Placeholder Image

字幕表 動画を再生する

  • 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: OK, so we're going to get started.

  • And we're going to continue on with folding.

  • So we had some introduction last time

  • about this module and thinking about in vitro

  • versus in vivo studies.

  • And where we're going to move on today

  • is discussing molecular chaperones.

  • And effectively, there'll be three case studies

  • over the next two to two and a half

  • lectures-- so trigger factor, GroEL/GroES, and DnaK/DnaJ.

  • And so first is some background.

  • We need to talk about what are molecular chaperones.

  • And so effectively, these are proteins

  • that influence protein folding within the cell.

  • And they can do this by a variety of ways.

  • So they can help to prevent aggregation and intermolecular

  • interactions between polypeptides.

  • They can facilitate folding by limiting conformational space

  • and preventing side reactions.

  • An important point to keep in mind throughout this

  • is that these chaperone proteins bind to proteins transiently

  • here.

  • What are the types of processes they can assist in?

  • A variety are listed here.

  • And we see that it's quite broad.

  • So they can help in de novo folding, so for instance,

  • folding of a nascent polypeptide chain emerging

  • from the ribosome.

  • They can assist in refolding.

  • So for instance, if proteins have unfolded or become

  • aggregated because of stress, they can help here.

  • They can assist in the assembly of oligomeric proteins

  • in protein transport, and they also

  • assist in proteolytic degradation here.

  • And so we can classify these chaperones

  • into three main groups depending on how they act,

  • and those are listed here.

  • So we can have holdases, foldases, and unfoldases.

  • So something you might want to ask yourself

  • as you see these different chaperone systems is to ask,

  • what is the role?

  • Is it one or multiple?

  • So holdases help to stabilize non-native confirmations.

  • So effectively, the chaperone will

  • bind a polypeptide and a non-native confirmation

  • and stabilize that for some period of time.

  • Foldases assist in folding of a polypeptide

  • to its native state.

  • And unfoldases, as the name indicates,

  • can help with unfolding proteins, so for instance,

  • if a protein has misfolded and that needs to be undone,

  • or maybe a protein needs to be extracted

  • from some aggregate that's formed in in multiple proteins

  • that's a problem.

  • And so we're going to think about the chaperones

  • in the cytoplasm in two main groups for the ones

  • that interact with newly synthesized polypeptides.

  • So first, we can think about trigger factor, which

  • is a chaperone that's associated with the ribosome,

  • as we'll see.

  • So trigger factor is involved in co-translational folding,

  • meaning the polypeptide is still associated with the ribosome

  • and de novo folding.

  • And then we'll examine some downstream cytosolic

  • chaperones.

  • So these are chaperones that do not bind to ribosome--

  • GroEL/GroES, DnaK/DnaJ.

  • And just as a general overview of this molecular

  • chaperone concept here--

  • so this is taken from the required reading--

  • effectively, what's shown in this scheme

  • is a variety of different states a polypeptide

  • can find itself in.

  • So here we see a partially folded protein.

  • This protein may form from an unfolded protein or maybe

  • a native protein.

  • We have an aggregate.

  • And if we look down here, we're seeing the effect

  • of some generalized chaperone.

  • OK, so one important point to make from this

  • is that the chaperone's not part of this final structure.

  • It's just helping the polypeptide

  • get to its native state.

  • OK, and we can think about different rate constants,

  • whether it be for folding or aggregation,

  • chaperone binding Kon, chaperone dissociation Koff.

  • So for instance, if we look here,

  • we have a partially folded polypeptide.

  • And imagine the chaperone binds that.

  • Or maybe the chaperone binds an unfolded polypeptide.

  • OK, it's going to act as a holdase or a foldase.

  • And what can we see down here--

  • or an unfoldase-- what we see down here

  • is an indication of an event that's

  • driven by ATP hydrolysis.

  • And so what we'll see and what's known

  • is that many of these chaperones switch

  • between low and high affinity states

  • for some substrate polypeptide.

  • And these low and high affinity states

  • are somehow regulated by the ATP binding and hydrolysis.

  • So here, for instance, we see that step.

  • And imagine a Koff getting us back into this direction

  • here, right?

  • So you can begin to ask yourself questions like,

  • under what conditions and terms of these rate constants

  • is folding efficient?

  • When would a chaperone act as a holdase?

