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  • 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?