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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?
So maybe that's helpful for initially identifying
that an interaction exists.
But in terms of getting more detailed information in terms
of how do these actually interact,
that may be tough here.
OK?
So easy to come by, but potential complications.
Just in terms of thinking about this in the linker,
why is it important to think about the linker
and your choice of some reagent here?
So what properties does the linker give?
AUDIENCE: [INAUDIBLE] to the link,
then I guess its flexibility will determine
how close the two proteins have to be in space for those to be
[INAUDIBLE].
ELIZABETH NOLAN: Yeah.
So there's some constraints imposed
by the linker in terms of how close together or far away
are groups that react.
What else comes with the linker?
How does it affect the properties of the molecule?
Alex?
AUDIENCE: I was going to say it can dictate how likely you
get a cross-linking on the same molecule between two amines.
If you make it short enough, so that it
can't reach the next lycine or something,
then it can prevent [INAUDIBLE]
ELIZABETH NOLAN: Yeah.
May be able to.
So what's an inherent property of a molecule?
AUDIENCE: It might affect solubility.
ELIZABETH NOLAN: Yeah.
Right.
It may affect solubility.
So linkers can be-- this is a bunch of CH2 groups,
relatively hydrophobic, right?
There can be more hydrophilic linkers or other strategies.
And then the question is, does that matter?
Does the solubility properties work
with your experiment or not?
But imagine if you want to do cross-linking in a live cell,
you need that cross-linker to get into the cell.
So you need to think about membrane permeability
and what happens after that.
Here.
So the linker is another critical aspect.
And so, if you're ever working with a cross-linker,
that's something you want to think about in addition to what
types of side chains or what types of biomolecules
do you want to modify.
So let's look at an example of a heterobifunctional linker.
It's not linker.
Yeah.
Well, it is cross-linker.
OK.
So this one will have a different type of spacer group.
So it will be with a cyclohexyl.
So what do we have in this case?
Steve?
AUDIENCE: So you have an NHS ester and also a maleimide.
And then the sulfonate group probably helps the solubility.
ELIZABETH NOLAN: Right.
So there's a bunch of interesting aspects
to this molecule.
So we have the NHS ester to react with an amine.
Right here we have a maleimide, which will react with thiols.
So heterobifunctional, because there's
two different reactive groups for different types of side
chains.
And then, as Steve mentioned, we have this group here.
And so, this is to improve water solubility.
OK.
And then what do we have in this linker region?
AUDIENCE: A cyclohexyl instead of the aliphatic--
ELIZABETH NOLAN: Yeah.
And what does that give?
AUDIENCE: Isn't it rigid?
ELIZABETH NOLAN: Yeah, exactly.
Like cyclohexyl, right?
Think about chair conformation, rather
than what I have done here.
But it will give a more rigid linker,
and also shorter than what we see up here.
So this is on the order of eight angstroms.
So how might this molecule be used?
What could you do with it that you can't do with this one?
AUDIENCE: Cross-link cysteine and lycine [INAUDIBLE]
ELIZABETH NOLAN: Yeah.
Well, that's the first point, right?
You can have two different groups.
One end will react with a cysteine.
One with some lysine.
So is this specific, or non-specific, or both?
AUDIENCE: Probably depends on the context.
ELIZABETH NOLAN: Yeah.
Right.
Could depend on the context.
And then from the standpoint of specific cross-linking--
which I would argue is a better use of this compound--
what can you do?
Just imagine you have some protein of interest
and maybe you want to label it here.
And you have some side chain.
So site-directed mutagenesis to put in a cysteine.
And then you can modify that there,
such that you have cross-linking reagent, right?
And then you can imagine whatever
your experiment is here.
So again, thinking about using this compound in, say,
a complicated mixture, like a cell lysate--
you want to see if there's any binding partners or whatever.
What's the limitation in terms of reactivity
of this amine group that you would use in that second step?
Where do you lack control?
AUDIENCE: You still can't control
for the alpha for the N-terminal reaction, right?
ELIZABETH NOLAN: What do you mean by that?
AUDIENCE: So if the [INAUDIBLE] is free,
then would you have comparable reactivity
between the N-terminals, and, for example, your desired
lycine?
ELIZABETH NOLAN: OK.
So that could be an issue.
So do lycines and N-terminal alpha amino groups
have different reactivity?
Do they have different PKAs?
And is that something you could control?
Maybe, maybe not.
But more broadly than that, so you
have an issue that it will react,
let's say, with any amine, right?
Can you control when it reacts?