  • When would aggregation occur?

  • So aggregation would occur if this Kagg

  • is much greater than, say, for instance, Kon here

  • to work systematically through this scheme.

  • And here are just some points and words

  • related to that scheme and things

  • to think about from a broader picture.

  • So in terms of the systems we're going

  • to examine in the cytoplasm, this is the overview slide.

  • And where we're going to begin in this overview

  • is with the ribosome.

  • And we see that in red here we have a nascent polypeptide

  • chain emerging.

  • OK, so what does this scheme indicate?

  • What we see is that here is the player trigger

  • factor, which is involved in co-translational folding.

  • And we see that about 70% of nascent polypeptides

  • interact with trigger factor.

  • And these can arrive in a native conformation.

  • We see there's two other systems here.

  • So on the right, we have GroES and GroEL.

  • So GroEL provides post-translational folding.

  • We look and see about 10% to 15% of peptides in the cell

  • interact with GroEL/GroES.

  • And as we'll see, it provides this folding chamber

  • on a protected space.

  • We also see here that this system uses ATP.

  • OK, and here we have another two players, DnaK

  • and its co-chaperone DnaJ.

  • And we see they're binding to some sort of polypeptide

  • in a manner that's different than GroEL/GroES.

  • OK, about 5% to 18% of polypeptides

  • come into contact with these two players.

  • We also see this system as ATP-dependent,

  • and there's another player, GrpE,

  • which we'll see is the nucleotide exchange

  • factor needed here.

  • OK, so we see that maybe there's some crosstalk here.

  • And here we have some needed native polypeptides.

  • So just some things to keep in mind--

  • it's important to think about concentrations

  • and some approximate concentrations are listed here.

  • If we're thinking about the ribosome

  • DnaK/DnaJ, GroEL, and trigger factor.

  • Just to note that many chaperones are also

  • called Heat Shock Proteins, and this

  • is because their expression increases with increased

  • temperature or stress.

  • So Hsp70, Hsp60, that's for heat shock protein.

  • So where we're going to begin is with an overview

  • of trigger factor.

  • Yes?

  • AUDIENCE: I wanted to ask.

  • What do you mean by native and this protein

  • exist in several conformations in this slide?

  • ELIZABETH NOLAN: So proteins are dynamic, right?

  • We know that.

  • So native means a native fold, so a native state

  • of this protein as opposed to the protein being unfolded

  • if it's supposed to be globular or being

  • some undesirable oligomer aggregate.

  • So when this polypeptide comes off the ribosome,

  • that's a linear sequence of amino acids.

  • And it needs to adopt its appropriate conformation

  • to do its job, OK?

  • And as I said last time, we're not

  • discussing natively unfolded proteins

  • in the context of this class.

  • AUDIENCE: So proteins can have many different native receptors

  • in this slide?

  • ELIZABETH NOLAN: Yes, they're dynamic.

  • But there is going to be, like if you need a beta sheet,

  • a domain that has beta sheets, for instance, that

  • needs to fold and form there.

  • So we can discuss further if you have more questions about that.

  • But think about ubiquitin, for instance,

  • from recitation week one.

  • That had a very defined shape, right?

  • So it's native fold from looking at that PDB file.

  • AUDIENCE: OK, I think I just need to understand

  • in that previous slide.

  • When I talk about one protein here, right?

  • 70% percent of the proteins--

  • ELIZABETH NOLAN: Yeah, this is thinking

  • about all the proteins and all the peptides in the cell.

  • AUDIENCE: OK, I thought it was one type of protein.

  • ELIZABETH NOLAN: No, this is looking

  • at proteins in broad terms.

  • And so what we'll see as we move forward

  • is that certain types of proteins

  • interact with GroEL where others don't, right?

  • Trigger factor interacts with many, many of them

  • because it's associated with the ribosome,

  • and the ribosome's synthesizing all polypeptide chains there.

  • OK, so we're going to start with trigger factor.

  • And the first thing just to be aware of when thinking about

  • trigger factor and where it acts--

  • so we saw it sitting on top of the exit tunnel of the 50S

  • in the prior slide--

  • is that there is a lot of things happening near the exit

  • tunnel of the ribosome.

  • OK, so here we have our 70S ribosome.

  • Here's the polypeptide coming out.

  • A few proteins are indicated.

