hi everyone this is the video uh for let's see the second video in week one section two all right so today we're going to be talking about how proteins interact with other molecules and how we measure and talk about the strength of that interaction all right so i will just go to the slides and then talk more okay okay so in this video we're going to discuss the bonds involved in protein ligand interactions um how the number and the strength of those bonds determines the affinity between the protein and sligand um and then we'll discuss how we measure affinity um and how we describe affinity so if we're talking about a protein and it's ligand how do we describe that affinity um you know we put a number to it basically so we'll we'll talk about how we come to that number i just want to point out that when i say protein and ligand those are really general terms i mean ligand is a really general term um and by ligand i mean it could be another protein um so protein protein interaction where one of those proteins is considered the ligand the ligand could be a piece of dna it could be a nucleotide you know pretty much anything that the protein binds to okay so a ligand is a very general term it's not necessarily just a small molecule okay so in the cell typically um a protein which is here shown in cartoon form in green uh we'll have um a binding pocket in which a ligand comes in binds now you know not all proteins i say over here that not all proteins um have a binding pocket there are plenty of protein especially protein protein interactions where the there's a more of a binding interface than there is like a binding pocket so it could be like a flat ish binding interface between the two molecules so um but the idea of a binding pocket is just really nice when we talk about how three-dimensional architecture really matters and things like that so we'll just use the example of a binding pocket okay and so we have some sort of molecule that binds in the binding pocket we call that molecule ligand here in yellow and then these little red lines are denoting non-covalent bonds okay now typically um and i don't i can't think of an example where this isn't true um the bonds that hold proteins and their ligands together are non-covalent and the reason they're non-covalent is because non-covalent bonds are weaker than covalent bonds plus in the cell and we'll talk about this more when we talk about enzymes in the next video typically um you need an enzyme to create or break a covalent bond um and so in the cell when you have all these molecules floating around and you need molecules to bump into each other and interact with one another then you're not going to always want an enzyme needed for that interaction so that could be one reason why these are generally non-covalent bonds the other reason is because the cell is a very dynamic place and so there's a lot going on things change rapidly the cell needs to be able to um change what pathways are activated what proteins are there what other molecules are there just depending on what's going on inside the cell what's going on inside outside what's going on outside the cell so it's a really dynamic place things are coming together things are coming apart other things are coming together other things are coming apart um and so because non-covalent bonds are weaker bonds it's they're just more dynamic interactions um and so that is another reason why they could be primarily um non-covalent interactions um i recommend watching movie 2.4 in the movies folder um it's just basically showing a picture of molecules moving around and vibrating and coming together and they talk a little bit about affinity which we're going to talk about here okay let's move on to the next slide okay so um starting over here you have a protein and you see the polypeptide backbone and then you see these little um balls that denote particular side chains which side chains which is another word for our group all right so when you're green yellow red green whatever okay so then your protein folds and you see these little balls sticking out into the binding pocket binding site um partly what they're trying to show here and partly what i'm going to try to drive home here is you know from a three-dimensional and chemistry point of view which r groups are sticking out into that active site and where they are in three-dimensional space really matters uh for making specific interactions with whatever is going to come in and bind in the active site or in the binding site uh it's an active site if it's an enzyme it's a binding site if it's not um so you know think about the fact that when a protein binds to its ligand these are specific interactions they can't just go and find anybody right there they're having specific shape and a specific chemistry to bind to that specific ligand and there is these binding interactions are dynamic you can measure them with dissociation or association constants we'll talk about the dissociation constant how we use that as a measure of affinity and then you have this term affinity and what affinity means is basically that as you have molecules moving around in space and they bump into each other if they form bonds with one another if there's good fit good chemistry fit and good three-dimensional fit then they're gonna form a number of non-covalent bonds and they're going to stay bound together for some period of time before they dissociate right and so when you're talking about like rate constants in chemistry you know you're measuring okay how long do you stay bound to one another before you dissociate and so if you stay bound together for a longer period of time you have um better affinity and the reason you have better affinity is because you're forming more bonds or stronger bonds okay if there's less affinity then that's because there are less bonds or less strong bonds and less affinity means that you're going to dissociate more rapidly now again don't get