't have any of these things because myoglobin is only in the muscles Myoglobin should never enter in your lungs. We don't want myoglobin anywhere near the lungs So if we now look at these four helices, uh, there are different parts of these structures and everything Remember that I said before about introns and exons in your genomes That there are lots of letters in your alphabet But not all of them code for amino acids and proteins If there are now five six helices here And then I will show you a genome again if you don't remember it In the DNA exon x stands for express and in here is inactive or something There are these strange regions They don't seem to do anything and we don't really know what they do The other strange thing is that these only occurs in higher organisms vertebrates bacteria I don't have introns bacteria. I'm sorry to break it again But if you think that you're a fancy organism bacteria are far more beautiful than you are They're far more efficient than we are We're basically we're the old model of a car that should have been replaced a long time ago and bacteria is the latest MacBook or whatever So can you imagine what these Express units correspond to? It would make a lot of sense to assume that they correspond to the secondary structure elements, right? And it's completely wrong In hemoglobin you see that they the first one is the red the second is the blue and the third is the yellow It has no correlation whatsoever to the secondary structure Because the intron parts they are cut out by gene scissors far before we even start to fold the protein And for a very long this has been a very intense research topic the last 20 years because we we obviously know Again, nothing in nature occurs by chance Those introns must be important But we haven't really known what they do and there have been lots of hypothesis There was a really cool paper that appeared roughly four days before we started this course In nature where Abou Lea's team showed that introns appear to be mediators of cell response to starvation And that's okay. So what happens with the bacteria when they starve? They die Or they simply don't replicate bacteria replicator, but bacteria is Bacteria is an extreme form of colony in the sense that the individual is not important, right? But for vertebrates and everything one difference that we are much better at adapting to changes in our environment Now that's good for us. You could argue for nature it might be more efficient if we just died And then there are new humans instead But this is a key difference in vertebrates that you don't have in bacteria and what they showed here is that by Doing fairly simple experiments and trying to cut out the introns and everything They can show that the the organisms are no longer able to respond as well to starvation And I'm sure that this is not the last story of it But just to show that the cool thing with all of these things is not that it happened recently It's happening as we speak. Uh, there's literally a paper that's four weeks old If this turns out to be right think of their ability to be a noble price in the future, but it's going to take 20 years for that Uh, I think I already spoke about those helix ridges and growths I'm not going to go through that in the interest of time, but I will go through this Remember the thing I mentioned that if you had two helices And if we put the hydrophobic residues on the inside they're going to form something very nice and stable And this is related to the hemorrhage thin and everything that I was the third example protein I showed you this works not only for two helices, but it works for three helices And if you can take a wild guess that it might actually work for four two, you are completely right So we can take four helices And then we somehow turn all the interior parts here into some sort of either hydrophobic Well to first approximation, let's make it hydrophobic But let's add just a few residues that combine say a heme group Now we have a small protein that is hydrophobic on the inside Hydrophilic on the outside to soluble in water it can bind a heme group. What can the heme group in turn bind? We just went through it three slides ago. So unless you were sleeping you should know the other oxygen So what could you use that protein for? You can imitate the oxygen binding properties of blood, and this is used not Again, this is still very heavy, but you can use it to try to create artificial blood And that is being sold in some parts of the world for two reasons At first there are some religions where people absolutely don't want to receive blood donations That's probably more of a parenthesis in the grand scheme of things But there are plenty of cases where it's simply it's difficult to get blood Human blood has all the problems with different antibodies Not to mention diseases like hiv and the Hepatitis c and everything that has spread from blood donations historically So if you could create artificial blood even it's not at all going to be as sufficient as our human blood because they don't have the fanciness of hemoglobin with the multiple states But again having something that binds oxygen so that you survive is way better than not having it You can use it for slightly less fancy things and imagine if we take four Helices like this and we forget about the hemoglobin But we take something that's water soluble on the outside and then we make them Fats or oil soluble on the inside so we can solve it a bit of fatty acids there What could you use that for? we could Get oil and put it in water right? So why what why would that be useful?  