  • In addition to trigger factor, just

  • be aware that there's other players here.

  • OK, one of these is Signal Recognition Protocol, which

  • I mentioned briefly last time.

  • This is involved in delivering membrane proteins

  • to their destination.

  • We also have enzymes that do work here,

  • whether it's an enzyme for removing

  • the N-terminal methionine, enzymes for deformulation

  • of that N-terminal methionine, et cetera.

  • So somehow, trigger factor needs to work

  • in the presence of these other constituents there.

  • OK, so when we think about trigger factor, what

  • you want to think about is a protein

  • of about 50-kDa that's shaped like a dragon.

  • OK, this is ATP-independent.

  • So what I said earlier about low and high affinity states

  • and switching between these states being driven

  • by ATP binding and hydrolysis, that

  • does not apply for trigger factor.

  • It's the exception in what will be presented in this course.

  • And it's associated with the ribosome.

  • And what trigger factor does is it provides a folding cavity

  • or cradle over the exit tunnel.

  • And by doing so, it gives this emerging polypeptide

  • a protected space to begin to fold,

  • so reduction of intermolecular interactions.

  • So if we take a look at trigger factor,

  • one depiction is shown here.

  • And as I said, think about a dragon.

  • And it's actually described as having a head, arms,

  • and a tail.

  • And so we can think about this also

  • in terms of N and C-terminus.

  • The region of trigger factor that

  • interacts with the ribosome and binds the ribosome

  • is down here in the tail region.

  • And so what trigger factor does and what's

  • indicated by the cartoon you've already seen

  • is that it associates with the translating ribosome

  • with a one-to-one stoichiometry.

  • So think about having one trigger factor over that exit

  • tunnel here.

  • And as you can see, these different domains

  • also have some additional activities.

  • And the main chaperone activity is

  • attributed to the C-terminal region here I've color-coded.

  • So what are some characteristics of trigger factor?

  • If we look at the surface and consider

  • where different types of amino acids are found,

  • so whether they have polar or non-polar residues,

  • that's depicted here.

  • OK, what do we see in this depiction?

  • Where are these residues?

  • Is a given type of residue clustered in any one spot?

  • Yeah, I see some heads shaking no.

  • No, right.

  • They're distributed all about.

  • We see non-polar and polar residues

  • distributed across the surface of trigger factor here.

  • And so why might this be?

  • What's thought is that, effectively, trigger factor

  • uses its entire inner cavity--

  • and you'll see how that forms in a minute--

  • for substrate accommodation.

  • And you can imagine that all of these different polypeptides

  • emerging from the ribosome have different amino acid

  • compositions, right?

  • So this allows it to be relatively

  • versatile from that perspective.

  • If we take another look at structure

  • and think about how trigger factor binds

  • to the ribosome, what's found here,

  • so we're looking at structures of trigger

  • factor bound in orange and unbound

  • in green to the ribosome.

  • And the ribosome's omitted for clarity.

  • There's a helix-loop-helix region

  • that is involved in that interaction.

  • And what's found is that when trigger factor is bound,

  • it's quite dynamic.

  • And it can swivel around this ribosome binding site

  • by about 10 degrees in every direction.

  • So why might that be important?

  • One, this flexibility may allow it

  • to accommodate many different polypeptides that

  • are emerging from the ribosome.

  • And it may also facilitate its coexistence

  • with those other proteins and enzymes

  • that are acting by the exit tunnel

  • that we saw on the prior slide.

  • OK, so here is just a model attempting

  • to show the different ways and degrees

  • to which trigger factor can move from various structural studies

  • when attached to this ribosome here.

  • So this N-terminal domain, I have protein L23 listed here.

  • It also contacts the 23S ribosomal RNA and the protein

  • L29.

  • And what's found is that some salt bridge interaction

  • is important.

  • And we'll see that in a moment.

  • Here, if we look at a depiction of trigger factor actually

  • bound to the 50S, so here we have the 50S.

  • Here's the exit tunnel.

  • And here's our dragon-shaped molecule sitting.

  • I like to say on top.

  • I guess it's on bottom here.

  • But anyhow, the polypeptide's coming out,

  • and it has this cavity where it's

  • protected from all of the other constituents in the cell.

  • And here's just a rotated view showing it

  • on top-- so tail region, head, and arms.

  • So we have this cradle over the exit tunnel.