stuck into this idea that a better affinity or poorer affinity is better or worse because it's not so apply the goldilocks hypothesis here where every protein ligand interactions has a particular affinity for its particular function so some you know when a protein comes together with its ligand sometimes um the cell needs that to be a stronger interaction and sometimes it needs it to be a more transient um interaction where they dissociate more rapidly so it's not that it's better for worse it just needs to be just right for that particular protein and ligand okay so if a protein and a ligand need to bind together for a long time and there's a mutation and they don't have such good affinity for each other anymore then that can be bad right but just because a protein and a ligand um don't have great affinity for one another doesn't necessarily mean that's a bad thing it just might mean that um for whatever those molecules are you know whatever pathway they're in that it needs to be a transient interaction and that's what the cell needs okay um so bonds non-covalent bonds right so number of bonds strength of bonds from chemistry you guys know that bond angle matters um bond distance matters um you know distance distance between atoms matters steric strain matters so you can't have you know if if a ligand is too big to fit into a binding site you know that's that's not going to work so it has to um has to fit basically um yeah so these are all things that i want you to be thinking about when we're thinking about how proteins bind to ligands so this particular slide gives you an example of a mythical protein and here you're just seeing the outline of the active site okay so there's there's protein here and it's showing you a number of r groups that are sticking into the binding site it's showing you that you could even have interactions between backbone atoms and the ligand so that's certainly possible for this particular protein you have a serine you have an arginine you have a backbone interaction you have glutamic acid you have threonine and serine and it's making interactions with a nucleotide called cyclic amp um which we'll talk about when we talk about cell signaling but cyclic amp is a pretty ubiquitous nucleotide primarily used as a signaling molecule both in prokaryotes and eukaryotes um and um yeah so it's a nucleotide so it has an adenosine it's got a sugar and it's a monophosphate meaning it's only got one phosphate and that's cyclized so the phosphate is actually cyclized with the sugar so it's connected to the sugar in two different places where um with another with a you know like an atp it would only be connected to the five prime carbon by one covalent bond um anyways that's not terribly important for what we're doing here um just what i want you to notice is like say with a phosphate here and your cyclic amp um your cyclic adenosine monophosphate you have an oxygen this oxygen has a negative dipole and it's interacting hydrogen bond with hydrogen that has a positive dipole because it's bound to the oxygen which is more electronegative you can have an ionic bond here which they're calling an electrostatic interaction between a charged arginine and then one of the oxygens on the phosphates typically charge so that can form an ionic bond you have a couple more hydrogen bonds occurring one is between backbone um here between the glutamic acid um and one of the hydrogens on the cyclic amp um so on and so forth okay so again um the architecture of this three-dimensional space really matters where the r groups are um how they where they stick out in three-dimensional space um bond angles um strength of bonds that sort of thing okay um so yeah so those are the things that i want you to think about when we think about protein ligand interactions um this next slide is just i found a picture on the internet showing a number of different protein ligand interactions some of these are proteins some of these are maybe they're all proteins um i'm not sure one of the things i wanted to point out was that you can look the modeling that they're using in this picture where they've got the protein um in space full view and then they've actually got the ligand uh in backbone view here um one of the things i just wanted to point out was to reiterate that sometimes this is kind of a flatter binding surface and then sometimes you really have a binding pocket um like here with uh ribonuclease inhibitor and ribonuclease a which is a um something that chews up rna um let's see and then if you're wondering um i believe they're showing a heat map of interactions so in the backbone model you can't see r groups and so they're just showing you where um on the ligand you're getting interactions with the with the protein using a using a heat map but more i just wanted to show you what different surface interactions might might look like okay um and finally um this is a little bit more complicated um but hopefully i can i could take you through this um there's a couple of readings i gave you um that might be helpful as well so i don't want to get really far into the biochemistry and the math and deriving this dissociation constant and all that stuff um i just want you to have a general understanding of what we mean when we talk about kd of a protein-like interaction um and in general how you how you find it um so kd is a dissociation constant and so what it's basically measuring is um how often um you know when when two molecules come together um how soon is it before they dissociate or how likely is it that they dissociate so first realize that this is sort of like the opposite of affinity and so when you're thinking about the relationship between affinity and kd they actually have um um there's a word i want but i can't think of it right now the opposite um relationship right so if affinity is higher than kd is lower if infinity is lower kd is higher excuse me because you're talking because kd is a dissociate a