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ELIZABETH NOLAN: We're going to move on with GroEL/GroES

and a few more comments about where we closed yesterday

and then talk about experiments that

were done to determine what polypeptides

are folded by this machinery.

So I'm just curious.

Has anyone stuck trigger factor or GroEL into PubMed

to see how many hits you get?

Yeah, so Rebecca's question yesterday, or on Monday,

about trigger factor and active versus passive folding

motivated me to take a look.

So just to give you some scope, if you

put trigger factor in PubMed, as of last night,

there's 11,810 hits there.

GroEL is closer to 2,000 to 3,000-- in that range.

If you put trigger factor active folding,

you end up with 34 hits.

Most of those are about using trigger factor

in protein overexpression.

So if you also express trigger factor, does that help?

And it looked like there was one paper in those 34

that suggests an active folding role for one of the domains.

But that is just looking at an abstract.

And so, the point there is there are many, many studies that

consider these chaperones and a huge literature to search.

So what we're able to cover here is really just the tip

of the iceberg for that.

There's also a new review out on GroEL/GroES,

which is not required reading, but we're

posting it on Stellar.

So it just came out last month, and I really

enjoyed reading this review.

I thought they did a very good job

of talking about current questions that are unanswered

yet in terms of models and presenting different models

for how this folding chamber works-- so passive

versus active, for instance.

And they also give a summary of the substrate scope--

so the experiment we'll talk about today.

So where we left off last time, we

went over the structure of this folding chamber

and here's just another depiction of the overview.

So effectively, we have to back-to-back heptamer rings

as shown here.

Some polypeptide in its non-native state can bind.

It initially binds up at the top by these apical domains,

and there are some hydrophobic interactions.

OK, ATP also binds, and we have all seven ATPs

found within one ring, the ring that has the polypeptide.

OK, we see the lid come on, and then this polypeptide

has some time, a residency time, in this chamber to fold.

And then after the residency time,

which is generally quoted on the order of 6 to 10 seconds,

the lid comes off, and it gets ejected.

And during that time, the ATPs are hydrolyzed.

So somehow, this ATP hydrolysis gives conformational changes

that drive this cycle.

OK, and then we see, again, we flip

to having function in the other ring.

So one point to make involved cooperativity,

so I hope you've all seen cooperativity before, probably

in the context of hemoglobin.

We have examples here of positive cooperativity

and negative cooperativity.

So within one heptamer ring, ATP binds to all seven subunits.

So that's positive cooperativity.

And then we can think about negative cooperativity

between the two rings, where we only

have ATPs bound to one ring.

So the other heptamer ring will not have ATP bound here.

So what is happening inside this chamber?

The polypeptide enters the chamber,

and it's given this protected environment to fold.

And we saw that when the GroES lid comes in

that the hydrophilic nature, hydrophobic nature

of the interior changes.

And it becomes more hydrophilic.

So I just want to point out-- and this also builds upon

Rebecca's question from last time--

is this passive folding in the chamber

so effectively in Anfinsen's cage,

where the primary sequence dictates the trajectory?

Or does the actual chamber itself play a role?

So that would be active folding.

And effectively, is there forced unfolding or refolding

by GroEL itself?

So perhaps the apical domains can

force unfolding before polypeptide

is released into the chamber.

And the cartoon that was just up indicated that to some degree.

Maybe the cavity walls are involved.

And what I would say is that the pendulum on this

has swayed quite a bit over the years in terms of

whether or not GroEL is a passive folding cage

or actively involved in folding.

And some of the debates in the literature

have resulted from experimental set-up

that may bias results to indicate

one thing or the other.

And that's something the community is striving

to work out these days.

And I'll talk about that a bit more on the next slide.

But I'll just note-- these questions are still there,

and the recent review I just noted

discusses these questions.

There was a study just a few years ago

that was performed with very dilute polypeptide substrate--

so below one nanomolar.

And what they conclude from this study

is that GroEL is involved in active folding

of a maltose-binding protein mutant.

One question I'll just spring up with this

is, maltose-binding protein is a nice model polypeptide,

but what happens for a native GroEL substrate?

And is there utility in studying those?

So why have I emphasized this dilute protein sample point

here?

So what happened in some early work,

in terms of studies that were done to try to differentiate

active or passive folding, is that there

were some complexities in in vitro studies.