  • It's giving a protecting space for folding

  • of that nascent polypeptide chain.

  • If we just look at a little more detail here, what do we see?

  • So there's a salt bridge between an arginine residue,

  • arginine 45 of trigger factor, and glutamate,

  • glutamate 13 of the ribosomal L23 that forms a salt bridge.

  • So in this depiction, we have trigger factor in red.

  • We have L23 in green.

  • OK, and so you need to be thinking

  • about the amino acid side chains here, and that's something,

  • too, Joanne and I want to stress a bit after recitation

  • last week is, really, in this course,

  • the importance of thinking back to the chemical structures

  • and properties of the molecules that

  • come up within this course--

  • so positive charge, negative charge, that interaction

  • here for that.

  • So what happens when a polypeptide is

  • emerging from the exit tunnel and it

  • encounters trigger factor?

  • There's many possibilities.

  • And as I said, trigger factor is dynamic.

  • And an interesting point about this protein

  • is that trigger factor can differentiate

  • between vacant and translating ribosomes,

  • OK, and so what's found from in vitro studies

  • is that the binding affinity, which

  • I'll describe as a dissociation constant which

  • is 1 over the Ka of trigger factor for the ribosome

  • varies by several orders of magnitude

  • depending on whether or not the ribosome's translating.

  • So we have the Kd measured on the order of 1 to 2 micromolar

  • if the ribosome is vacant, and a Kd of about 40 to 70 nanomolar

  • for a translating ribosome.

  • OK, and just in recitation 10, we'll

  • talk about binding studies more.

  • But just if needed for review, if we're

  • thinking about A plus B going to AB, we have Kon and Koff.

  • And Kd is Koff over Kon, and Kd is 1 over the Ka here.

  • So let's look at some aspects of a model for a trigger factor

  • dynamics during translation.

  • So as I said, it can differentiate

  • the vacant and translating ribosomes.

  • What's been found from in vitro studies

  • is that the mean residence time on the ribosome

  • is about 10 seconds.

  • So what are the possibilities?

  • One, trigger factor can bind to a vacant ribosome,

  • and that's shown here.

  • And it can bind to a translating ribosome,

  • and it does this with higher affinity, so greater Kon.

  • So what happens after trigger factor binds

  • to a translating ribosome?

  • What we see is that the nascent polypeptide

  • chain is coming out.

  • And in this cartoon, what's depicted

  • is that it's beginning to fold in this protected region here

  • made by the trigger factor cradle.

  • And what we see from this point is that there's

  • three possibilities.

  • So if we look first on the left, what happens?

  • Trigger factor dissociated from that polypeptide that's

  • emerging from the ribosome.

  • So recall these chaperones bind and release the polypeptides.

  • In this case, it's left.

  • There's some folding that's happened,

  • and this peptide is still associated with the ribosome.

  • So what might happen next?

  • Maybe this polypeptide has the ability

  • to reach its native state without the help of trigger

  • factor anymore.

  • So that's shown here.

  • It's released, and it's folded.

  • Maybe some other chaperones in the cytoplasm helped with that,

  • but it's not shown here.

  • Alternatively, maybe trigger factor binds again.

  • So maybe this is one domain, and then somewhere else,

  • there's some other region that needs some help with folding.

  • And we see that here.

  • So it can bind and release the same polypeptide

  • more than once.

  • What are our other options?

  • So maybe trigger factor, after being here,

  • remains bound to the ribosome, and the polypeptide

  • is released.

  • Or look what happens here.

  • We have trigger factor bound.

  • We see release of the polypeptide

  • with trigger factor bound, or here we

  • see that there's even two trigger

  • factors bound to the same polypeptide emerging

  • from the ribosome.

  • And just thinking about this from the perspective

  • of the number of different polypeptides

  • that are synthesized by an organism,

  • all different lengths, all different levels of complexity,

  • it's not too surprising that there's various possibilities

  • here.

  • So again, if you're presented with data,

  • you need to ask, what does the data say?

  • And what type of particular aspect of this model

  • does it support?

  • Yeah?

  • AUDIENCE: How often is the ribosome actually vacant?

  • ELIZABETH NOLAN: How often is the ribosome vacant?

  • Yeah, I don't know how often the ribosome is vacant.

  • So in vivo, in your test tube, you

  • can completely control that, which

  • is what's going to give some of these data here.