AUDIENCE: To some extent [INAUDIBLE] pH.
ELIZABETH NOLAN: So what are you thinking?
AUDIENCE: If you--
ELIZABETH NOLAN: So if you think about just experimental design,
right?
And say you were to try to use pH to control reactivity--
and I'm defining this broadly-- reacting with any amino group.
So we're not going to try to do something to selectively
label one, right?
This is reactive.
It will react, right?
So would pH change your whole buffer?
Or pH change the cell lysate, and then
switch to turn on reactivity?
Probably not.
Probably not, right?
That, I'd say is not very likely.
So the issue I'm getting at here is
that you have little temporal control or spatial control
of an NHS ester.
It will react with an amine provided your conditions are
appropriate.
So just getting back to this pH issue and a little digression,
if you want to use something like an NHS ester
in a test tube experiment, what you
need to think about beyond pH?
So what do you need to think about with the buffer?
AUDIENCE: You don't have something [INAUDIBLE] buffer
[INAUDIBLE] so you might want to use the phosphate buffer,
something that doesn't [AUDIO OUT]
ELIZABETH NOLAN: So this is a key point.
You need to think about cross-reactivity
with the buffer.
So if you have tris buffer, you have amine.
If you have a buffer that's like glycine, there's amine, right?
And your buffer concentration in most instances
is much higher than whatever the concentration is
of the molecule you want to actually modify, right?
If you think about 10 million molar tris or 75 million
molar tris compared to micromolar or nanomolar
of some protein, so you need to have
a buffer that's not reactive.
You need to have an appropriate PKA.
Those are important considerations.
You need to know that your reagent is good.
Sorry, appropriate pH.
What about the thiol here?
What do you need to think about if you're doing a test tube
experiment and want to modify a thiol with a maleimide
or something else, like iodoacetamide
that we saw last time?
AUDIENCE: Buffers need to avoid DDT.
ELIZABETH NOLAN: So what's DDT?
AUDIENCE: [INAUDIBLE]
ELIZABETH NOLAN: Right.
Or BME, beta mercapto ethanol.
Right.
Even before that step, what you need to make sure?
So what if there's multiple cysteines?
AUDIENCE: That they're not [INAUDIBLE]..
ELIZABETH NOLAN: Right.
So either inter- or intramolecular, right?
So if a reducing agent's added and the reducing agent
is thiol-based, again, you're going
to have much more reducing agent than your protein of interest,
right?
So you don't want your thiol-reactive probe
to react with the reducing agent in the buffer here.
So that needs to be removed.
And then if you remove it, you need to ask,
does the thiol stay reduced or is it susceptible
to air oxidation?
So these are just all practical considerations to keep in mind.
If a reaction doesn't work, why doesn't it?
And was it something that wasn't right with the buffers there?
OK.
So back to this issue of not having much control
about timing control for reactivity of these types
of groups, what could be done to overcome that?
So what other types of cross-linkers are out there?
Yeah.
Photo-active.
Photo-reactive cross-linkers.
So what's the idea here?
AUDIENCE: [INAUDIBLE] the appropriate [INAUDIBLE]
ELIZABETH NOLAN: Yeah.
So what do we have?
And what can we do?
So just the first point to make is
that we want to think about specific labeling here.
So we can attach site-specifically to a protein
or some other biomolecule, maybe it's bi-cysteine modification
with something like a maleimide.
Maybe it's unnatural amino acid incorporation.
And it's chemically inert locally until irradiated.
OK?
And so, basically irradiating this photo-reactive
cross-linker will activate the photo-reactive group,
and then you get s-linking.
OK.
So this type of approach is often
used to capture binding partners.
It can be used in the test tube or in cells.
What are the types of photo-reactive cross-linkers?
AUDIENCE: Aryl azides.
ELIZABETH NOLAN: Yeah.
So aryl azides are one type.
What's one we saw in class?
Although we, didn't talk about photochemistry.
Yeah, benzophenone.
And there's some other examples.
So what's another example?
AUDIENCE: Fluorinated [INAUDIBLE]
ELIZABETH NOLAN: Yeah.
So they fall in here, right?
So we can think about, either just
phenyl azides or fluorinated phenyl azides.
So another way to do this is to generate
carbenes via diazirines here.
We'll pretty much focus on these types, which are major types.
So where did this idea come from?
How new is this type of work to stick a photo-reactive group
on a protein, and then use it in a cross-linking application?
And where did the idea come from in the first place?
What types of chemists often study photochemistry?