It doesn't But if you take this type of proteins if you know either but you can have it does contain fat right? But you can have fat in water so you can mix in a much larger fraction of water in the fat By having these so called there is actually emulsifiers And you might have seen what happens If you take at least some low calorie food products low calorie margarine or something and you try to say you try to stir fry in it It used to happen 10 years ago at least Something strange happens in the pan. It just turns to water or something You can't fry it So what happens with protein when you heat it? It breaks down it unfolds And suddenly you release all the water right so you now have an oil You have an oil fat mixture in the pan together with some Unfolded protein that will burn So quite a lot of research I think particularly even lund if I recall correctly I certainly try to create proteins like this that are more stable Because if you can make this protein so stable that it can withstand boiling You can suddenly boil low calorie products And I think Arla did that a few years ago. So the latest generation of low calorie products Cream fresh for instance, you can actually boil it And again, that looks like we can't you imagine the amount of money that it was for these companies And you're basically talking about protein design industrial protein design on a very large scale It's certainly possible to now we're talking about pure alpha helix and pure beta sheet domains It's certainly possible to mix them to and there are pretty much just two ways of doing that we can have Either they can be separate So you have one part that's just helices and one part that's a sheet or you can alternate them And when we alternate them we typically write that as alpha slash beta And the only difference is if you alternate them you would have something like this that you would have a helix one strand of the helix Go down and then you need to have a sorry one strand of a sheet go down And then you would need a helix to go up on the outside And then it's another strand of the sheet to go down so that they're parallel Because if you want parallel sheets, you need something to you basically need something to go back to the start of the The other alternative is that you could have some like this alcohol di dehydrogenase When you're here, too, you see that this parallel beta sheets the beta sheets And then alpha helix go down beta sheet up alpha helix down etc What does alcohol dehydrogenase do? It breaks down ethanol So there's a there's a large genetic variations in this all of the globe So a large part of the asian population is a bit deficient in adh And that's why they get very intoxicated. They're not as good as you there I would actually it's probably us. We are a freak mutation Because in the rest in the rest of the world you didn't have to drink that much alcohol But in the cold north it was important to be able to drink both milk and alcohol But seriously because it's it's energy, right? But in the rest of the world it's not that important That's actually that's actually why we have a much higher tolerance for lactose Most other in the rest of the world Again, you stop You stop breastfeeding when you're six months old You don't need it after that But we came up with the idea that we can steal the energy from the cows There are a number of folds like this and I'm not going to go through them in all detail But I think that for historical reasons they're beautiful and there was in the 1970s There was this booming era when we found all these the alpha Just as we found the sequence and the genetic code This is basically the structural code of all the proteins And this is a very really old love story for mine And that's why I have some of the images but Maybe it's not the most important thing to know the folds by heart But the part that is important to know is that These structures are not random And throughout these structures you have binding sites and everything And virtually all these binding sites they they occur on the surface Because otherwise you can't bind things But they also they tend to occur on the edges of these domains Where you frequently have unpaired hydrogen bonds Or you have several helices pointing to a place Where you can really put something charged on top of the helices So if you were to guess where a binding site or anything is Then virtually never going to occur right in the middle of the protein And they're never going to occur right in the middle of a secondary structure element But typically on edges of secondary structure elements The other alternative is that you have structures that contain One part that's helix and one part that's beta sheets So here we have sheet up down up down up down And then you go to the backside there's a helix helix You can also have here a helix helix helix and then a couple of sheets And then one more helix at the end A beautiful protein that is a tata