  • Joanne, do you know?

  • No.

  • Yeah, anybody know?

  • I don't know, right?

  • So does it make sense to have many vacant ribosomes?

  • AUDIENCE: Are there more vacant ribosomes maybe

  • like floating around than there are

  • membranes bound [INAUDIBLE]?

  • ELIZABETH NOLAN: So I think that's a can of worms we're not

  • going to go down right now in terms

  • of where the ribosome is here.

  • So let's look at a functional cycle.

  • This is just another depiction of a potential functional cycle

  • where we have the ribosome bound to mRNA.

  • There's a nascent chain.

  • Here we see several trigger factors bound,

  • and we see options.

  • So either the native fold, or maybe there

  • needs to be some work of downstream chaperones, right?

  • And at some point, triggered factor will be dissociated,

  • and it can come around and rebind again.

  • So there is some evidence the formation of a trigger factor

  • dimer when it is not with the ribosome.

  • We don't need to worry about that detail too much

  • for our thinking about what's happening here,

  • because this is a one-to-one stoichiometry.

  • So how is trigger factor influencing

  • the folding process?

  • If we think about foldase, unfoldase, and holdase,

  • so these cartoons show that some folding is happening

  • in that cradle, especially the ones we saw before, right?

  • And that's perfectly reasonable that somehow trigger factor

  • is allowing or accelerating productive co-translational

  • folding of that polypeptide.

  • So from that perspective, it would be a foldase.

  • Is it possible that it's also a holdase?

  • And could trigger factor, in certain cases,

  • keep nascent chains unfolded?

  • Maybe to help prevent premature folding that

  • would be an error during polypeptide synthesis, that's

  • another possibility.

  • And they're not mutually exclusive, right?

  • So again, it's a question of an individual system

  • and looking at the data.

  • So the behavior may depend on the circumstance

  • in the polypeptide chain.

  • Rebecca?

  • AUDIENCE: I'm just curious.

  • So when we're talking about it acting

  • as a foldase, mechanistically, is the trigger factor

  • physically interacting with and promoting

  • a certain conformation?

  • Or is it just providing a space where

  • everything else is isolated?

  • Or do we even know?

  • ELIZABETH NOLAN: Yeah, so do we even know?

  • So this is something we'll talk more about in the context

  • of the chamber GroEL.

  • But what are the possibilities?

  • One is that trigger factor is just

  • providing a safe place for this polypeptide

  • to fold to its native conformation.

  • Because recall last time, we discussed the primary sequence

  • and how primary sequence can dictate the fold

  • and what's thermodynamically most stable, right?

  • But in the cell, the cell is very crowded, right?

  • So trigger factor can protect this polypeptide

  • from all those other constituents in the cell that

  • might cause unwanted intermolecular

  • interactions, for instance, and cause

  • a different folding trajectory.

  • Is it possible that the cavity wall of trigger factor

  • could influence that energy landscape?

  • So that's the other aspect of your question.

  • Is it an Anfinsen cage, so just allowing folding?

  • Or is it actually affecting the landscape?

  • I don't know if we're suggesting that it

  • influences the landscape, the energy landscape.

  • But that doesn't mean that literature is not

  • out there for that.

  • So I think of it typically as a cradle.

  • And as I said, we'll come back to this idea with GroEL

  • where there have been studies and people arguing one

  • over the other.

  • OK, so with that, we're going to leave trigger factor

  • and move to the macromolecular machine, GroEL/GroES.

  • And so GroEL falls into a subset of chaperones

  • that are called chaperonins.

  • OK, and these are chaperones that

  • are essential for viability in all tested cases.

  • OK, so that tells you this machinery

  • is really important for the cell and must

  • be involved in folding of some important players here.

  • So in terms of GroEL/GroES, what do we have?

  • I can describe this as bullet-shaped.

  • a bullet-shaped folding machine.

  • And so GroEL is the chaperone, and GroES is the co-chaperone.

  • And they work together.

  • And so what we have if we draw this in cartoon form is

  • we have GroES, and we can describe GroES

  • as the lid of the folding chamber.

  • And here we have GroEL.

  • OK, and what GroEL is, this gives us cavities for folding.

  • OK, and we can think of it like a barrel.

  • And as drawn, we see two pieces here.

  • And as we'll look further, we'll see that these

  • are two heptameric rings.

