how our proteins fold in order to look at how proteins fold we actually need to first talk about protein denaturation so denaturation is going to be disrupting the native confirmation of a protein if you've ever cooked eggs you have disrupted um the native confirmation and cause denaturation so you'll notice that the egg white is very clear but once you start cooking it it becomes cloudy until it becomes white and that is because you're denaturing the protein using heat and now that protein is no longer soluble in water and comes out of solution as cooked protein so heat is commonly used to denature proteins but there are other things that you can use uh we've got some examples down here you could use Ura you could use um potassium um cyanate you could use a detergent like SDS you can even use acid in order to denature your proteins in fact that's how cheeses are made is by adding a lot of cheeses are made um it would be using acid denaturation so maybe lemon juice or vinegar added all right when we are looking at denatured protein it's going to have what is called a TM this is the melting temperature of the protein and this is going to be where you have half folded protein and half unfolded protein and you can actually make a plot like this of your protein denaturation uh so here we've got fully folded protein and as we are increasing the temperature so this would be look looking at heat um to denature the protein as we add heat a greater percentage of your protein is going to unfold until you're at 50% folded 50% unfolded you continue to add heat and you will be fully unfolded most proteins and biological systems are going to have a TM of between 40 to 60° C now if you have a thermophile that's a a organism that loves heat the um full the TM for those proteins tends to be a lot higher greater than 100° C which is how they're able to withstand that heat because their proteins don't denature all right so let's say we have a protein um we're going to look at ribonuclease and you are you denature it um let's say using Ura which is a common way to denature a protein without getting it to crash out of solution crash out means to precipitate uh so much like cooking eggs you're precipitating those proteins um so so let's say we use ribonucleases a and we unfold it um it will take about one minute for the protein to spontaneously go back to its native structure this is absolutely amazing so let's think about this we've got this long chain of amino acids with nearly infinite um possibilities of how it could organize itself so we looked at at the Ramic condrin plot so we know that it's not near infinite but there are a lot of still different possibilities for every single amino acid of how it could form so for ribonucleases to put this in perspective if each combination we sampled for 1 * 10-3 seconds it would take us 1 * 10 to the 30th years in order for this one single protein to get to the correct structure and have sampled every single possible confirmation so clearly a protein doesn't just sample different confirmations until it finds a good one there's got to be more of a method to its Madness so how do proteins fold so quickly so there's a lot of factors that are going to drive our protein folding and and we're going to go back to thermodynamics now we remember that Delta G is Delta H minus t Delta s so this Delta s is our entropy Delta H is our INF alpy and Delta G is our Gibs free energy that's our whether we're spontaneous so we want a negative value for Delta G in order to be spontaneous now if we are looking at our protein folding that is overall going to be a positive Delta s uh which means that it is not very um sorry not a positive Delta s that's going to be a negative Delta s it's negative because we're decreasing our entropy going from this random polypeptide chain to a nice neat folded protein is not is is decreasing entropy it's not increasing entropy so this is going to be an unfavorable process so a negative Delta s with a negative temperature here is going to lead to a positive value so that's a confirmational entropy now what we're going to be getting out of this though is we're going to be getting out intermolecular forces intermolecular forces are actually going to be a negative Delta H it's going to be exothermic to form these intermolecular forces so we're going to get some hydrogen bonding we're going to get some dipole dipole interaction we're going to get some Vander walls interactions those are going to be a favorable Delta H so we're going to get a negative Delta H from those non-covalent interactions and then when we fold our protein the water that was associated with this entire polypeptide chain is now going to be excluded from the protein it's going to be going to the outside this is going to result in free water it was hydrogen bound um to the polypeptide now it is accessible and free so because it's being excluded from the protein that is going to be favorable in terms of entropy we're creating more disorder by releasing these water molecules so we're going to have this hydrophobic effect which will be a favorable uh Delta s so if we add these up our overall protein folding is going to be a spontaneous Delta G but remember spontaneous does not mean instantaneous it just means that our protein folding is a favorable interaction overall okay so let's look at then how does a protein fold so first thing is that our proteins are not going to be our globular proteins are not static so that means that there is movement in them they don't get into their correct form and then freeze they are moving they are dissolved in water and so there's going to be some movement of the protein so this protein is going to fold towards its lowest energy confirmation so um this pictures how they use to kind of illustrate what that means so I'm actually going to use this so this is looking at it from the top looking down this is looking at it from the side so this would be our top view so this would be where we have fully unfolded protein it's just our polypeptide chain every time your protein folds a little bit more more it is going to be a lower energy structure because remember that Delta G overall is is negative for protein folding uh so maybe it folds and it will get to this point right here so it's now in a lower energy form when it moves and refolds it will go maybe to here which might be a new lower energy form and then finally it might get to its final form which is its lowest energy form but the point is no matter how it folds it's being funneled down into its lowest energy form which is why we don't have to sample 10 to the 50th different confirmations the first fold that it does is leading it towards its final fold now in general there actually is some of these like lower energy intermediates that it could get trapped in and there are enzymes whose job is to help untrap the proteins out of those false folding low energy intermediates and funnel them into the ultimate lowest energy structure which is generally the native structure of the protein so this is just another view we've got this um polypeptide chain unfolded and it can rapidly sample these different confirmations and this is just a single folding event it's just one single confirmation um so these are rapid reversible secondary structure forms all we have is Alpha Helix and beta sheeps loots and turns we have actually formed any domains now so one of those may lead us into now our energy well so for example this particular reversible secondary structure form can lead us into this folding of this um these two domains which can then lead us folding into our final native protein so this would be our final bottom part of our energy well so um again unfolded protein and we're going to decrease the entropy of we go down but we're increasing our stability all leading us down into our final native structure so for example once we formed this it might stay that might be this confirmation here which goes to this confirmation which may be here which goes to here and then our final native structure all right there are proteins that help facilitate this process so one of those proteins is called chaperonins so these are a protein that help promote protein folding so they can prevent protein misfolding or aggregation of proteins so um what we would have is this unfolded protein it's going to go into your chaperon protein and it is going to prevent some of these um low energy intermediates that are not favorable or protein aggregation from occurring and it is a energetically required process so we're going to have to put in ATP that's our energy source um our our energy molecule in biological systems so we have to put in the ATP and when we release that ATP is ADP then we will get our natively folded protein so the ATP is helping make sure that the protein is folding in the correct uh position and the chaperonins are essentially making it so that the Mis misfolds don't occur as often okay there are diseases that are caused by protein misfolding so some some of them are caused by just simple mutations in your polypeptide chain and so your protein can't fold properly because of the fact that you have a mutation uh but some of them are related to just proteins misfolding in general has nothing to do with the mutation in the polypeptide chain so here we see some diseases that are caused by proteins misfolding and often these are amalo related diseases which means that they are forming Aggregates when they shouldn't be forming aggregates and so the the idea of being able to stop protein misfolding and stop the Aggregates or these amals uh from forming is actually really important uh for um human health