Subtitles section Play video Print subtitles The following content is provided under a Creative Commons license. Your support will help MIT OpenCourseWare continue to offer high-quality educational resources for free. To make a donation or view additional materials from hundreds of MIT courses, visit MIT OpenCourseWare at ocw.mit.edu. ELIZABETH NOLAN: What we're going to do today is just discuss a few aspects of cross linking. So we decided it was important to introduce this within recitations this year, because cross-linking comes up time and time again. And there's different ways to do this, and different strengths and limitations to different approaches. So I guess in just thinking about this, what is cross-linking? So if you say, oh, I'm going to use a cross-linker for my experiment, what does that mean? AUDIENCE: Forming a covalent linkage between two molecules of study. ELIZABETH NOLAN: Yeah. So there's going to be formation of some sort of covalent linkage between two or maybe more-- right? Because some cross-linkers can have more than two reactive groups, OK, of study, right? So we're chemically joining two or more molecules. So why might we want to do this? What are possible applications? AUDIENCE: Study protein-protein interactions. ELIZABETH NOLAN: So that's one. So protein-protein interactions, right. And that could be identifying unknown protein-protein interactions or maybe you know two proteins interact, act but you don't know how, right? And you decide to use cross-linking as a way to probe that. So how might cross-linking help with studying a known protein-protein interaction? AUDIENCE: Start getting an idea of where the proteins are actually interacting or which residues [INAUDIBLE] ELIZABETH NOLAN: Yeah. AUDIENCE: It could allow you to isolate them. [INAUDIBLE] ELIZABETH NOLAN: Right. So maybe there's an unknown one, and you fish that out, because a cross-linker was used, right? And you know what one of them are. Or maybe, say, we know that these two proteins interact somehow, but we don't know how. So is it on an interface on this side versus maybe the other side versus maybe behind the board, et cetera. And so, there's many ways to study protein-protein interaction. And really, how I'll present cross-linking today is in the context of this particular application, but there are many others. But if we just think, we've seen a lot of protein-protein interactions in this course, right? So just even today, ClpXP is an example, right? We saw protein nucleotide interaction with the ribosome GroEL GroES is an example of protein-protein interactions, right? And they've been studied by many other methods, like crystallography for instance. But sometimes maybe it's not possible to get a structure, right? And you want to define an interaction surface or know exactly what residues are important. So here, say, is protein-protein. But that could be generalized to any other type of molecule, like RNA, DNA, right? What about a single protein? So can you use cross-linking to learn more about tertiary structure, quaternary structure? So imagine for instance, rather than two separate proteins, we have one protein where there's some flexible linker. And we have reason to believe these different domains interact. But how do they interact? Again, is it something like this undergoes some conformational change and they're like this versus other possibilities here? So what about just other applications of cross-linking chemistry before we look at some examples of molecules? So we can capture and identify binding partners, as Lindsey indicated. We can study known interactions. Where else could this come up? While it wasn't defined in this way, we've seen certain technology that takes advantage of cross-linking chemistry often. AUDIENCE: Within the realm of biological things, it's used for-- I mean, if you want to find a functional root. So like bioaccumulation or general bioconjugate chemistry for [INAUDIBLE] ELIZABETH NOLAN: Right. So general. Exactly, general bioconjugate or conjugation chemistry. So maybe you want to attach a tag to a purified protein. Maybe you want to modify an antibody. Similar chemistry can be employed. And likewise, even like from application standpoint, a mobilization. So say you need to make your own resin to do some sort of affinity chromatography and you want to attach a protein or an antibody to that, you can use the types of chemistry shown here. So we're going to talk about a few different types of cross-linker and the chemistry, and pros and cons. And just as a general overview, I'll describe types. So we just heard the word homobifunctional. So homobifunctional versus heterobifunctional. OK. And this refers to the reactive groups. So we need to talk about what types of chemistry is going to be used to do cross-linking. So this refers to reactive groups. And then another classification will be non-specific versus specific. And so, this doesn't refer to, say, the chemical reaction between the cross-linker and