  • The ring that has the lid attached

  • is called the cis ring.

  • Or sorry-- the chamber or heptamer with the lid attached

  • is cis, and the one below is trans.

  • And this is huge.

  • So this whole thing is on the order of 184 angstroms just

  • to give some scale.

  • So EL is the chaperonin, and ES is the co here.

  • So what we'll do is look at the structural characteristics

  • of GroEL and GroES individually and then

  • think about function here.

  • So for GroEL, what we have are to have to heptameric rings.

  • OK, and so if we look from the top here, what we have are

  • the seven subunits arranged in this ring.

  • OK, and what we see is that there's

  • an inner cavity that's about 45 angstroms in diameter, OK?

  • And each subunit is about 60 kilodaltons, which

  • is why this is called Hsp70.

  • And so if we take a look in this structural depiction

  • here, in the middle, this is the top view.

  • OK, and the different subunits have been color-coded.

  • They're all the same polypeptide.

  • They're just differentiating them

  • here so it's easy to see each one.

  • And here's that inner cavity.

  • If we look at the side view again,

  • we need to consider a little more detail.

  • OK, so each one of these is a 7-mer.

  • OK, and each subunit of GroEL three

  • domains that are organized A, I, E--

  • so apical domain, intermediate domain, and equatorial domain.

  • And then if we look at this bottom ring here,

  • they're organized like that.

  • OK, so effectively, what we have is a back-to-back arrangement.

  • OK, so we have 14 subunits and two back-to-back rings.

  • And so if we take a look again at this depiction, what's

  • been done is that in this top 7-mer ring,

  • for one of the subunits, the three domains

  • have been colored.

  • OK, so we see that the apical domain

  • is an orange, the intermediate domain in yellow,

  • and this equatorial domain in red shown here.

  • And this is one isolated subunit again with this

  • color-coding here.

  • So what happens when the lid binds?

  • So we're currently looking at this as just GroEL

  • the to heptamers.

  • But we need to begin to think about GroEL with its lid.

  • What happens when the lid binds is that there's

  • a conformational change.

  • OK, and so I'll just draw this, and then we'll

  • look at the structure.

  • OK, so imagine that this is one GroEL

  • subunit of the 7-mer ring, OK?

  • When GroES binds to that ring, what happens

  • is that the GroEL subunits change

  • from a closed conformation, which

  • I'm kind of showing as closed, to something that's open.

  • OK, so effectively, it's like opening up a hinge.

  • OK, and so the consequence of this

  • is that when the lid binds to this cis cavity,

  • the size of the central cavity expands dramatically.

  • So it basically doubles.

  • And that's something that's not clearly indicated here.

  • So we can modify the cartoon.

  • OK, so let's take a look and then talk

  • about why that's important.

  • So here are two depictions where we have GroEL/GroES,

  • and we can look at a GroEL subunit that

  • does not have GroES bound, so with the trans ring.

  • Or we can look at a GroEL subunit

  • where the GroES lid is bound, so in the cis.

  • OK, and so this is actual structural depiction of what

  • I tried to indicate on the board here where we have closed

  • and open.

  • And so this opening is making this cis ring much larger

  • in terms of its central cavity, OK?

  • So these are major conformational changes

  • and details of which are described here.

  • But effectively, the two points to keep in mind

  • is, one, that diameter and size of this central cavity doubles.

  • And we can think about why that might

  • be important in terms of accommodating a larger

  • polypeptide as we get towards the functional cycle of this.

  • And also, what we'll see as we move forward

  • is that the distribution of hydrophobic and hydrophilic

  • residues on the interior of this cavity

  • changes dramatically when GroES binds here.

  • So briefly, to look at GroES, what

  • does that look like from a structural perspective?

  • So GroES is also a heptamer.

  • OK, each subunit is only about 10 kilodaltons.

  • It's about 30 angstroms in height

  • and about 75 angstroms across here.

  • And what we see if we look at the structure

  • of an individual GroES, so again, here, what we see

  • is that there is a beta sheet region.

  • And there is this region here that's

  • described as a mobile loop.

  • And if you look, the beta sheet region's on top,

  • and this mobile loop is down where it binds to GroEL.

  • OK, so effectively, when GroES docks onto GroEL,

  • these mobile loops bind to hydrophobic peptide binding

  • pockets that are on the top of this heptamer there.

