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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.

  • Right, because the methionine has been depleted

  • from the culture medium.

  • And so effectively, the methionine

  • that these organisms are seeing are the S35-labeled methionine.

  • And all polypeptides have an informal methionine

  • from the initiator tRNA and what other methionines

  • are in the sequence.

  • So, if we think about doing this for 15 seconds,

  • and we think about the translation rate, which