whatever it's hitting, but rather whether or not the cross-linking reagent is site-specifically attached to a protein or biomolecule of interest or not. If we just think about this non-specific versus specific, if we want to attach a cross-linker at some specific site in a protein, how can we do that? So think back to the ribosome discussion, where unnatural amino acid incorporation was not attached, but was introduced. So that's one possibility. If you have an amino azyl tRNA synthetase and a tRNA that can allow some sort of cross-linker to be introduced site-specifically, and it works for your experimental situation, you can do that. So we saw benzophenone, which is a cross-linker and the evolution of that orthogonal ribosome ribo-x. But let's say you can't do that, right? So for instance as far as I know, there's no tRNA AARS pair for benzophenone in a eukaryotic cell, right? Or maybe in some circumstance. What is something just using standard biochemistry you could do? So what type of residues can be modified in a protein? AUDIENCE: Cysteine. ELIZABETH NOLAN: Yeah. So cysteine, lysine. These are common side chains that are modified. And what would you say is more typically employed if you want to introduce a site-specific modification using chemistry? AUDIENCE: Cysteine. ELIZABETH NOLAN: Cysteine, right? So if you have an individual cysteine that's in the protein or maybe you use site-directed mutagenesis, you know where that cysteine is, and then you can modify it with some reagent there. We'll come back to that in a minute. So in terms of reactive groups then on the protein, we can think about lysines, right? We have the epsilon amino group, cysteines. We have the thiol. What do we need to think about for our chemistry when thinking about these types of side chains and wanting to do a reaction? So under what conditions do we have a good nucleophile? Pardon? AUDIENCE: [INAUDIBLE] ELIZABETH NOLAN: Yeah. So we need to think about the basicity, right? The PKA of these groups, right? That's very key here for that. What else do we need to think about? What other factors might govern reactivity, just thinking broadly? So PKA. For your amine, it will be type of amine. For a cysteine, redox will play a role, right? You can't have your cysteine and a disulfide. It needs to be the free thiol form. So these are all things to keep in mind. So Alex has used a homobifunctional cross-linker. Why did you use a homobifunctional cross-linker? AUDIENCE: It was to stabilize a nanoparticle. ELIZABETH NOLAN: To stabilize a nanoparticle. OK. So very different type of application here. AUDIENCE: Yeah, that's why I didn't mention it. ELIZABETH NOLAN: That's fine. Yeah. We're not doing much with nanoparticles here. But let's say we want to use a non-specific homobifunction. So this was non-specific cross-linker to look at some protein-protein interaction, right? So if we just suppose, for instance, we have some protein A and we think it interacts somehow with protein B, how can we use cross-linkers to study this? So let's take a look at an example of a homobifunctional cross-linker in terms of design. So this one will be amine reactive. And its name is DSS here. So effectively, if we want to dissect this structure into different components, what do we have? AUDIENCE: Two leaving groups kind of linking. ELIZABETH NOLAN: So we have two reactive groups, or leaving group, separated by a linker. And in this case, we have two NHS or 6-cinnamyl esters, right? That are amine reactive. So what's the product of reacting an alpha amino group or a lysine epsilon amino group with an NHS ester? What do we get? AUDIENCE: Amide. ELIZABETH NOLAN: An amide, right? We get an amide bond. And then we have this linker or spacer region. OK? Here. So two amine reactive groups and a linker, or spacer. And in this particular case, this linker or spacer is about 11 angstroms and it's flexible. And it's stable and cannot be cleaved. So in the case of Alex's project, this was used to stabilize a nanoparticle. Did you have a pure nanoparticle? Or was this in a very complicated mixture? AUDIENCE: It's very not in this course. ELIZABETH NOLAN: So what's going to happen if this reagent, say, is added to cell lysate? What are you going to get? AUDIENCE: Random cross-linking with a bunch of different lysate proteins [INAUDIBLE].. ELIZABETH NOLAN: Yeah. So there's a high, high likelihood of a lot of different cross-links, right? So potentially a big mess, right? High likelihood, right? Because you have no control over where these reactive groups are going to hit. And do most proteins have lysine residues? Yeah. Do all proteins have an alpha amino group? Yeah. Well, some might be modified, but anyhow. You have very little control with this type of reagent. So then the question is, if you use it, how are you going to fish out your desired protein-protein interaction? Or even if you're working with two purified proteins and they have multiple lysines, you can end up getting multiple cross-links, right?