So, here, I just have a cartoon of folding in the chamber.

And if we think about only one polypeptide within the GroEL

chamber, it's folding in isolation.

So there's no possibility for it to form

an aggregate or a ligamer with other polypeptides.

It's all alone here.

So this folding in the chamber avoids the complications

of the folding landscape we talked

about in the introductory lecture to this module.

So what happens in aqueous solution, right?

There's the possibility that, depending on your conditions,

maybe there's some sort of aggregate that forms.

And if this aggregate forms, what does that

mean in terms of what you see?

And so, in earlier work, there were

some in vitro kinetic studies that

indicated GroEL accelerates folding relative to folding

in dilute aqueous solution.

But some of these comparisons weren't appropriate,

because as it turns out, oligomerization might compete

with what you're watching for.

And so, if there's some oligomerization happening,

it might indicate that the rate is slower than you think.

So there's ways to monitor for this.

And it's just a point in terms of what control studies do

you need to do to make sure your experimental setup is

appropriate there.

I think it'll be exciting to see what's

to come in future years about this question

and what kinds of biophysical techniques

are applied, including single-molecule studies here.

So where we're going to go, moving on,

is to think about what actually are the substrates for GroEL.

So what polypeptides get folded in this chamber?

And how do we begin to address that question

from the standpoint of what's happening in the cell?

OK, so first, we're just going to consider some observations.

And then we're going to go into the experiments here.

So here are some observations.

So the first one is that polypeptides,

up to 60 kilodaltons, can fold in this chamber.

So that's quite big--

60 kilodaltons.

Some proteins or polypeptides need

to enter the GroEL multiple times to be folded.

So that means the chaperone has the ability to bind and release

and re-bind the polypeptide here.

So when studies are done in vitro, what's found

is that almost all polypeptides interact with GroEL.

So you just saw even an example of that

in terms of this non-native maltose-binding protein.

So many polypeptides will interact.

And this really contrasts what's observed in the cell,

where, in vivo GroEL is involved in only folding about 10% of E.

coli proteins here.

OK, so what observations three and four suggest

is that GroEL has some preference

for particular endogenous polypeptides.

And what we want to answer is, what are these polypeptides,

and what are their properties here?

OK, so Hartl's group did some nice studies

to look at this, what needs to be done.

First of all, there needs to be a way

to isolate the polypeptides that are interacting

with GroEL in the cell.

And then, once these polypeptides are isolated,

they need to be analyzed in order

to learn about their identity and properties.

OK, so we're going to look at experiments

that were done to address this.

And they involve pulse-chase labeling

of newly synthesized proteins, amino precipitation,

and analysis here.

So in terms of addressing what are these substrates,

we're going to begin with pulse-chase labeling.

OK, so basically, the goal of this experiment

and why we're starting here is we

want to determine which proteins interact with GroEL.

And, in addition to which proteins,

we want to determine how long they interact.

OK, so what is the experiment?

These experiments are going to be

done with like E. coli cells.

So we want to know what's happening in the cell.

So imagine we have an E. coli.

And so these bacteria are grown in some culture medium.

And the trick here is that they're

going to be grown in medium that's depleted in methionine.

So incubate, or grow, in medium with no methionine.

OK, so effectively, we're depleting them

of that amino acid.

OK, so then after some period of growth,

what are we going to do?

We're going to spike the culture with radiolabeled methionine.

And this is the pulse.

So we're going to add 35S methionine.

And we're then going to incubate for 15 seconds.

OK, and so that's the pulse with a radiolabeled amino acid.

Then what are we going to do?

And after we go through the steps, we'll go through why.

After this stage, we're going to add

excess unlabeled methionine.

And we're going to then continue this culture for 10 minutes.

OK, this is the chase here.

And during this chase period, basically, samples

will be taken at varying time points.

OK, and then, at some point, we're just going to stop this.

OK, so just say, stop culture and experiment.

So what's happening in each of these steps?

And why are we doing this?

So what we want to do is think about newly translated

polypeptides.

OK, so we have a living E. coli.

It has ribosomes.

And these ribosomes are going to be synthesizing polypeptides

over the course of this experiment.

So during the pulse period, all proteins, or all polypeptides,

synthesized are radiolabeled.