  • OK, here's another depiction.

  • So you're seeing the beta sheet parts on top,

  • and here are the mobile loops that

  • can bind to peptide binding grooves of GroEL.

  • AUDIENCE: So does that mean the inner cavity of the cis

  • part of GroEL is always open I guess after GroES binds?

  • And trans is always closed?

  • Because it looks like just from the way we've drawn it.

  • Does GroES bind to the other side also?

  • ELIZABETH NOLAN: Yeah, so right, this is how we've drawn it.

  • We've drawn it like a bullet.

  • And so does GroES bind to the other side?

  • And how do these two chambers function?

  • OK, and as we move forward getting

  • to the functional cycle, what we'll see

  • is that both rings are functional,

  • but they're functional at different points in the cycle.

  • OK, so GroES, yes, can bind to either one,

  • but it's this bullet type of shape that

  • is considered to be functional.

  • So you might ask, what about a football?

  • If we stick another GroES on the bottom,

  • we get a football-shaped species.

  • And there are some in vitro studies

  • that show a formation of a football with two GroEL rings

  • and two GroES rings, but those are found at very high ATP

  • concentrations.

  • And so it's thought that they may not be significant,

  • that they're a transient species effectively

  • of unknown significance that at least in the test tube,

  • you can form at very high ATP.

  • OK, yeah?

  • AUDIENCE: And then is the cis and trans, it's not predefined,

  • just it depends on wherever the GroES makes it?

  • ELIZABETH NOLAN: Right.

  • It's going to depend on wherever the GroES is.

  • OK, and what we'll see as we move forward

  • is we need to think about also how ATP binds.

  • And ATP binding will also happen in one or the other,

  • depending at the point in the functional cycle here, OK?

  • So we just want to get the structural aspects

  • under control before we look at the functional cycle.

  • So this is one last slide on the structure.

  • And so I find this to be a really beautiful machine.

  • Here we have the bullet-shaped two GroELs and one GroES.

  • And here we have the different domains colored.

  • And here what we have is a cutaway view

  • to look at the interior of the chambers.

  • And so we have the cis chamber on top,

  • the trans chamber on bottom.

  • And in the color-coding here for this cross-section, what

  • we have in yellow are hydrophobic residues,

  • and in cyan, hydrophilic residues.

  • OK, and so what's important to do

  • is take a look at the cis chamber and the trans chamber

  • and ask, what's going on in the interior?

  • And why might that be important?

  • So what do we see comparing the distribution

  • of yellow and cyan, or hydrophobic and hydrophilic?

  • Lindsay?

  • AUDIENCE: It's much more hydrophobic

  • in the trans chamber.

  • ELIZABETH NOLAN: Yeah, right.

  • The trans chamber is much more hydrophobic

  • in terms of that lining than the cis.

  • So the cis chamber, as we'll see in a minute,

  • is where the polypeptide will be folding.

  • So a polypeptide will end up in the cis chamber,

  • and the lid will be on top.

  • So why might this be an important feature--

  • not only that we need this cavity size to grow,

  • but we need a change in the lining to be more hydrophilic?

  • AUDIENCE: Because if it's assisting folding,

  • it's likely that the hydrophobic residues are

  • more likely to be buried in the center

  • the protein in the polypeptide.

  • So you'd want to facilitate favoring

  • the hydrophilic residues to be interacting

  • on the outside of the protein?

  • ELIZABETH NOLAN: Yeah, so often, that's right to think about.

  • Where do we find different types of residues,

  • say, in a protein with a complex fold?

  • And typically, we think about hydrophobic residues

  • on the interior and hydrophilic residues on the exterior.

  • So for instance, there is a model

  • of folding called hydrophobic collapse.

  • And effectively, you have hydrophobic interactions,

  • and then the rest of folding occurs, right?

  • So you'd imagine there's a benefit to having

  • a hydrophilic exterior if you want

  • the exterior of the protein to be hydrophilic here.

  • So what is the functional cycle?

  • And in thinking about this, we need

  • to think about ATPs and ATP hydrolysis.

  • And what you'll find as you read is that often the model is

  • drawn a bit differently depending on the paper

  • you read.

  • And that's because they're just some uncertainties out there.

  • So don't get hung up on that.

  • I have two different examples within the lecture slides here.