binding protein This small protein This is the protein that finds the genes in your DNA So this will all these beta sheets They contain hydrogen bonds right So that the beta sheets will bind they will recognize certain bases in the DNA And in particular the the reason why it's called tata There's a t-a-t-a-a region And that's where we should initiate transcription That's a new gene It's the start of a book in the library Let's start to read this book So nature does everything that happens in your body cell If you don't know what's done it's a protein doing it So all these proteins and transcription factors everything that really they're pretty much They're just stupid molecules that are more or less diffusing a First they diffuse in three-dimensional space and then they bind to DNA And then they diffuse along the DNA And suddenly they find a pattern and then they bind to this pattern And then that starts a chain reaction where something else binds with this protein And then eventually you split the strands and start scrapping DNA And that has happened a couple of billion times since we started this lecture Uh finally that although these are the most common structures There are a bunch of other structure these horrible irregular ones This is another neurotoxin on Brazilian scorpion In some cases and I think this is one of them The reason why it's so irregular is likely that this protein will likely form structure Once it's binding to my nervous system Which I hope it won't Um now but because if this protein is not very stable itself right But once this is binding to say my channels or something If it can form a more stable structure when it's bound to my channels Again, it's not that's a very strong downhill driving force Remember what I said that we need we have some sort of changes in free energy And assume that it's a minus 50 minus 50 kilojoules per mole Binding advantage if this one is binding to my protein It's going to bind to my protein and it will never go uphill again Because uphill barriers too far And then it's bound and it's not going to knock out my cellular response My neural response Brazilian scorpion I can probably survive Something slightly more severe is cobra toxin I hope Luz is going to tell you about ligand gated iron channels on Monday Or I will have to I will be upset because those are my pet channels Cobra toxin they bind to your ligand gated iron channels and they block them So that they can't open Doesn't matter how much how much ligands you have try to activate them And that literally means that at the end of one cell When the signal is supposed to jump to the next nerve cell The response stops you literally kill the nerve signal Which is okay if it's just my finger or something right But this involves your breathing your heartbeats and everything And that's why it has so great consequences Again, it's a beautiful small proteins I think it's amazing how the animal has been able to do this Which leads to another problem If these proteins are so potent and everything A beautiful example is these poisonous frogs Do you know them inside of America?  Because the frog again a frog also has a nervous system Maybe not as it also has yours But so how would nature do it? So remember what I said that this binds to some sort of protein right And there is a binding side We don't know what it is in this case But forget about that for a second And there are like three or four residues That are important residues for binding this toxin And that's the site that this toxin targets So if you were the frog what would nature do in the frog? Or mutate them right? So the frogs have mutations in these sites So the frog has slightly different signaling in the nervous system So the frog of course still needs signaling But the frog's own nervous system is mutated a bit So that the frog doesn't bind to its own toxins So it can't knock out the nervous system in the frog And it's possibly the same in the cobras So I think it's two residues that are mutated A tiny change And the reason for that is that suddenly it's not advantageous In terms of free energy It would be an uphill barrier to bind And then it's not going to happen at all So it also comes down to the free energy And you might at this point think that this is insanely complicated And in one way it is But there was a famous paper in the early 90s That although we discover more and more proteins There are like more than 100,000 structures known And hundreds of millions of sequences The amount of completely separate folds Is actually surprisingly small The Cyrus Schottje wrote a paper called A Thousand Folds for the Molecular Biologist And let's turn out to be wrong If you think about folders Just the general pattern in which we're doing things There might be 1,500 maybe 2,000 or something But as many proteins and as many sequences we can have And as many mutations you can have in the frog Overall there isn't that much diversity And remember let's say that it's 2,000 These 2,000 completely different patterns of building That's what creates all the diversity in nature From the frogs to the Christmas trees to you And the color of your hair And that nature tends to reuse folds all over the place So that's why you see for the four helical bundles They can bind the oxygen