AUDIENCE: DNA [INAUDIBLE]
ELIZABETH NOLAN: More broadly.
So physical organic chemistry, right?
There's a whole component of photochemistry there.
Let's take a vote.
2000?
First photo cross-linker.
1990?
'80?
'70?
'60?
Just no clue?
So around 1962 was the first paper
using a photoreactive group on a protein here, Westheimer.
And then Jeremy Nulls in 1969 was the first example
of an aryl azide.
OK?
So this work came out of physical organic chemistry
and at a time where physical organic chemists were
transitioning into enzymology.
So we don't have time to go into a lot of the photochemistry
of these different moieties, but it was quite rich there.
So how does this work?
What types of reactions and groups
get modified here in the cross-linking?
So let's think about them.
So let's consider an aryl azide.
So what happens when aryl azides are irradiated with UV light?
AUDIENCE: Took all of the nitrogen gas.
Get a nitrene.
ELIZABETH NOLAN: Get a nitrene.
Yeah.
So if we just think about nitrenes for a minute,
what types of chemistry do nitrenes do?
Are they reactive?
So can they insert into C-H bonds?
N-H bonds?
Add to double bonds?
Can they do other things as well?
OK.
So here we have our protein.
What's going to happen?
As Steve said, we're going to generate a nitrene.
So how does that happen?
We irradiate with light to get our nitrene.
So what happens with these aryl azides
is some interesting photochemistry when you're
at, say, room temperature.
So rather than this nitrene reacting, say,
with a C-H bond or an N-H bond, it actually
undergoes a ring expansion.
So what we get-- and this is very fast.
So on the order of 10 to 100 picoseconds.
So this happens before it has a chance
to react with something else.
OK.
To give us this intermediate.
OK.
And so, this species has very different chemistry
than a nitrene.
And what happens is it will react with nucleophiles.
So imagine our amino group to give the cross-link.
So this pathway is the dominant pathway
if just an aryl azide is used here.
To think about from the standpoint
of wanting to do cross-linking.
So let's say you attach this aryl azide
to a protein of interest, and then you
irradiate with light and look for it
to cross-link with something, is this an issue?
It will form a cross-link.
Would you rather have a nitrene reactor or this seven member
reaction with a seven membered ring?
AUDIENCE: [INAUDIBLE] nitrene, but it
would depend on what you're actually looking at, like what
you were investigating.
ELIZABETH NOLAN: OK.
So why would you argue for the nitrene?
AUDIENCE: Because we were talking about the nitrene
does have the capacity to do a [INAUDIBLE] So
if you wanted to do something like that kind of chemistry,
then having this be the dominant pathway would be inefficient.
ELIZABETH NOLAN: Yeah.
There's a lot more C-H bonds than there
are lysines or N-termini.
So that's one aspect.
We've lost that chemistry.
And then to another point, how well did these reactions work?
So nitrene reactions are very fast.
Relatively speaking, this is kind of sluggish there.
And so the question is, what can be done in order
to improve upon this?
OK.
And Steve mentioned these fluorinated phenyl azides
there.
And so, photochemical work, unrelated to any sort
of biological cross-linking chemistry,
showed that if you fluorinate aryl azides
you can get nitrene reactivity, rather than this other pathway
here.
OK?
And so, if we just take a look at that, what happens?
For instance, imagine we have this tetrafluoro analog here.
We can imagine irradiating this and getting to our nitrene.
I'm going to skip the steps.
OK.
Now what can happen?
Imagine we have some C-H bond nearby.
We get this cross-link.
And this reaction is very, very fast here.
Very, very fast.
Can ring expansion occur in this situation?
OK.
So I'm pointing this out, because the language
in the packet was a little strong.
If there is something for this nitrene to react with nearby,
it will react.
But this can undergo ring expansion.
It's just much slower than the case above.
So the studies I've read say about 170-fold slower there.
So it's not that the pathway is completely blocked.
It also too depends on the experimental conditions.
But anyhow, this is quoted to be near diffusion controlled here
for that.
I mean, this is pretty interesting when
you think about it, right?
Because aryl azides, they can be fed to cells
to do unnatural amino acid incorporation, right?
They're used in click chemistry for instance,
types of conjugation chemistry.
But here, the photochemistry can be taken advantage of
to give a cross-linker that can be controlled
in a temporal manner there.
So what about the benzophenone?
What does benzophenone react with after being
irradiated with light?
So imagine you have your protein.
And maybe in this case you did unnatural amino acid
incorporation to site-specifically attach
a benzophenone.