measure of dissociation an affinity is a measure of essentially how long they're going to stay together or how how much they like each other um and then general way in which you find kd for any particular protein ligand interaction is to do this okay you set up a series of of test tubes or a series of experiments and you decide on a particular um concentration of protein and then you titrate in ligand meaning that you use the same amount of protein but then you do use varying concentrations of ligand from low to high you know from zero to whatever okay and but probably not zero but you get what i mean so you vary your ligand concentration okay and then you mix your whatever amount of protein you have and whatever amount of ligand you have and you're going to have lots of test tubes because you're going to vary your ligand concentration all right you're going to mix them together and you're going to let them kind of diffuse around and then and diffusing around they're going to bind together and they're going to stay together for a while and they're going to come apart and um you know they're going to let them basically go to a dynamic equilibrium to equilibrium okay and so at any given moment in time you know your protein your ligand are going to be wiggling around in your solution you know maybe some will be bumping into each other and not binding some will be bumping into each other and staying bound um and so what you want to be able to measure is actually how much protein is in complex with ligand so you want to be able to measure at equilibrium um the concentration of complex uh complexed protein all right protein in complex ligand which is your c in the equation here okay so um let's see so then what you're looking for is in your titration of ligand at what concentration is your protein in the half bound state and so what does that mean half bound state means that at what concentration adi sorry at what concentration of ligand at equilibrium is half your protein free and half your protein in complex with ligand okay um understand you keep ligand at a much higher concentration than protein um and so anyways we don't measure the relative decrease of ligand that's in concentration um we just assume that we have a basically a um consistent concentration of ligand no matter what's happening all right but what you're looking at is the concentration of protein and the concentration of complex so say for example you put in 100 protein you're not actually going to put in 100 molecules of protein it'll be way more than that but for purposes of just the example we'll say we threw in 100 molecules of protein so what you're asking is is at what concentration at what ligand concentration at equilibrium do you have 50 of your protein free unbound and 50 of your protein in complex with ligand okay because in your equation where concentration of p the concentration of l divided by the concentration of c the point at which with a ligand concentration at which at equilibrium your concentration of p is equal to your concentration of complex because 50 of your proteins are free and 50 of your proteins are complex so equal concentrations your concentration of p is equal to your concentration of c and they sit one and over one over the other in your um numerator and denominator in your um equation and so the same number on top and the same number on bottom and the fraction equals one right so 50 over 50 say that means that the point at which um your protein is in the half bound state where half is free and half as in complex is the point at which your kd is equal to your lycan concentration because p and c are canceling each other out or equaling one essentially and that's what we describe as the kd of a particular protein ligand concentration okay uh we use molar units because that's how you do concentration all right and um in the cell typically your molar units are going to be in either micromolar or nanomolar all right micromolar is more than nanomolar and if your kd is in the micro molar range um it's an okay affinity if it's in an animal or range it's a pretty strong affinity what this means is that again the smaller your kd the tighter the protein and ligand binding interactions so the better affinity okay and conceptually this makes sense because if you can get to the half bound state with less ligand right because think about it when you're doing these sorts of experiments concentration matters because the more of something you have floating around in your test tube the more often they're going to bump into each other and the more likely you are to get an association okay and so if you have a lower ligand concentration and you're reaching the half bound state now having a lower light concentration means that the protein and ligand aren't bumping into each other more often because there's less ligand around um so if you can get to the half bound state with less ligand it means that when they are bumping into each other which is less often then um and you're reaching that half bound state and they're binding to each other they're staying bound longer whereas if it takes more ligand to reach the half pound state you have more ligand running around right they're bumping into each other you know maybe it's saying bouncing dissociated it takes with a lower affinity it takes a higher ligand concentration to reach that half bound state hopefully that made sense if not i'm always happy to explain it again and of course madison will be totally willing to help explain it as well again i'm more i want you to understand this conceptually and then be able to use the terminology to know that kd is a dissociation constant it's a measure of the strength of the interaction between a protein and its ligand um and that um a lower affinity means a higher kd and that a higher affinity means a lower kd okay all right that is it for this section and so the next video will be talking about enzyme function okay i will see you then