in the blood They can be the coat of the tobacco mosaic virus Or they can be either cytochrome folds So there are things that are small and simple And neat building blocks from a physics point of view Tend to be reused by nature And then in general we will come back to that At bifematics that if you have a reasonable number Of sequences you're going to maintain this fold Just because you change one residue Doesn't mean that it's going to change So typically I would say If 30% of the residue in two proteins are the same You're going to have the same fold That is in general true But remember what I said about molecular biology and examples There was a famous example in 97 by Lynn Regan They say roughly 50% of the residue is in a protein And then they could have it create a different fold And this is also a bit related to the prions I spoke about and everything I think where I'm pretty much done today But I'm going to take two more minutes here So why did I go through all this protein structure? Well proteins are important And you're going to see proteins Even if you want to go into experimental biophysics study spectroscopy What you're usually doing in spectroscopy Is you want to understand what a protein is interacting with Where in the body is this protein located? And then you would take this small protein And I say attack this more fluorescent probe to it Or you could add Let's see that these were two different proteins Then you can attach one probe here and one probe here And if these now happen to be close to each other in space You would then see a signal That one would convert energy to the other So it's not just that I'm a geek Can't happen to be interested in proteins For this closure I am But these are literally the building blocks for everything in bio Just that the atoms is the building blocks in atomic physics And you need to understand the spectra Almost everything we do has to do with these building blocks And they are created entirely from eugenium But that is not quite the entire story So on Monday Lucy is going to speak about membranes and membrane proteins So where do you get membranes from? I already showed you some hints about lipid bilayers Right and cell walls and everything And the proteins we get from your DNA But where do you get the membranes? Because it's the phospholipids Where do those molecules come from? Yeah So literally you are what you eat So that if you eat a different diet You will literally change the composition of your body And your cellular membranes So if you eat lots of cholesterol Your membranes will become slightly stiffer Which is perfectly fine in your age But by the time you're to my age You should probably start to think of your cholesterol intake So literally you can change the composition of your body With your food You can't change the proteins That's much more difficult But the cell walls and everything will change Depending on what you eat Which is pretty fun and scary And that's actually used in a couple of diseases That you can actually treat diseases Like epilepsy and everything By altering the food Thereby altering the properties of your membranes Which is pretty cool There are a bunch of study questions That I won't go through in detail I think I Many of my figures here are from Finkelstein But this is reasonably well covered in Nordland too So make sure I think you need to have a gut feeling About proteins are And how all the structural diversity And everything is related to free energy Because every single structure The reason a structure occurs Is because of free energy The reason why they bind Or not bind something The frog is because of free energy So try to think of these biological features In terms of physics Because it's all governed by very simple laws In particular the partition function And the Boltzmann distribution With that Four minutes early Almost five Have a nice weekend


Hey, this is Brett, and this 2017 Ford F-250 Crew Cab Short Box Platinum Edition is stock number 14242Z. I am here at Summit Automotive in Fond du Lac, Wisconsin, near New and used heavy-duty truck headquarters. This 2017 Ford F-250 has the 6.2-liter V8 gas engine, puts out 385 horsepower. It's paired up with the 6-speed automatic transmission. This truck has been fully-safetyed and inspected by our service shop. Has a fresh oil and filter change, all the fluids have been checked and topped off. This thing is 100% ready to go. Really a clean truck all the way around, and that's exactly what I'm going to do in this video. I'm going to go all the way around inside, underneath, start it up, take a look under the hood, show you all the options on the interior, and give the most accurate representation that I can of the vehicle. White gold metallic is the color issue to all my videos in 4K. If you like the video, you can subscribe to the YouTube channel, click the bell notifications, get updates on the videos I do each and every day, as well as having access to one of the largest catalogs of vehicle and heavy-duty truck walk-arounds on YouTube. This one comes with the 20-inch polished aluminum wheels in there in fantastic condition. No major scuffs, scrapes, or corrosion on there, and it comes with Michelin LTX-AT LT27565R20 tires. I would say these tires have right around 50% to 