  • OK, but if we just think about the functional cycle--

  • and I'll just draw a little bit, and then we'll go to the board.

  • So imagine we have one GroEL here, OK?

  • And as drawn here, there's no GroES.

  • There's no peptide, and there's no ATP.

  • So imagine some peptide comes in that

  • needs to be folded by this machinery and ATP.

  • OK, and these end up inside of the chamber.

  • And then we can have our lid come in.

  • OK, so now this polypeptide is in this protected cavity,

  • and ATP can bind in the equatorial domain of GroEL.

  • So each GroEL monomer will bind one ATP.

  • So there's seven ATP bound in one heptamer

  • if it's in the ATP-bound form, OK?

  • And so let's take a look in a little more detail.

  • So what do we see?

  • And the thing to keep in mind, as I said before,

  • is that both chambers are active and functional.

  • They're just functional at different points

  • within this overall cycle.

  • OK, so if we begin here, what do we see?

  • This top GroEL heptamer has no cap.

  • What we see here is that we have the bottom GroEL bound

  • to ADP and GroES.

  • Some unfolded polypeptide comes along.

  • It binds, so maybe there's some hydrophobic interaction

  • between the top of GroEL and some region

  • of this polypeptide.

  • What do we see happening?

  • The ATPs come in, so I indicated them together, there's

  • some timing where there's questions.

  • These ADPs from the bottom chamber are ejected.

  • We see ATP binding, so there's seven--

  • one per subunit.

  • The polypeptide binds, and here comes GroES.

  • OK, and so once this polypeptide is

  • encapsulated in this chamber, there's some residency time.

  • And this is often quoted on the order of 10 seconds.

  • Also note here.

  • Look what happened at the bottom ring.

  • GroES got ejected.

  • OK, so with GroES binding here, there

  • was ejection of GroES from the bottom and loss of these ADPs.

  • OK, there's ATP hydrolysis during this time.

  • The polypeptide is trying to find its fold.

  • And then look what happens here.

  • We see GroES coming into the bottom.

  • Again, we have release of ADPs, release of GroES,

  • and this polypeptide kicked out, which may or may not

  • be in its native fold, OK?

  • If we take a look showing this as a complete cycle here--

  • and again, I said before there can be some differences

  • from depiction to depiction--

  • but here, we are seeing GroEL.

  • We have the top one and the bottom heptamer.

  • Here's some polypeptide that needs to be folded.

  • It's initially grabbed by the top part of GroEL.

  • ATP comes in.

  • We have this ATP-bound form.

  • Here comes GroES.

  • The polypeptide gets pushed into this chamber,

  • and now it's closed.

  • We have ATPase activity, so ATP hydrolysis

  • to give the ADP-bound form.

  • OK, and then what happens here?

  • OK, what we're seeing now, this bottom ring

  • is becoming functional.

  • ATP binds another polypeptide.

  • OK, and then we have release of GroES in the polypeptide

  • from the top chamber.

  • OK, and then you can flip this and work around the cycle

  • again.

  • OK, so this is a case where we can

  • think about the affinities of the ATP

  • and the ADP-bound forms of GroEL and what that

  • means GroES binding here, OK?

  • And so the ADP-bound form of GroEL

  • has a lower affinity for GroES than the ATP-bound form

  • here for that.

  • So each GroEL heptamer acts as a single functional unit,

  • and both rings are active as shown here

  • but in different points of the cycle.

  • OK, and so the thinking is that ATP binding and hydrolysis

  • drives uni-directional progression through this cycle.

  • With that said, there's a lot of questions as to how.

  • So what is it about this ATP binding and hydrolysis event

  • that allows this work to happen?

  • That's a question that I see as still pretty open.

  • And so I'll close with that here now.

  • I suggest to review this cycle before next time.

  • And what we'll address on Wednesday

  • is experiments that have been done

  • to sort out what are the polypeptide substrates

  • for GroEL/GroES.

  • So we know they must be some important players given

  • that these are essential for viability.

  • What are they?

  • And how is that determined?

The following content is provided under a Creative

字幕と単語

ワンタップで英和辞典検索 単語をクリックすると、意味が表示されます

B1 中級

9.タンパク質の折りたたみ 2 (9. Protein Folding 2)

  • 1 0
    林宜悉 に公開 2021 年 01 月 14 日
動画の中の単語