What happens?
What happens is that there's formation
of a triplet diradical.
And what will this do?
Here, it's going to react with some C-H bond
to get the cross-link.
Let's say you have this guy here and you
want to do a cross-linking experiment.
So we can imagine some different possibilities.
What do you think you'll get out?
Are you only going to get out your desired cross-link?
What might happen?
2:00, 1:50 on a Friday.
Let's get some jumping jacks.
Come on.
Should I dismiss all of you, because there is a major energy
low today, I have to say there.
Yeah.
Do you think you'll get one product, 10 products, 100?
AUDIENCE: There will be lots of side reactions.
ELIZABETH NOLAN: Right.
There's still the possibility for many side reactions, right?
And you always need to be aware of that.
So if you cross-link something, the next question is,
is this something that's actually relevant or not?
Or is it an artifact there?
So the analysis can be very complicated.
And so, that's just something to think about.
Say you have cross-linked species from cell lysate,
what are you going to do to analyze that?
Just think about some of the things that have come up
in other contexts here.
We talked about protease digest and mass spec
for looking at substrates of GroEL GroES, that's
something that can be applied.
And there's many sophisticated new tools
to get a lot of information out of the mass spec, which
we won't talk about.
But having tags within the cross-linker, right?
So then you need to ask, how well is the coverage going
to be?
So even after this step, there's a lot more work,
which we won't go into details in this recitation today.
What about inherent efficiency of cross-linking
in terms of these benzophenone versus the aryl azides?
We want to think about relative cross-linking efficiency.
Any sense of that?
AUDIENCE: I think the benzophenone compared
to the diazirine is a lot less efficient.
I don't really know [INAUDIBLE]
AUDIENCE: I have a question.
When we're talking about efficiency,
is it purely based on the speed of this reactivity?
Or is it also taking into account
the different cross-reactions that could occur?
Because it seems like there are more possibilities
for more cross-reactions.
Even though it might be more reactive, it's not--
ELIZABETH NOLAN: Yeah.
The former, right?
Just thinking about the reaction.
There's the possibility of cross-reactions
for all of these.
They're highly reactive.
A nitrene is highly reactive.
The benzophenone triplet diradical is highly reactive.
A carbene, if you're going to get that from some diazirine
is very reactive.
And yes, it's something important to think
about in terms of your experiment.
What is the relative efficiency of the reaction?
So I said that aryl azide is a little sluggish
compared to the others.
Something to consider, right?
You know what is the timescale of whatever it
is you're trying to trap.
So the wavelengths.
What is it about these wavelengths
that might be undesirable?
AUDIENCE: For in vivo studies, one shifting towards UV
means that you can have issues undesirable,
like DNA cross-linking stuff, but also it
means that it's not going to have
deep penetrants [INAUDIBLE] shift towards [INAUDIBLE]..
ELIZABETH NOLAN: What wavelength would you like?
JOANNE STUBBE: I would like it around 650.
These are all UV visible interface.
And you have hundreds of things that
absorb length are very incredible inefficient.
[INAUDIBLE] Most people never identify what
they get out of the other side.
They just see two things stuck together,
and that's the extent of it.
They never describe the molecular details.
ELIZABETH NOLAN: So let's actually just close--
JOANNE STUBBE: [INAUDIBLE]
ELIZABETH NOLAN: Right.
So one of the questions I asked in the discussion section,
is it worth the effort if you're going to site-specifically put
in a cross-linker?
And imagine you find this protein-protein interaction,
if one chooses, you can do quite a bit
more experiments in terms of where
you place this cross-linker and mapping out that interaction
region there.
And so, that's I think also just a take-home is often
you need to put your reactive group in more than one place
to really get at the answer to the question you're asking.
And so, there's folks around doing that there.
But is it 20 positions?
Is it 10?
Is it 50?
Because if you don't know at the beginning,
you may need to do a lot of just systematic trial and error
for that.
Yeah.
So I think you should all read the packet.
And there are some suggestions for reading
if you're curious to learn more, one of which
is a manual from Thermo.
So often, the companies give a lot of good general background
information, and there's many different types of chemistry
included in that as well.
I'll also point out, Ed is here for those of you
who don't know Ed.
So he'll be presenting next week on cryo-EM.
And you should definitely read the fatty acid synthase paper
beforehand.
The structures are incredible.
And fatty acid synthase serves as a base
for our discussions of polyketide and polyketide
synthases, which is where we'll begin module four in thinking
about the biosynthesis of natural products there.
OK.
Have a good weekend.