60% of the tread left. You can see you get the Rancho Shocks part of that FX4 off-road suspension package. Framing underbody is extremely clean on this truck. This is a one-owner clean title history out of Iowa. Front bumper fender is in excellent condition. Comes with the LED headlamps, LED running lights, and the LED fog lamps. We'll turn all those on at the end of the video so you can see just how bright they are. With the chrome tow hooks, note that the lower valance is in excellent shape. No scuffs or cracks on that. You get the platinum-finished grille. The Ford logo, the front camera, this one comes with a nice bug shield, and the hood is in fantastic condition as well. I didn't see any major dents or dings on there. Passenger side front fender is in great shape. Passenger side front wheel, no major scuffs or scrapes on there. And white gold metallics, obviously got some metallic in it. It's a really fine metallic, really good looking color, and the paint is in excellent condition as well. As you go down this side of this 2017 Ford F-250, take note of how clean that body is, how reflective and mirror-like that paint is. I take these HD videos so if you're far away or even if you're close by and you just cannot make the trip down, but you're still interested in purchasing the vehicle, you can see the truck, hear the truck, and have confidence in what you're looking at before you even get here. So when you do get here, there's absolutely no surprises, and you can make a smart and informed buying decision or decision to come take a look at the truck from wherever you're at. Now if this video helps you make that buying decision or the decision to come look at the truck, let your salesmen know that you saw the video that I was helpful and that Brett sent you, and then I can keep doing these videos for our great customers all over the U.S. each and every day. Back tires have just as much tread as the front tires. Frame and underbody back here is in very nice condition as well, has all the remaining factory exhaust, so it hasn't been altered in any way. And the lower rockers and cab corners all look great. This was the first year of the aluminum body on the Superduties. You can see it has the power running boards and they work nicely. Get the FX4 decals, LED tail lamps, chrome-tipped exhaust, rear bumpers in great shape, no dents or dings in there. It does have the back of parking sensors, full towing package, which includes a receiver hitch, four-pin and seven-pin wiring. The platinum tailgate, that's an excellent condition. You get an access soft roll-up tonneau cover. That's a really nice tonneau cover, and it's in really nice condition at NCME rips or tears on that. This one does have the tailgate step assist. You get a Linux spray and bed liner in here. Side box tie-downs, LED bed lights, seven-pin wiring, and it comes with a companion fifth-wheel hitch. It also has the fifth-wheel and goose neck prep package. And the bed is just in phenomenal condition. It also comes with a toolbox up there. Didn't see any major dents or anything. That spray and bed liner goes on to the tailgate, and the tailgate does have the tailgate step assist, which I think I already told you. Close that up. Shuts nice and solidly, and as we go down the driver side, just as clean as that passenger side, no dents or dings on the box here. And for full disclosure, this back wheel is in excellent shape as well. Down the rest of the side, cabin doors look really good. Does come with the power scope tow mirrors. They have line-spot monitoring, built-in directional signals, line-spot mirrors, 360 cam, they power telescope out, they power fold in. I'll show you how those work. In just a second, this one does have the driver side door code entry. We do have the code for this truck. If you ever want to figure out what your code is, for your 17 to 22 super-duty, in the upper right-hand part, so you can get a video showing you how to find that. Back seats are in very nice condition. They are heated on the outboard side. No rips or tears back here. It does have the power sliding rear window with the built-in rear defrost. These seats fold up for extra storage. This does have the, I believe this is the goose neck ball in here. Receiver hitch reducer. Lots of storage space. This piece collapses down. Has a Husky all-weather floor mat back here. You do get the heated seat buttons, 110 volt, 400 watt max plug-in, and a 12 volt power point. These seats fold down like so. And you do get the child safety locks on the back doors. Inside and bottoms of the doors all look fantastic as well. Get the tire and loading information sticker as well as the VIN sticker. No Canadian trucks here.  No rips, no tears on these seats. They are in fantastic condition. You get the platinum badge on the backrest. And both these seats are heated and cooled. Husky all-weather floor mats up here. Power drop-down tailgate, power pedals, auto headlamps, LED side lights, power telescopic and tilt steering wheel. See that works nicely. Power windows locks, mirrors, memory driver seat, these mirrors do power fold in. Ignition's gotta be on. Wood grain trim on the doors. And you can see that those power running boards work nicely on the driver's side as well. Hop inside, check out the miles, the radio, and everything that this truck has to offer on the interior. You get the digital speedometer, 22,346 miles on this truck, very low miles on here. Instrument cluster's very nice and clean. Smells very clean inside this truck. I don't think it's ever been smoked in. Steering wheel's in nice shape. No rips or tears on there. Does have the Bluetooth and audio controls on the right. Cruise controls and adaptive cruise controls on the left. Information center controls there as well. Back to these mirrors, they do power fold in. I was like showing both sides. So you know that both sides are working properly. And then they do power telescope out and in. And out and in. Yeah, the first couple of years of this body style you had to have the ignition on for those mirrors to work. Six-speed automatic transmission with the optional tap shift. Factory brake control turned out four wheel drive. So when it comes with the synced eight inch radio, you get the factory navigation system on here. There is highway 41, so that is working nicely. You get AM, FM, Sirius, XM radio capabilities, CD player and Bluetooth capabilities. Here are your climate controls. This one does have a heated steering wheel and your dual climate controls there. You get all your different apps and then you get your settings in here. This one also has Android Auto and Apple CarPlay. And my favorite feature on the Platinum Editions is the massaging seat. You can set those up and the seats will massage the upper and lower part of the seat. Very nice system on there. Stability control, hazard lights, downhill assist control, lane departure warning, and then of course your 360 camera, which is right there. You get a front camera as well. See if we can go to a wide view. And then of course you can do a close up back to the 360 and there's a couple other cameras you can do but I think the truck's gotta be started for those. So we'll start it up in just a second. Sony premium sound system, CD player, more tactile volume tune and climb controls turned out for a drive factory brake controller, heated cool C buttons, two USBs. You get a storage area down here, ambient lighting in the cup holders. And my trick is you get two cup holders, abracadabra four cup holders. So that's my magic trick. Keyless entry with remote start power drop down tailgate, get dual glove boxes, 12 volt power point, 110 volt, 400 watt max plugin. Husky all weather format down there looks good. The passenger side of floor mats in really nice shape and this one does have the second real fixed glass and power sunroof in the front. Power sign rear window controls, home link buttons for your garage door security systems and lane systems, map lights, sunroof and sunshade controls and let's start it up. Starts right up, no check engine lights or anything like that. There is your backup camera. Now you can zoom in on that receiver hitch to get hooked up to your camper boat trailer first time, every time. Then you do get the cargo cam and that is working nicely as well. But the truck's got to be on and you have to have it in reverse to access that camera. So that's why I did it that way. Turn all those lights on. I would personally like to thank you for checking out the video today. There are those LED side lights and hopefully from this HD video you've been able to verify the quality condition options and of course, cleanliness of this truck all the way around. All those lights are working nicely and very bright. Really a very clean truck all the way around on here. Under the hood we have the 6.2 liter V8 gas engine engine base, very clean, runs very smooth. Once again, this truck has been fully safety to and inspected by our service shop. Has a fresh oil and filter change all the fluids have been checked and topped off and this truck is 100% ready to go. There is the emissions sticker. Shocks are doing a nice job holding that hood up and I would highly recommend this truck from a quality and condition standpoint. I'd ship this one to California, Texas, New York, Florida. Whoever's gonna get this truck's gonna absolutely love it and to see more pictures of this truck or one of our other 550 new and used cars, trucks, SUVs, minivans, rain glas, half tons, three quarter tons, one tons. You name it, we gotta go to the website right there summitauto.com. Full pictures and descriptions of every single vehicle all at summitauto.com. If you'd like to check out more HD videos you can go to youtube.com slash summitauto. Click the bell notifications, get updates in the videos I do each and every day as well as having access to one of the largest catalogs of vehicle and having you do truck walkarounds. On YouTube, almost 9,000 videos in county. In fact, in a second you will see a link to subscribe to my YouTube channel in the upper left. A link to all of the F-250 videos I've ever done in the upper right. Link to this vehicle on our website in the lower left and a link to one of our latest YouTube videos in the lower right. Click those check us out. We're super excited to help you with this ultra clean 2017 Ford F-250 crew cab short box, platinum and white gold metallic. Thanks again for checking out the video. Remember to like, subscribe and share on the YouTube channel. I really appreciate it. Thanks again.